ELSEVIER
NEOPLASTIC DISEASE
Immunohistochemical Detection of Pax8 and Napsin A in Canine Thyroid Tumours: Comparison with Thyroglobulin, Calcitonin and Thyroid Transcription Factor 1
J. A. Ramos-Vara”, C. B. Frank, D. DuSold* and M. A. Miller*
* Department of Comparative Pathobiology, Purdue University, 406 South University St., West Lafayette, IN and Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA
Summary
Expression of thyroid transcription factor (TTF)-1 corroborates a thyroid origin of neoplasms. Thyroglobulin and calcitonin immunohistochemistry (IHC) can distinguish between a follicular and C-cell origin of thyroid tumours, respectively. Pax8 (expressed by normal canine thyroid follicular cells) and napsin A (expressed mainly by C-cells) labelling was compared with labelling for TTF-1, thyroglobulin and calcitonin in 114 canine proliferative thyroid lesions. All 81 follicular tumours expressed thyroglobulin and were negative for calcitonin; 79/81 (98%) of these tumours expressed TTF-1 and Pax8 and 60/81 (74%) expressed napsin A. All 25 C-cell lesions expressed calcitonin and were negative for expression of thyroglobulin; 22 (88%) were pos- itive for TTF-1, 13 (57%) for Pax8 and 24/24 for napsin A. Six mixed follicular-medullary carcinomas ex- pressed all five markers. Both carcinosarcomas expressed TTF-1 and napsin A, and one each of these tumours expressed thyroglobulin, calcitonin or Pax8. Pax8 expression was also detected in epididymal cells, endometrial cells and vas deferens epithelium, in Sertoli-like ovarian cells, and in some cases of ovarian ade- noma, pancreatic carcinoma, renal cell carcinoma and Sertoli cell tumour. Napsin A was also detected in adre- nocortical cells, ovarian granulosa cells, epididymal and endometrial cells, as well as in some renal cell carcinomas, pulmonary adenocarcinomas and Sertoli cell tumours. In summary, Pax8 was as sensitive as TTF-1 and slightly less sensitive than thyroglobulin for identification of follicular tumours, but had low sensi- tivity for C-cell tumours. Napsin A was as sensitive as calcitonin for C-cell neoplasms, but was less sensitive than thyroglobulin for follicular neoplasms. Thus, these markers are sensitive and, except for renal cell carcinoma (for Pax8, napsin A) and pulmonary adenocarcinoma (for napsin A), are specific thyroid tumour markers.
@ 2016 Elsevier Ltd. All rights reserved.
Keywords: dog; napsin A; Pax 8; thyroid neoplasms
Introduction
An estimated 1.1-3.8% of all canine tumours are of thyroid origin (Lunn and Page, 2013). Using inva- siveness or metastasis as criteria of malignancy, most canine thyroid tumours are classified as carcinomas (Ramos-Vara et al., 2002). The histological diagnosis of canine thyroid neoplasms can be straightforward,
but immunohistochemistry (IHC) is needed to distin- guish some follicular tumours, particularly compact (solid) tumours, from medullary tumours (Ramos- Vara et al., 2002). Thyroglobulin and calcitonin, products of follicular and medullary C cells, respec- tively, are two commonly used immunohistochemical markers. Thyroglobulin is detected in 90-100% of canine thyroid carcinomas and calcitonin in 70-100% of medullary carcinomas (Ramos-Vara et al., 2002).
Correspondence to: J. A. Ramos-Vara (e-mail: ramosja@purdue.edu). 0021-9975/$ - see front matter
http://dx.doi.org/10.1016/j.jcpa.2016.07.009
Please cite this article in press as: Ramos-Vara JA, et al., Immunohistochemical Detection of Pax8 and Napsin A in Canine Thyroid Tumours: Comparison with Thyroglobulin, Calcitonin and … , Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.07.009
J.A. Ramos-Vara et al.
