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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: Hum Pathol. 2021 Aug 18;117:88–100. doi: 10.1016/j.humpath.2021.08.004

Somatostatin receptor 2 (SSTR2) is highly sensitive and specific for Epstein-Barr Virus-associated nasopharyngeal carcinoma

Kartik Viswanathan 1,2, Peter M Sadow 1
PMCID: PMC8511208  NIHMSID: NIHMS1734354  PMID: 34416258

Abstract

Viruses are known drivers of head and neck squamous cell carcinomas (HNSCC), particularly Epstein-Barr virus (EBV) and human papilloma virus (HPV). Both EBV-positive nasopharyngeal carcinoma (EBVNPC) and HPV-positive oropharyngeal SCC (OPSCC) can have overlapping histomorphology and molecular signatures, including nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) pathway mutations. A recent study showed that NFKB activation in EBVNPC drives somatostatin receptor 2 (SSTR2) expression that is detectable by immunohistochemistry and by imaging with 68-Gadolinium-DOTA-peptide radioconjugate. However, whether a similar NFKB-SSTR2 signaling mechanism exists for other virus-positive HNSCC, namely HPV-positive sinonasal carcinomas and OPSCC, remains unclear. Here we examined SSTR2 expression in a cohort of EBV-positive, HPV-positive, and virus-negative HNSCC with immunohistochemistry.

SSTR2 immunohistochemistry was performed on our cohort of primary and/or metastatic EBVNPC, HPV-positive sinonasal SCC, OPSCC, HPV-negative sinonasal and oral cavity SCC, and benign tonsil and adenoid tissue. For SSTR2 staining, the extent was categorized as focal, multifocal or diffuse, and the intensity was categorized as weak, moderate or strong. Multifocal/diffuse SSTR2 staining of any intensity was considered positive.

Among primary, recurrent and/or undifferentiated NPC, 90% showed multifocal to diffuse strong SSTR2 expression. One HPV-positive sinonasal carcinoma showed patchy SSTR2 staining. None of the remaining HPV-positive sinonasal carcinomas, OPSCC, or oral cavity HNSCC showed significant SSTR2 staining. Overall, SSTR2 is highly sensitive and specific for EBVNPC and could represent a surrogate biomarker. Among HNSCC assessed here, we recommend testing primary NPCs for SSTR2 due its relevance for diagnosis, associated imaging modalities and its therapeutic implications for patient care.

Keywords: SSTR2, immunohistochemistry, head and neck malignancy, EBV, Epstein-Barr, HPV, human papillomavirus, squamous cell carcinoma, nasopharyngeal carcinoma, nasopharynx, oropharynx

Introduction

Head and neck squamous cell carcinomas (HNSCC) represent the sixth most common malignancy in the world with a wide geographic prevalence and accounting for >750,000 new cases with an approximate 40–50% mortality rate in 2018 (1, 2). HNSCC comprises a diverse array of morphologic patterns depending on the anatomic site and the inciting etiologic risk factor (carcinogen or viral). With the decline in tobacco in recent decades, virally driven HNSCC now account for a more visible subset of new HNSCC cases with human papilloma virus (HPV) and Epstein-Barr virus (EBV) being most commonly recognized. Site-specific infections with HPV in the sinonasal tract and oropharynx seemingly promote HPV-positive sinonasal SCC and HPV-positive oropharyngeal SCC (OPSCC), whereas EBV infection, which most often flourishes in the fossa of Rosenmüller, leads to EBV-positive nasopharyngeal carcinoma (EBVNPC). Virally driven and non-viral HNSCC may show significant histomorphologic overlap and often require ancillary studies to ensure accurate diagnostic classification, prognostication and therapeutic triage. Molecular studies have furthered our understanding of the genetic drivers of malignancy in these tumors. EBVNPC and HPV-positive HNSCC show morphologic overlap but carry unique molecular signatures (3–5). Aberrations in the nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) signaling pathway is notably over-represented in virally driven HNSCC compared to non-viral HNSCC. However, despite the progress on the molecular signatures of these various HNSCC, survival outcomes have yet to show significant shifts (1). Hence, there is still a continued need to identify novel biomarkers that could have potential clinical impact on prognostic or treatment decisions.

In EBVNPC, a recent study by Lechner et al. elucidated a mechanistic link between EBV-mediated NFKB activation and somatostatin receptor 2 (SSTR2) expression (6). SSTR2 is a G-protein coupled receptor that plays an important role in development and remains strongly expressed in adulthood. SSTR2 expression is widely distributed in the body, but particularly in the central nervous system, meningothelial cells, neural crest-derived cells and in neuroendocrine cells (7). In the head and neck, SSTR2 expression is enriched in similar tumor types, with neuroendocrine tumors, meningiomas and olfactory neuroblastomas being the most common (8–11). The marked overexpression of SSTR2 in these tumor types compared to the surrounding normal tissues makes them amenable to imaging with somatostatin analogs bound with radionuclide (68-Gadolinium-DOTA-peptide radioconjugate) (12). For the same reason, somatostatin analogs, such as octreotide, or those associated with particle-emitting compounds, such as 177-lutetium, are used in the treatment of these SSTR2-expressing tumors (13, 14).

