Immunohistochemical Reactivity of Prostate-Specific Markers for Salivary Duct Carcinoma

Salivary duct carcinoma (SDC) is a relatively rare salivary gland malignancy [1‒3]. Its histological characteristics are generally similar to those of invasive ductal carcinoma of the breast [1‒3]. Most cases occur de novo, although some represent the malignant component of carcinoma ex pleomorphic adenoma (PA). Immunohistochemically, SDCs are typically positive for androgen receptor (AR), which serves as a confirmative diagnostic marker [4]. AR plays essential roles in the development and progression of prostate cancer [5, 6]. A recent study has shown that immunohistochemistry staining NKX3.1 has diagnostic value in prostatic adenocarcinomas; this marker is related to androgen expression [7]. NKX3.1 is an androgen-regulated homeodomain gene whose expression is predominantly localized to the prostate epithelium [7]. According to previous studies, NKX3.1 expression is significantly decreased in prostate cancer patients compared to those with benign prostate hyperplasia [8]. Further, loss of NKX3.1 expression is strongly associated with hormone-refractory disease and advanced tumor stage in prostate cancer [9]. We hypothesized that NKX3.1 is a new immunohistochemical marker for SDC; however, to the best of our knowledge, few studies have examined this prediction [10]. Therefore, the purpose of this study was to investigate whether NKX3.1 staining in combination with other immunomarkers of prostate carcinoma could have a diagnostic and prognostic value in SDC.

In total, 42 SDCs were resected at Oita University and Oita Prefectural Hospital between February 2004 and June 2018. The patients included 36 men and 6 women, with a mean age of 67.8 years (range, 49–89 years). The primary sites of SDCs were the parotid gland in 27 cases, the submandibular gland in 12 cases, and the minor salivary glands in 3 cases. Histological features were assessed using hematoxylin-eosin-stained sections from formalin-fixed, paraffin-embedded tissues by N.T. and H.N. For immunohistochemical analyses, representative tissue blocks were selected. Immunohistochemical staining was performed manually. Primary antibodies and antigen retrieval methods for immunostaining are indicated in Table 1. The sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase activity was abolished by incubation with 3% hydrogen peroxide for 20 min at room temperature. The antigens were retrieved according to the manufacturer’s instructions. The slides were incubated with the first antibody and stained using the streptavidin-labeled biotin peroxidase complex system (Nichirei, Tokyo, Japan). They were visualized using diaminobenzidine tetra­hy­drochloride (DAB) solution. More than 30% reactivity in tumor cells and higher staining intensity than that of normal ductal or acinar cells indicated a positive result. Differences were analyzed using Fisher’s probability test; p < 0.05 was considered statistically significant.

The clinical findings for SDC are summarized in Table 2. Of the 42 cases diagnosed as SDC, 29 (69.0%) cases were de novo SDC and 13 cases (31.0%) were SDC ex PA. Both types were most common in the parotid gland and in elderly men. Nodal metastasis was observed in 17 (60.7%) of 28 cases with lymph node resection. A total of 14 (70.0%) of 20 de novo SDC cases with lymph node resection showed nodal metastasis, while 3 (37.5%) of 8 ex PA cases showed nodal metastasis (p = 0.123). Eight (19.0%) patients died of the disease, with a mean survival of 17 months after diagnosis (range, 2–28 months), and 7 cases (87.5%) of these 8 cases were de novo. Regarding the histological findings of SDC ex PA, 7 of 13 cases were intracapsular carcinoma. The remaining 6 cases included 3 minimally invasive carcinomas and 3 invasive carcinomas. Eleven cases (7 intracapsular carcinomas, 2 minimally invasive carcinomas, and 2 invasive carcinoma) were non-recurrence cases. Two cases were locally recurrent (1 intracapsular carcinoma and 1 invasive carcinoma), of which the latter died of the disease. No significant differences were noted in terms of age, sex, regional nodal metastasis at operation, or recurrence during follow-up between the two clinicopathologic forms, but distant metastasis during follow-up showed a significant difference (p < 0.0027).