Thyroid transcription factor (TTF)-1, a member of the NKX-2 gene family of homeodomain-containing transcription nuclear factors involved in morphogen- esis and cytodifferentiation (Pelosi et al., 2001), is de- tected in the thyroid, brain and lung during embryogenesis and in postnatal thyroid and bron- chioloalveolar cells (Pelosi et al., 2001; Ramos-Vara et al., 2002, 2004; Ordóñez, 2012a). TTF-1 is ex- pressed by follicular cells and C cells of the normal thyroid gland in a variety of animal species (Fabbro et al., 1994; Holzinger et al., 1996; Bejarano et al., 2000; Katoh et al., 2000; Ordóñez, 2000; Zane et al., 2016). Human thyroid follicular and C-cell tumours also express this marker (Bejarano et al., 2000; Hinze et al., 2000; Katoh et al., 2000). Canine follicular and medullary thyroid tumours are also positive for TTF-1, with a sensitivity of 90% and 50%, respectively (Ramos-Vara et al., 2002).
Pax8 is a transcription factor with a significant role in the organogenesis of the kidney, thyroid, nervous system and organs derived from mesonephric and müllerian ducts (Chi and Epstein, 2002; Ordóñez, 2013; Zane et al., 2016). Pax8 is used in human oncology mainly in the diagnosis of renal and ovarian carcinomas and those of müllerian duct origin (Nonaka et al., 2008a; Lotan et al., 2009; Laury et al., 2010, 2011; Sangoi et al., 2010, 2011; Tong et al., 2009, 2010; Wiseman et al., 2011; Tacha
et al., 2011; Hu et al., 2012; Knoepp et al., 2012; Ordóñez, 2012b, 2013; Ozcan et al., 2012; Zhao et al., 2012; Clayton et al., 2013; Mentrikoski et al., 2014; Zhang et al., 2014). Pax8 has been used sporadically as a marker in the diagnosis of human thyroid neoplasms, with a sensitivity of 50-91%, this sensitivity being higher for papillary or follicular tumours than for medullary tumours (Nonaka et al., 2008a,b; Tacha et al., 2011; Ye et al., 2012).
Napsin A is an aspartic proteinase detected in normal type II pneumocytes, alveolar macrophages, renal proximal tubular epithelium and exocrine pancreatic cells (Chuman et al., 1999; Schauer- Vukasinovic et al., 1999; Hirano et al., 2000), as well as many pulmonary and renal neoplasms (Chuman et al., 1999; Schauer-Vukasinovic et al., 1999; Hirano et al., 2000; Ye et al., 2011; Ordóñez, 2012c; Whithaus et al., 2012; Matsukuma et al., 2013; Li et al., 2014; Sun et al., 2014; Xu et al., 2014; Zhu et al., 2015). Napsin A has also been detected in human ovarian clear cell carcinomas (Yamashita et al., 2015). It is expressed variably in up to 50% of human thyroid carcinomas, mostly in those of papil- lary origin (Suzuki et al., 2005; Bishop et al., 2010; Mukhopadhyay and Kantzenstein, 2012; Turner et al., 2012; Kadivar and Boozari, 2013; Xue et al., 2013 Daniel et al., 2015).
| Table 1 Summary of immunohistochemical data | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Calcitonin | Napsin A* | Pax 8 | Thyroglobulin | TTF-11 | ||||||||||||||||
| - | 1+ | 2+ | 3+ | - | 1+ | 2+ | 3 3+ | e | 1+ | 2+ | 3+ | e | 1+ | 2+ | 3+ | - | 1+ | 2+ | 3+ | |
| Carcinosarcoma (n = 2) | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 2 |
| C-cell adenoma (n = 1) | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
| Compact carcinoma (n=11) | 11 | 0 | 0 | 0 | 4 | 2 | 2 | 3 | 0 | 0 | 0 | 11 | 0 | 0 | 3 | 8 | 0 | 0 | 0 | 11 |
| Follicular carcinoma (n = 18) | 18 | 0 | 0 | 0 | 3 | 6 | 3 | 6 | 0 | 0 | 0 | 18 | 0 | 1 | 0 | 17 | 0 | 0 | 0 | 18 |
| Follicular compact carcinoma (n = 47) | 47 | 0 | 0 | 0 | 12 | 10 | 12 | 13 | 1 | 0 | 0 | 46 | 0 | 3 | 7 | 37 | 0 | 0 | 3 | 44 |
| Follicular papillary carcinoma (n = 2) | 2 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 2 |
| Medullary carcinoma (n = 23) | 0 | 1 | 2 | 20 | 0 | 4 | 4 | 14 | 10 | 1 | 3 | 9 | 23 | 0 | 0 | 0 | 2 | 1 | 2 | 18 |
| Medullary hyperplasia (n=1) | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| Mixed | 0 | 1 | 0 | 5 | 0 | 0 | 2 | 4 | 0 | 2 | 1 | 3 | 0 | 3 | 0 | 3 | 0 | 3 | 0 | 3 |
| follicular-medullary carcinoma (n = 6) | ||||||||||||||||||||
| Papillary carcinoma (n=3) | 3 | 0 | 0 | 0 | 0 | 0 | 1 | 2 | 2 | 0 | 1 | 0 | 0 | 0 | 1 | 2 | 2 | 0 | 1 | 0 |
Tumours are scored for immunoreactivity (% of positive cells): - , <5% of cells labelled; 1+, 5-15% of cells labelled; 2+, 16-50% of cells labelled; 3+, 50-100% of cells labelled.
