CD73 expression in normal, hyperplastic, and neoplastic thyroid: a systematic evaluation revealing CD73 overexpression as a feature of papillary carcinomas

CD73 converts AMP to adenosine, an immunosuppressive metabolite that promotes tumorigenesis. This study presents a systematic evaluation of CD73 expression in benign, hyperplastic, and neoplastic thyroid. CD73 expression was assessed by immunohistochemistry in 142 thyroid samples. CD73 was expressed in normal thyroid (3/6) and goiter (5/6), with an apical pattern and mild intensity. Apical and mild CD73 expression was also present in oncocytic cell adenomas/carcinomas (9/10; 5/8) and in follicular adenomas/carcinomas (12/18; 23/27). In contrast, papillary thyroid carcinomas featured extensive and intense CD73 staining (49/50) (vs. normal thyroid/goiter, p < 0.001). Seven of nine anaplastic carcinomas were CD73-positive with heterogeneous extensiveness of staining. Medullary and poorly differentiated carcinomas were mostly CD73-negative (1/6; 2/2). These results were corroborated by NT5E mRNA profiling. Papillary carcinomas feature enhanced CD73 protein and mRNA expression with distinct and intense staining, more pronounced in the invasive fronts of the tumors. Supplementary Information The online version contains supplementary material available at 10.1007/s00428-021-03100-x.


Introduction
CD73, encoded by the NT5E gene, is an ectonucleotidase which produces adenosine [1]. Adenosine is often present in the tumor microenvironment and can lead to immunosuppression and epithelial-mesenchymal transition of tumor cells, correlating with poor prognosis in several tumor types [1,2]. Also, higher levels of CD73 correlate with refractoriness to cancer treatments [1,3].
New evidence pointing the possibility to restore immune response via CD73 blockade has triggered a characterization of CD73 expression in human cancers [4].
Few data are available on CD73 expression in thyroid cancer. Increased CD73 expression and enzymatic activity in papillary thyroid carcinoma (PTC, n = 3) vs. normal thyroid, goiter, and follicular adenoma (n = 9) was reported [5]. Another report described increased NT5E mRNA and protein levels in PTC vs. normal thyroid cell lines [6]. The same group showed that the PTC/adjacent paired expression ratio of NT5E mRNA was associated with metastatic lymph nodes and tumor size and that all PTC (n = 29) were CD73-positive by immunohistochemistry (IHC) [7]. Inês Monteiro, Edoardo Missiaglia, and Amedeo Sciarra contributed equally to this work.
In the present study, we examine CD73 expression using both IHC and mRNA profiling in a large series of normal, hyperplastic, and neoplastic thyroid. This study aims to complete and nuance the early data regarding CD73 expression in thyroid neoplasms.

Methods
Formalin-fixed paraffin-embedded surgical thyroid specimens were retrieved from the archives of the Institute of Pathology of the Lausanne University Hospital (1995-2019). Cases with the desired coded diagnosis, patient consent, and available material were chosen. Six cases each of "normal thyroid" and "goiter" were chosen. For the remaining diagnostic categories, a total of 130 eligible cases were found. One block per patient was selected. All diagnoses were confirmed.
Immunohistochemistry, RNA profiling using HTG EdgeSeq Oncology Biomarker Panel, and quantification of tumor-infiltrating mononuclear cells (TIMC) are described in Supplementary Methods.

CD73 immunohistochemical expression
Results are summarized in Table 1.
In oncocytic adenomas and carcinomas, CD73 was not expressed by cells arranged in a trabecular pattern; nonetheless, follicular areas presented a mild apical staining (9/10, 90%, median H-score 11.25; 5/8, 63%, median H-score 17.5, respectively) (Fig. 1c). Only one case of oncocytic adenoma presented foci of tumor cells staining with strong intensity. The H-scores of oncocytic tumors did not significantly differ from those of normal thyroid and goiter (p = 0.781).
Interestingly, PTC with follicular morphology (n = 17) presented a staining pattern similar to that of classical PTC (staining extending to the basolateral membrane in 11/17 cases, of moderate/strong intensity in 12/17 cases) ( Fig. 1e-f).
Remarkably, all cases of primary PTC with an invasive front (n = 23) distinctly comprised a higher proportion of tumor cells staining and/or higher intensity of staining in the invasive front, in comparison to the rest of the tumor (median H-score of the invasive fronts = 225, median global H-score of the respective tumors = 140, p < 0.001) ( Fig. 1j-l).
Two cases of poorly differentiated carcinomas expressed CD73 with mild apical staining (2/2, 100%, median H-score 8.75) but only in small areas where cells were organized in follicles (Fig. 1g).
The majority of anaplastic carcinomas were CD73-positive (7/9, 78%, median H-score 50), presenting membrane and cytoplasmic staining (Fig. 1h). Inter-and intralesional staining was heterogeneous in terms of percentage of CD73-positive cells and staining intensity. This heterogeneity was reflected by an H-score of the positive cases (n = 7) ranging from 10 to 237.5 (Fig. 2a). The H-scores of anaplastic carcinomas did not significantly differ from those of normal thyroid and goiter (p = 0.113).
Finally, most medullary carcinomas were CD73-negative ( Fig. 1i) as was also the case with hyperplastic parafollicular C-cells identified in the adjacent thyroid parenchyma (confirmed with calcitonin staining) in one case of multiple endocrine neoplasia. Only one medullary carcinoma contained a small proportion (5%) of tumor cells with faint CD73 staining (1/6, 17%, median H-score 0). The H-score of medullary carcinomas was significantly lower compared to normal thyroid/ goiter (p = 0.035).