The representative panel of the immunoreactivity of prostate carcinoma markers in normal salivary gland, SDC ex PA, and de novo SDC is shown in Figure 1. The results of immunohistochemical analysis for the PA area in SDC ex PA, SDC de novo, and SDC ex PA are presented in Table 3. NKX3.1 and AR were expressed in the nuclei, and α-methylacyl-CoA racemase (AMACR), prostatic acid phosphatase (PAP), and prostate-specific antigen (PSA) were detected in the cytoplasm. With respect to immunoreactivity in normal salivary glands, serous acini (mucinous acini were negative) and ducts showed positivity for NKX3.1. AR, AMACR, PAP, and PSA were negative in normal samples. In the immunoreactivity of the PA area in SDC ex PA cases, epithelial and myoepithelial components showed positivity for NKX3.1 (92.3%), whereas AR and AMACR tended to show weak positivity compared with the SDC area. The AR-positive cases only showed positivity in the epithelial component. PAP and PSA were negative in the PA area. Among NKX3.1-positive PA cases, 12 out of 13 were negative for NKX3.1 in the SDC area. AR- and AMACR-positive PA cases were also positive in the SDC area.

Among all SDC cases, 81.0, 35.7, 58.5, 33.3%, and 0% were positive for AR, NKX3.1, AMACR, PAP, and PSA, respectively. The incidences of these marker-positive de novo SDC cases were higher than those of SDC ex PA cases, although the difference was not statistically significant. Comparisons of immunohistochemical staining results for cases with local recurrence and distant metastasis during follow-up are shown in Table 4. The cases were divided into three groups, namely, cases with local recurrence, distant metastasis, and no local recurrence or distant metastasis (no recurrence). In total, 33.3% of cases with local recurrence, 30.8% of cases with distant metastasis, and 40.9% of cases with no recurrence were positive for NKX3.1. The positive rates for AMACR of the local recurrence, distant metastasis, and no recurrence groups were 83.3, 66.7, and 45.4%, respectively, and the differences were significant (p < 0.05; local recurrence: p = 0.0352, distant metastasis: p = 0.0205). Similarly, with respect to positivity for PAP, cases with no recurrence (22.7%) were less frequent than those with local recurrence (41.7%) and distant metastasis (50.0%). There were no significant differences in the immunoreactivity of AR, NKX3.1, AMACR, PAP, and PSA between de novo SDC and SDC ex PA, including intracapsular, minimally invasive, and invasive carcinoma, and between invasive and noninvasive areas.

NKX3.1, located on chromosome 8p21.2, is an androgen-regulated homeodomain gene that is predominantly expressed in the prostate epithelium [7, 11]. NKX3.1 directly regulates AR-dependent genes, which are overexpressed in prostate carcinoma, deregulated in advanced prostate cancer, and enriched in recurrent prostate cancer [7]. Moreover, NKX3.1 likely regulates genes that are active in promoting cell survival or preventing cell apoptosis [11]. Considering this, it is likely that NKX3.1 plays a pivotal role in prostate tumorigenicity. Using immunohistochemistry, Irer et al. [12] reported that NKX3.1 expression was elevated in benign prostate hyperplasia tissues compared with that in normal tissues. On the other hand, Bowen et al. [9] reported that the loss of NKX3.1 expression was strongly associated with hormone-refractory disease and advanced tumor stage in prostate cancer. In general, SDCs are typically positive for AR in immunohistochemical examination [4]; thus, the expression of NKX3.1 was estimated to be relevant to SDC tumorigenicity. The role of NKX3.1 gene expression during mouse salivary gland development has been reported by Schneider et al. [13]. In our study, NKX3.1 immunoreactivity was detected in PA (92.3%) as well as in normal salivary glands, such as in the ducts and acini. The expression pattern of NKX3.1 in normal salivary gland or its lesions was similar to that in the prostate gland. However, NKX3.1 expression in SDC cases (35.7%) was decreased compared with that in PA or in normal tissues. As SDC is a high-grade salivary gland malignancy, the lower immunohistochemical stain score for NKX3.1 is similar to that in previous reports showing loss of NKX3.1 expression in advanced prostate cancer [9, 14]. As both SDC and prostatic adenocarcinoma express AR, SDC seems to share the AR-controlled pathway related to NKX3.1 with prostatic cancer in its tumorigenesis. However, in human prostatic lesions including hyperplasia and adenocarcinoma, the role of NKX3.1 in prostatic cancer is still controversial [9, 8, 12, 14, 15]. Further consideration is needed to yield any findings about the role of NKX3.1 in the prostate as well as in the salivary gland.