One medullary carcinoma was not tested for napsin A.
TTF-1, thyroid transcription factor-1.
Pax8 and Napsin A in Canine Thyroid Tumours
The aims of this study were: (1) to evaluate the immunohistochemical expression of Pax8 and napsin A in canine thyroid neoplasms; (2) to compare Pax8 and napsin A immunoreactivities with those of thyro- globulin, calcitonin and TTF-1; and (3) to evaluate the cross-reactivity of Pax8 and napsin A in normal and neoplastic canine tissues.
Materials and Methods
Case Selection and Histopathological Analysis
A search of the Animal Disease Diagnostic Labora- tory formalin-fixed, paraffin wax-embedded tissue archive for cases between 2004 and 2014 was per- formed. A total of 114 cases of canine thyroid lesions
were selected based on the quality of samples avail- able for IHC. All tumours were reviewed and re- classified independently by the authors, based on the World Health Organization histological classifi- cation of thyroid neoplasms (Kiupel et al., 2008). The tumours included two carcinosarcomas, one C- cell adenoma, one example of C-cell hyperplasia, 11 compact carcinomas, 18 follicular carcinomas, 47 follicular compact carcinomas, two follicular papil- lary carcinomas, 23 medullary carcinomas, six mixed follicular-medullary carcinomas and three papillary carcinomas (Table 1). Follicular or medullary origin was confirmed by IHC: strong immunoreactivity for thyroglobulin and no reactivity for calcitonin was in- terpreted as the tumour having a thyroid follicular
| Table 2 Immunohistochemical labelling of non-thyroid tumours | ||||
|---|---|---|---|---|
| Tumor | Organ | No. cases | Pax8 positive, (n %) | Napsin A positive, (n %) |
| Adenoma | Adrenal cortex | 1 | 0 | 0 |
| Adenoma | Ovary | 1 | 1 (100) | 0 |
| Adenoma | Parathyroid | 5 | 0 | 0 |
| Adenosquamous carcinoma | Mammary | 1 | 0 | 0 |
| Basal cell carcinoma | Skin | 3 | 0 | 0 |
| Carcinoma | Adrenal cortex | 3 | 0 | 0 |
| Carcinoma | Anal gland | 4 | 0 | 0 |
| Carcinoma | Kidney | 5 | 3 (60) | 2 (40) |
| Carcinoma | Large intestine | 6 | 0 | 0 |
| Carcinoma | Lung | 5 | 0 | 5 (100) |
| Carcinoma | Mammary | 5 | 0 | 0 |
| Carcinoma | Pancreas | 5 | 1 (20) | 0 |
| Carcinoma | Parathyroid | 2 | 0 | 0 |
| Carcinoma | Salivary gland | 2 | 0 | 0 |
| Carcinoma | Small intestine | 5 | 0 | 0 |
| Carcinoma | Stomach | 8 | 0 | 0 |
| Ceruminous carcinoma | Skin | 4 | 0 | 0 |
| Dysgerminoma | Ovary | 1 | 0 | 0 |
| Granulosa cell tumour | Ovary | 2 | 0 | 0 |
| Hemangiosarcoma | Various organs | 5 | 0 | 0 |
| Hepatocellular carcinoma | Liver | 4 | 0 | 0 |
| Hepatoid gland carcinoma | Skin | 4 | 0 | 0 |
| Histiocytic cell sarcoma | Spleen | 3 | 0 | 0 |
| Hyperplasia | Lymph node | 1 | 0 | 0 |
| Hyperplasia | Parathyroid | 1 | 0 | 0 |
| Interstitial cell tumour | Testis | 2 | 0 | 0 |
| Lymphoma | Lymph node | 8 | 0 | 0 |
| Melanoma | Oral | 5 | 0 | 0 |
| Mesothelioma | Various organs | 3 | 0 | 0 |
| Osteosarcoma | Oral | 3 | 0 | 0 |
| Pheochromocytoma | Adrenal gland | 2 | 0 | 0 |
| Sebaceous carcinoma | Skin | 4 | 0 | 0 |
| Seminoma | Testis | 4 | 0 | 0 |