Tumor-infiltrating immune cells
Adenosine leads to immunosuppression by several mechanisms, namely by hampering immune effector cell functions and contributing to T cell exhaustion [8]. First, we seeked to characterize the density of TIMC in the primary malignant lesions (n = 85) and the eventual correlation with CD73 expression. Most lesions presented no or few TIMC. Only anaplastic carcinomas presented mostly a moderate or abundant quantity of TIMC. The abundance of TIMC did not correlate with CD73 expression in the tumor cells (one-way ANOVA p = 0.776) (Supplementary Fig. S1a).

Discussion
In this report, we evaluated CD73 expression in a series acc o u n t i n g f o r m o s t t h y r o i d l e s i o n s , u s i n g a n immunohistochemistry assay corroborated by an mRNA analysis in a subset of cases.
Strikingly, we observed that PTC showed enhanced CD73 expression beyond the apical membrane, into the basolateral membrane and the cytoplasm, and that this phenomenon was even more prominent in the invasive front. This might reflect a functional role for CD73 in PTC invasiveness. Indeed, a recent study demonstrated that CD73 inhibition suppressed PTC cell migration in vitro and that CD73 expression was associated with lymph node metastasis, suggesting that CD73 is involved in the dissemination of PTC [9].
This extended pattern of staining was preserved in PTC with follicular morphology. This feature suggests that CD73 IHC could be of use in diagnostic practice to help distinguish follicular-patterned lesions which may pose the differential diagnosis between follicular versus papillary carcinoma.
The theory that anaplastic carcinomas usually develop from dedifferentiation of differentiated thyroid carcinomas is generally accepted [10]. Given the heterogeneity of CD73 expression in these carcinomas, we conjecture that tumors with higher H-scores could potentially have developed from PTC, while negative or faintly positive tumors could have developed from follicular carcinomas.  Mild staining intensity of 1 or 1.5, moderate staining intensity of 2, strong staining intensity of 2.5 or 3 CD73 overexpression seen in papillary and anaplastic tumors might relate to the tumor microenvironment. It has been reported that mesenchymal stromal cells increase CD73 expression in tumor cells through TGF-β1 production [11]. Interestingly, in the 21 samples analyzed by HTG EdgeSeq, NT5E and TGF-β1 mRNA were positively correlated (Spearman's rank correlation p = 0.04) (data not shown).
Most medullary carcinomas and non-neoplastic parafollicular C-cells were CD73-negative, suggesting an absent CD73 expression in normal and neoplastic thyroid endocrine cells. A similar observation was made in our previous study regarding pancreatic neuroendocrine tumors [12].
Our results regarding CD73 expression at the protein and mRNA levels are corroborated by the NT5E gene analysis retrieved from the Gene Expression Omnibus (GEO) database (GSE27155 set) [13] (Supplementary information Fig. S2).
No relationship between CD73 expression in the primary tumor and either the density of TIMC or the mRNA expression of T cell markers was found. In accordance with our findings, a recent study found no correlation between CD73 and CD8 transcript levels. However, a positive correlation between the expression of CTLA4, CD274, PDCD1, and CD73 transcript levels was reported [9]. The number and distribution of tumor-infiltrating immune cells are determined by several factors, namely, the neovascular permeability, the Hematoxylin and eosin (upper panels) and corresponding CD73 IHC (lower panels) at various magnifications of an invasive front of a papillary thyroid carcinoma; j panoramic view of a papillary thyroid carcinoma with a stronger CD73 staining along the invasion front (left edge) compared to the central portion of the lesion; k (inset of (j), rotated) strong and complete membrane and cytoplasmic staining of the invasive front; l (inset of (j), rotated) less intense apical membrane staining of the central part of the lesion. Images a-i and respective insets were taken at an original magnification of × 2 and × 18, respectively. Images j-l were taken at an original magnification of × 0.23, × 6.40, and × 6.40, respectively gradients of various cytokines, and the composition of the extracellular matrix [14,15]. Adenosine is likely to act locally, impairing immune cells in a paracrine fashion. Still, functional studies are needed to characterize the effect of CD73 expression in tumor-infiltrating immune cells.
In conclusion, CD73 is highly expressed in PTC, presenting a distinct IHC pattern. The high CD73 expression observed in PTC encourages to further explore the use of therapies targeting the CD73-adenosine pathway in this entity.