AMACR and PAP, which have been established as markers for prostate carcinoma, had positive detection rates of 58.5 and 33.3%, respectively, in our SDC immunohistochemical results. AMACR, also known as P504S, plays a role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives [16]. Jiang et al. [17] reported that carcinomas arising from tissues that normally do not express AMACR, were also positive for AMACR, including 94% of prostate carcinomas, 31% of urothelial carcinomas, and 27% of gastric adenocarcinomas. Zhou et al. [18] reported that more than 60% of ovarian carcinomas and 30% of breast cancers and melanomas overexpress AMACR. Our results are consistent with these reports. AMACR is expressed in SDC, but not in the normal salivary gland. Thus, it is suggested that AMACR plays a pathogenic role in SDC. Additionally, PAP is a prostatic epithelium-specific differentiation antigen that has been implicated in the growth of prostatic carcinoma cells and this process is regulated by androgen [19, 20]. Fan et al. [21] reported that 58.3% of SDCs were positive for PAP, whereas PSA was detected in 30% of SDCs. These findings might be related to the tumoral/neoplastic expression of AMACR or PAP in SDC tumorigenesis.

SDC is a clinically aggressive malignant tumor, but our results suggest that SDC ex PA has a better prognosis than de novo SDC. Significant prognostic factors in carcinoma ex PA include tumor stage, grade, proportion of carcinoma, extent of invasion, and proliferation index [22]. Generally, carcinoma ex PA has a relatively good prognosis, regardless of the carcinoma subtype, compared with that of other malignant tumors in salivary glands. We tested the utility of NKX3.1, AMACR, and PAP as prognostic factors for SDC, because the prognostic value of prostatic carcinoma markers in SDC has not been evaluated. Interestingly, AMACR and PAP immunoreactivity rates were higher in recurrence cases (local recurrence and distant metastasis) than in cases with no recurrence, especially for PAP. He et al. [23] showed that high expression of AMACR was significantly associated with an aggressive phenotype, with advanced tumor staging, increased vascular invasion, and increased perineural invasion in oral squamous cell carcinoma. Zha et al. [24] reported that AMACR is essential for prostatic cancer cell proliferation in vitro and that its enzymatic activity is elevated in prostatic cancer tissue. Xu et al. [25] reported that PAP-positive cases exhibited higher metastasis rates, larger tumors, more localized tumors, and higher pathological grades than did PAP-negative cases. Furthermore, the survival duration was also shorter in the PAP-positive group, as reported previously, suggesting that PAP has a prognostic value in prostate cancer [25]. Based on these reports, the immunohistochemistry of AMACR and PAP is possibly used as a prognosticator in SDC. Accordingly, SDC with local recurrence or distant metastasis showed higher rates of AMACR and PAP positivity. Thus, immunoreactivity of AMACR and PAP could be useful for assessing prognosis in SDCs. Although our clinical data indicate somewhat better prognosis in SDC ex PA than in de novo SDC, the immunohistochemical profile of the examined markers showed no significant difference. Thus, other factors may affect the difference in the biology of these two types of tumors.

Our results showed that SDC frequently expresses AR (81.0%), NKX3.1 (35.7%), and AMACR (58.5%) and occasionally expresses PAP (33.3%), indicating a close immunophenotypic homology with prostatic adenocarcinoma, except for PSA (0%). With regard to clinical pathology, our data show that the immunohistochemical staining of prostate-specific markers should be interpreted with caution, in the appropriate morphological context, when determining whether a metastatic tumor is of prostate origin, especially when patients have a history of SDC. PSA and histopathological findings might be useful for distinguishing SDC from prostatic carcinoma.

The standard treatment for SDC is surgical resection with or without radiation therapy. The tumor is resistant to chemotherapy and the mortality rate is high [26]. Currently, there are only few treatment modalities available for recurrent and metastatic SDC in practice [27]. Recently, androgen deprivation therapy (ADT), a mainstay treatment for metastatic prostate cancer, has been used for the treatment of metastatic and recurrent AR-positive SDC [28‒30]. Case series have reported a clinical benefit of ADT for recurrent and/or metastatic AR-positive salivary gland cancer; most of these cases were SDC or adenocarcinoma not otherwise specified [28‒30]. Based on the resistance of SDC to chemotherapy, ADT might become a useful treatment option.

In conclusion, we revealed the immunohistochemical reaction of some prostate cancer markers for SDC. NKX3.1 expression is useful for SDC diagnosis. NKX3.1 expression is significantly decreased in SDC compared to PA; however, it was not correlated with SDC progression or advanced stage. The immunoreactivity of AMACR and PAP could be useful for assessing prognosis in SDCs, but with regard to pathology practice, immunohistochemical staining of prostate-specific markers should be interpreted with caution when determining whether a metastatic tumor is of prostate origin, especially when patients have a history of SDC.

This study was approved by an ethics committee and institutional review board (number: 1637).

The authors have no financial conflicts of interest.