| Sertoli cell tumour | Testis | 5 | 1 (20) | 1 (20) |
| Squamous cell carcinoma | Skin | 4 | 0 | 0 |
| Thymoma | Thymus | 1 | 0 | 0 |
| Trichoblastoma | Skin | 1 | 0 | 0 |
| Urothelial carcinoma | Urinary bladder | 5 | 0 | 0 |
| Total | 133 | |||
| Target antigen | Clone | Manufacturer | Dilution | Antigen retrieval method* | Incubation time (min) |
|---|---|---|---|---|---|
| Calcitonin | Polyclonal | Dako, Carpinteria, California, USA | 1 in 800 | Reveal+ | 30± |
| Napsin A | ACI 3043 | Biocare Medical, Concord, California, USA | 1 in 100 | Divas | 50 |
| Pax 8 | BC12 | Biocare Medical | 1 in 200 | Diva | 60 |
| Thyroglobulin | Polyclonal | Thermo Shandon Immunon, Waltham, Massachusetts, USA | 1 in 600 | Proteinase K | 60 |
| TTF1 | 8G7/G3/1 | Dako | 1 in 800 | Diva | 60 |
*Antigen retrieval was performed in a decloaker chamber for all antibodies.
+Reveal (citrate buffer, pH 6.0).
*All incubations except for antigen retrieval at room temperature.
$Diva (Tris buffer, pH 6.0).
origin; conversely, strong immunoreactivity for calci- tonin and no reactivity for thyroglobulin was consis- tent with a medullary origin.
A total of 243 formalin-fixed, paraffin wax- embedded non-thyroid canine tissue samples were used to evaluate the cross-reactivity of Pax8 and nap- sin A antibodies. These samples comprised of 108 normal tissues, including: adrenal gland (n = 6), fetal tissues (n=1), gallbladder (n=2), heart (n=1), large intestine (n = 7), liver (n = 4), small intestine (n=8), lymph node (n = 4), lung (n = 2), mammary gland (n= 3), ovary (n=2), pancreas (n=9), parathyroid gland (n = 5), pituitary (n = 1), prostate (n = 2), sali- vary gland (n=1), skin (n= 14), spleen (n= 7), stom- ach (n = 7), testis (n = 7), thymus (n = 1), thyroid (n = 8), tonsil (n=2), urinary bladder (n=1) and uterus (n = 3). In addition, non-thyroid neoplasms (n = 133) including carcinomas, sarcomas, lym- phomas and melanomas were examined (Table 2).
Immunohistochemistry
Samples from surgical biopsy or necropsy examina- tions were fixed at room temperature in neutral buff- ered formalin for 1-3 days and embedded in paraffin wax, following standard procedures. Sections (4 um) were placed on positively-charged glass slides. A non- avidin-biotin peroxidase system (Promark™M, Bio- care Medical, Concord, California, USA) was applied using an automated stainer (Intellipath™, Biocare Medical) and reagent incubations were at room temperature. Labelling was ‘visualized’ by incubating the slides in 3,3’-diaminobenzidine (Bio- care Medical). All thyroid tumours were evaluated for immunoreactivity to antibodies to thyroglobulin, calcitonin, TTF-1, Pax8 and napsin A (one medul- lary tumour was not tested for napsin A). The details
of antibodies, dilutions, antigen retrieval procedures and incubation times are given in Table 3. Positive controls were normal canine thyroid gland for calci- tonin, thyroglobulin and TTF-1, and normal canine kidney for Pax8 and napsin A. The negative reagent control was a polymer negative control serum (Bio- care Medical) for both monoclonal and polyclonal antibodies.
Analysis was performed by evaluating neoplastic areas of each sample. Reactions were scored by per- centage of labelled neoplastic cells: negative, <5% of cells labelled; 1+, 5-15% of cells labelled; 2+, 16-50% of cells labelled; or 3+, >50% of cells labelled. Positive labelling for calcitonin, thyroglob- ulin and napsin A was cytoplasmic; for TTF-1 and Pax8, the labelling was nuclear.
Effect of Prolonged Fixation on Pax8 and Napsin A Immunoreactivity
The exact fixation time of many of the thyroid or other tumours was unknown. Therefore, we evalu- ated the effect of prolonged fixation on Pax 8 and nap- sin A immunoreactivity in normal kidney and lung, which are well-documented positive tissue controls in human diagnostics for Pax8 and napsin A, respec- tively. Samples from the same tissue specimen were fixed for 2 days, and 1, 2, 3, 4 and 5 weeks. Immuno- labelling was compared between these samples by semiquantitative evaluation of the percentage of labelled cells and subjective grading of intensity (Ramos-Vara et al., 2002).
Results
Based on the microscopical features and immunohis- tochemical profile for thyroglobulin and calcitonin, there were 81 tumours of follicular origin (18
Pax8 and Napsin A in Canine Thyroid Tumours
follicular carcinomas, 47 follicular compact carci- nomas, two follicular papillary carcinomas, 11 compact carcinomas and three papillary carci- nomas), 25 of medullary origin (one case of C-cell hy- perplasia, one C-cell adenoma and 23 medullary carcinomas), two carcinosarcomas and six mixed fol- licular-medullary carcinomas (Table 1). Eight of the samples examined were metastatic thyroid tumours.
Pax8 was expressed by normal thyroid follicular cells (Fig. 1), while napsin A expression was detected in interfollicular C cells and much less commonly in cells lining follicles (Fig. 2). Immunoreactivity for
*
Pax8 was weaker than that for TTF-1 in most medul- lary carcinomas, but Pax8 labelling was stronger in other thyroid tumours. Nuclear labelling for Pax8 was slightly heterogeneous within the same specimen and this was more apparent with napsin A. Labelling intensity for Pax8 was typically stronger in normal en- trapped follicular epithelial cells when compared with tumour cells.
The overall immunoreactivity for Pax8, napsin A and TTF-1 for canine thyroid tumours was 99/114 (87%), 70/113 (62%) and 109/114 (96%), respec- tively. All follicular tumours were positive for thyro- globulin (n = 81); TTF-1 and Pax8 expression was present in all tumours except for two papillary carci- nomas (Figs. 3-6). Napsin A expression in follicular tumours was much lower than for the above mentioned markers, with only 60 (74%) of the tumours showing immunoreactivity; papillary carcinomas were consistently positive (100%) for napsin A, while the two follicular papillary carcinomas were negative. Napsin A immunoreactivity for other follicular tumours ranged from 64 to 83% (Table 1). The overall immu- noreactivity for medullary tumours for TTF-1, Pax8 and napsin A was 88% (n = 22), 57% (n = 13) and 100% (n = 24), respectively (Figs. 7-10). One medullary carcinoma was not tested for napsin A. All (n = 6) mixed follicular-medullary carcinomas expressed thyroglobulin, calcitonin, TTF-1, Pax8 and napsin A. The two carcinosarcomas were positive for TTF-1 and napsin A, while one was positive for thyroglobulin and Pax8; the other carcinosarcoma was positive for calcitonin and negative for Pax8.
Of the eight metastatic tumours, seven were consid- ered to be of follicular origin by morphology and/or thyroglobulin/calcitonin phenotyping (results not shown). All eight metastases were positive for TTF1 and Pax8, and four were also positive for napsin A, including the tumour of medullary origin.
0
Pax8 and Napsin A Expression in Non-thyroid Normal and Neoplastic Tissues
For normal tissues, strong Pax8 immunoreactivity was observed in clusters of Sertoli-like cells in one of two ovaries. Nuclear labelling in the epididymal epithelium (strong and diffuse) and vas deferens was observed in six of seven and one of seven samples,
Please cite this article in press as: Ramos-Vara JA, et al., Immunohistochemical Detection of Pax8 and Napsin A in Canine Thyroid Tumours: Comparison with Thyroglobulin, Calcitonin and … , Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.07.009
respectively (Fig. 11). The endometrial epithelium was consistently and strongly labelled (3/3 tissues). Thyroid follicular cells were consistently positive in all cases. Normal tissues positive for napsin A included
one of six adrenal cortices (scattered and weakly- positive cells), strong labelling of fetal small airway epithelium (1/1 tissue), one of two ovarian follicular granulosa cells, variable cytoplasmic labelling of epididymal epithelium (six of seven tissues), strong labelling of thyroid parafollicular (C) cells (six of eight tissues) and uterine endometrial cells (one of three tissues) (Figs. 12 and 13). Pneumocytes and some bronchiolar epithelial cells were positive for napsin A (2/2 tissues).
For the 133 non-thyroid neoplasms evaluated, only six were positive for Pax8, including three renal cell carcinomas, one ovarian adenoma, one pancreatic
carcinoma and very few and weakly-labelled cells in one Sertoli cell tumour (Fig. 14). Tumours expressing napsin A included pulmonary carcinomas (5/5 tis- sues), renal cell carcinoma (2/5 tissues) and weak cytoplasmic labelling in about 30% of neoplastic cells in one of five Sertoli cell tumours (Fig. 15).
Effect of Prolonged Fixation on Pax8 and Napsin A Immunoreactivity
The percentage labelling and labelling intensity for Pax8 was slightly reduced after 1 week of fixation; this reduction was more apparent after 2 and 3 weeks of fixation and was marked thereafter. There was no reduction in immunoreactivity for napsin A during the period of this study.
Discussion
This study has evaluated the immunoreactivity of Pax8 and napsin A in canine thyroid tumours and compared the expression of these markers with that of thyroglobulin, calcitonin and TTF-1. Pax8 and napsin A are expressed in a different proportion of canine thyroid follicular and medullary neoplasms.
Please cite this article in press as: Ramos-Vara JA, et al., Immunohistochemical Detection of Pax8 and Napsin A in Canine Thyroid Tumours: Comparison with Thyroglobulin, Calcitonin and … , Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.07.009
Pax8 and Napsin A in Canine Thyroid Tumours
Overall, Pax8 was more sensitive than napsin A, but less sensitive than TTF-1 in labelling of these tu- mours.
In mammals, nine PAX genes have been identified, with homologues in flies, worms, frogs, fish and birds (Chi and Epstein, 2002; Blake and Ziman, 2014). These transcription factors play a critical role in organogenesis and lineage determination, including the maintenance of a stem-like phenotype and possibly of the cancer stem cell phenotype (Blake and Ziman, 2014). PAX8 plays an important role in thyroid development and is necessary for endodermal differentiation into follicular cells (Mansouri et al., 1999). In the adult thyroid, PAX8 regulates thyro- globulin, thyroid peroxidase and sodium/iodine sym- porter expression (Li and Eccles, 2012). Pax8 is expressed in human cancers of the ovary, placenta, kidney, thyroid and in Wilms tumour (Robson et al., 2006). PAX8 chromosomal rearrangements with the peroxisome proliferator-activated receptor (PPARG) have been detected in one-third of thyroid follicular carcinomas (Li and Eccles, 2012; Nikiforova and Nikiforov, 2016).
In normal human thyroid, Pax8 is expressed by follicular cells (Laury et al., 2011; Tacha et al., 2011, 2013). We observed the same reactivity in normal canine thyroid, with consistent Pax8 immunoreactivity in canine tumours of follicular origin and less common expression in medullary tumours. Pax8 expression in human thyroid tumours has been reported, with papillary carcinomas being the thyroid tumour subtype with more consistent (100%) Pax8 expression (Nonaka et al., 2008b; Laury et al., 2011; Tacha et al., 2011). Other subtypes, in which >90% of cases express Pax8, are follicular adenoma and carcinoma (Nonaka et al., 2008a,b; Laury et al., 2011; Tacha et al., 2011). Many anaplastic thyroid carcinomas also express Pax8 (Nonaka et al., 2008b; Laury et al., 2011). Only 51 human medullary thyroid carcinomas have been examined for Pax8 immunoreactivity (Ordóñez, 2012b); 33% were pos- itive, typically with <50% labelled cells (Nonaka et al., 2008a,b; Laury et al., 2011; Tacha et al., 2011). In the present series, Pax8 was detected in 57% of canine medullary carcinomas.
Although napsin A-negative status has been considered useful to distinguish thyroid carcinoma from pulmonary carcinoma (Ye et al., 2011; Xue et al., 2013; Daniel et al., 2015), this marker is expressed in thyroid carcinomas, particularly of the papillary type (Kadivar and Boozari, 2013). In the present series, napsin A was less sensitive (62%) than Pax8 or TTF-1 (87% and 96%, respectively) in labelling canine thyroid tumours. However, when
only canine medullary tumours were considered, nap- sin A had 100% sensitivity, while Pax8 and TTF-1 were detected in 57% and 88% of tumours, respec- tively. Napsin A was also more sensitive than Pax8 in carcinosarcomas and mixed follicular-medullary carcinomas. In human follicular tumours, napsin A is less sensitive than TTF-1 (Bishop et al., 2010; Turner et al., 2012). We did not find studies regarding the expression of napsin A in human medullary thyroid tumours.
Napsin A has been detected in up to 50% of meta- static thyroid carcinomas (Mukhopadhyay and Kantzenstein, 2012). Only one metastatic medullary carcinoma was available in the present series and it was both Pax8 and napsin A positive. All other meta- static thyroid tumours were positive for Pax8.
We also evaluated the immunoreactivity of Pax8 and napsin A in normal and neoplastic canine tissues. In normal tissues, Pax8 was strongly expressed by endometrial epithelium, renal tubules and parietal cells of the Bowman’s capsule, thyroid follicular epithelium and epididymal epithelium. Pax8 reac- tivity in normal human tissues is similar, with the exception of the testis, B lymphocytes, renal pelvis ur- othelium and pancreatic islet cells. Parathyroid cells also express Pax8 (Tong et al., 2009; Ozcan et al., 2011; Tacha et al., 2011; Li and Eccles, 2012). Some Pax8 polyclonal antibodies have cross- reactivity with Pax5; this broad cross-reactivity is not observed with the Pax8 monoclonal BC12 (Moretti et al., 2012; Tacha et al., 2013). To our knowledge, Pax8 immunoreactivity in parathyroid glands has not been examined with monoclonal antibodies. Additionally, pancreatic islets do not express Pax8 when examined immunohistochemically with monoclonal antibodies or by molecular methods (Lorenzo et al., 2011; Tacha et al., 2013). In the current study, using mouse monoclonal BC12, reactivity was not detected in normal lymphoid tissue, pancreas, parathyroid gland or urothelium of the bladder. The differences between the current study and the human literature might be the result of the Pax8 antibody used (polyclonal versus monoclonal) or just interspecies differences.
Pax8 expression in human neoplasms follows that in normal tissues with some exceptions. Thyroid tu- mours, renal cell carcinomas, ovarian serous carci- nomas and endometrial carcinomas are consistently Pax8 positive (Nonaka et al., 2008a; Tong et al., 2009, 2010; Laury et al., 2010, 2011; Wiseman et al., 2011; Tacha et al., 2011, 2013; Hu et al., 2012; Knoepp et al., 2012; Ozcan et al., 2012; Ye et al., 2012; Zhao et al., 2012; Ordóñez, 2013; Mentrikoski et al., 2014). Variable Pax8 immunoreactivity has
been observed in parathyroid adenomas, pancreatic endocrine tumours, seminomas, cholangiocarcino- mas, thymomas, thymic carcinomas and parathyroid adenomas using a polyclonal antibody (Nonaka et al., 2008b; Laury et al., 2011; Tacha et al., 2011, 2013; Ordóñez, 2012b). The only non-thyroid canine neoplasms positive for Pax8 in the present series were renal cell carcinomas and, with much less common and patchy labelling, one pancreatic carcinoma, one ovarian adenoma and one Sertoli cell tumour. Of the human soft tissue tumours examined, only rhabdo- myosarcomas (about 40% of cases) expressed Pax8 (Tacha et al., 2011). We examined fewer sarcomas, including lymphomas, osteosarcomas and melanomas, and none were Pax8 positive. In summary, as mentioned for normal tissues, comparison of results be- tween the current study and those in the human liter- ature may be invalid due to the use of different primary antibodies.
Napsin A immunoreactivity in non-thyroid normal tissues was mostly in the lung (type II pneumocytes, macrophages and some bronchiolar epithelial cells) and kidney (tubular epithelium of the cortex and deep medulla). Ovarian granulosa cells had moderate labelling in one case. This observed immunoreactivity in canine lung and kidney is similar to that in man, in which the main organ expressing napsin A is the lung (type II pneumocytes, alveolar macrophages [as a result of phagocytosis] and bronchiolar epithelium) (Chuman et al., 1999; Schauer-Vukasinovic et al., 1999; Hirano et al., 2000). Human renal tubular epithelial cells, mainly of cortical proximal convoluted and straight tubules, express napsin A; less intense labelling occurs in collecting ducts and loops of Henle (Schauer-Vukasinovic et al., 1999; Hirano et al., 2000).
Canine pulmonary and, less commonly, renal cell carcinomas were positive for napsin A. In addition, one of five Sertoli cell tumours was also positive. Nap- sin A is one of the most common markers used to iden- tify human pulmonary carcinomas and to distinguish primary lung tumours from metastatic carcinomas (Mukhopadhyay and Kantzenstein, 2012). The sensi- tivity and specificity in the diagnosis of pulmonary carcinomas is enhanced when napsin A is used in com- bination with TTF-1 (Ye et al., 2011; Turner et al., 2012). In the present series, napsin A was detected in all (5/5) pulmonary carcinomas examined. Forty percent of canine renal cell carcinomas in this series were positive for napsin A. This marker is consistently expressed in human papillary renal cell carcinoma and, less frequently, in clear cell carcinoma (Ordóñez, 2012c; Xu et al., 2014; Zhu et al., 2015). Recently, napsin A has been detected in numerous ovarian clear cell adenocarcinomas, but
not in other ovarian carcinomas (Yamashita et al., 2015). We did not examine ovarian carcinomas, and the only ovarian adenoma evaluated was napsin A negative.
In the present study, the results of Pax8 and napsin A immunolabelling in both canine thyroid neoplasms and non-thyroid tumours were similar to those re- ported in the human literature. Some of the differ- ences might be the result of subtle species variation in the expression of these antigens or the use of a different antibody that recognizes different epitopes.
In the current series, TTF-1 was expressed by 98% of thyroid follicular carcinomas, which is slightly higher than in a previous report (Ramos-Vara et al., 2002). The percentage of TTF-1-positive medullary carcinomas in the present series was also higher than in the previous report (100% and 45%, respec- tively). These differences might be the result of tumour selection or antigen retrieval and detection method used, both of them different from the previous study. In man, TTF-1 expression in medullary carci- nomas can be as high as 100% (Katoh et al., 2000).
Although napsin A immunoreactivity was not affected by prolonged formalin fixation, Pax8 had variably reduced reactivity after 1 week in formalin. This reduction in immunoreactivity may not affect interpretation in most diagnostic cases in which tis- sues are typically processed within 1 week of being placed in fixative.
In summary, Pax8 and napsin A are expressed by 87% and 62% of canine thyroid tumours, respec- tively. Although overall TTF-1 is more sensitive than Pax8 or napsin A in the identification of canine thyroid tumours, Pax8 and TTF-1 have similar sensi- tivity for follicular tumours, so their combined use might help to distinguish primary pulmonary (Pax8 negative) from metastatic tumours. However, due to the reduced number of metastases in this series, further studies are necessary. Pax8 is a less sensitive and less specific marker than thyroglobulin for follic- ular tumours; however, it does not produce the false- positive diffusion artefact observed typically with thyroglobulin after inadequate fixation (Ramos- Vara and Miller, 2014). Napsin A, on the other hand, is as sensitive as calcitonin and more sensitive than TTF-1 and Pax8 for tumours of medullary dif- ferentiation and, with the exception of pulmonary, renal and thyroid follicular tumours, it can be used to identify metastatic medullary thyroid carcinomas. Regarding non-thyroid tumours, Pax8 appears to be a good marker for identification of canine renal carci- nomas; napsin A appears to be very sensitive for pul- monary carcinomas and less so for renal cell carcinomas. Additional studies to confirm these con- clusions are in progress.
Pax8 and Napsin A in Canine Thyroid Tumours
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[ Received, June 10th, 2016
1 Accepted, July 18th, 2016