Lechner et al. demonstrated that SSTR2 expression correlated strongly with EBV-associated malignancies, including EBVNPC, EBV-positive pulmonary lymphoepithelial carcinoma and EBV-positive Hodgkin lymphoma (6). The upregulation of SSTR2 was detectable with immunohistochemistry, and the extent and intensity of expression in EBVNPC correlated with 68-Gadolinium-DOTA-peptide radioconjugate imaging intensity and susceptibility to somatostatin analogues with attached cytotoxic moieties (e.g., FEN-221) (6). However, while the study demonstrated that EBVNPC are enriched for SSTR2, the specificity of this marker among other virally-driven HNSCC remains unclear. Given the shared genetic alterations in TRAF3 and CYLD, known tumor suppressors that inhibit the NFKB pathway (3–5, 15), among subsets of OPSCC and EBVNPC, we hypothesized that a similar NFKB-SSTR2 signaling pathway might be active. In this study, to further explore this aspect, we investigated SSTR2 expression with immunohistochemistry in various primary and/or metastatic virally driven and non-viral HNSCC.

Material and Methods

Study Cohort

The study was approved by the institutional review board at Massachusetts General Hospital (2012P001024, PMS). Tumors consisted of primary and/or metastatic EBV-positive nasopharyngeal carcinoma (n=9), EBV-negative undifferentiated nasopharyngeal carcinoma (n=1), HPV-positive sinonasal carcinoma, (n=10 SCC and 1 multiphenotypic sinonasal carcinoma), HPV-positive oropharyngeal carcinoma (n=10), and HPV-negative sinonasal SCC (n=9), virus-negative keratinizing squamous cell carcinoma of the oral cavity and tongue (n=2). Palatine tonsil (n=10) and adenoid tissue (n=10) from patients treated for non-neoplastic conditions, such as tonsillar hypertrophy or deviated nasal septum, were included as controls. Cases were selected after review of the hematoxylin and eosin (H&E) stained biopsy and/or surgical resection slides by the authors (KV and PMS) using the current World Health Organization (WHO) diagnostic criteria (16). All tumor cases had appropriate ancillary studies, including immunohistochemistry and chromogenic in situ hybridization, to support or exclude the diagnosis.

Immunohistochemistry and chromogenic in situ hybridization

Chromogenic in situ hybridization for HPV and EBER, and IHC for p40 and p16 have been described previously (17–19). For SSTR2, IHC was performed using SSTR2 antibody (1:800, Rabbit monoclonal antibody, Clone UMB-7, Abcam, City, State) on formalin fixed paraffin-embedded tissue sections on a Leica Bond III system (Buffalo Grove, IL). The section was pre-treated using heat mediated antigen retrieval with Bond ER1 antigen retrieval buffer for 30 minutes and incubated with the antibodies for 15 min at room temperature. SSTR2 was detected with Leica Bond Refine detection system, and each section was counterstained with hematoxylin. The extent of staining is categorized as focal, multifocal, or diffuse, and the intensity of the staining is categorized as weak, moderate or strong. Staining of rare tumor cells (<1%) was considered negative. Focal to diffuse staining of any intensity was considered positive and the number of positive cases in each tumor subset was determined.

Results

Among EBVNPC, 33% (n=3) were primary in nasopharynx, 22% (n=2) were recurrent EBVNPC, and the remainder 44% (n=4) were metastatic to extrapharyngeal sites (e.g., lung). The median age of the patients was 56 years (range 33–75 years) with a male to female ratio of 2:1. All EBV-positive NPC had documented Epstein-Barr encoded mRNA (EBER) in situ hybridization confirming the diagnosis. The single EBV-negative NPC specimen tested was a recurrent tumor. Compared to the HPV-positive OPSCC and HPV-negative sinonasal carcinomas and the virus-negative HNSCC, the EBV-positive and EBV-negative NPC and the HPV-positive sinonasal carcinomas more often presented with advanced stage. EBV-positive NPC were most often associated with Asian descent (7/9, 78%) compared to the HPV-positive head and neck carcinomas (none in this cohort) and virus-negative sinonasal and oral carcinomas (none in this cohort). The clinicopathologic parameters for the entire tumor cohort are summarized in Table 1.

Table 1.

Cohort clinicopathologic characteristics

Tumor type SSTR2 immunostain Age Gender Race/Descent Location Size (cm) Tumor tissue tested (primarv, recurrence, metastasis) Clinical course Stage at presentation (Clinical or Pathological) Outcome (NED, AWD, DOD) Time to follow-up from initial diagnosis (months)
EBV-positive NPC (n=9)
Case
1 Positive 56 M Caucasian Lvmph node 2.4 M Patient underwent chemoradiation in 2009, relapse in 2015 on sacrum with repeat chemoradiation, now with metastases to bones and paraesophageal lvmph nodes in 2020 TxN2M0 AWD 146
2 Positive 58 F Asian Left nasopharynx NA R Locallv recurrent nasopharvngeal cancer status post definitive chemotherapv and radiation in 2011. 2015 relapse managed with chemoradiation with second local relapse in Februarv 2020 rcT2cN0cM0 AWD 126
3 Positive 73 M Asian Right nasal mass 4.4 cm primary tumor in 2016; current NA R T3N3M0 right-sided type 3 undifferentiated nasopharvngael cancer with involvement of C1 bone and supraclavicular node status-post chemoradiation in 2016, with local recurrence in April 2020 T3N3M0 AWD 63
4 Positive 33 M Caucasian Left superoposterior nasopharynx NA P Diagnosed in 2020 with liver lesions suspicious for metastases cT2N2M0 AWD 7
5 Positive 47 F Asian Lymph node, left neck 3.7 M right-sided EBV-positive T4N2M0 nasopharyngeal carcinoma status-post chemoradiation in April 2019 with contralateral late metastasis to the left neck status-post left neck dissection in June 2020. No adjuvant treatment indicated T4N2M0 NED 20
6 Positive 75 M Asian Left nasopharvnx 3.8 P locoregionally advanced nasopharyngeal carcinoma now on chemoradiation Stage II-IVB disease AWD 8
7 Positive 48 M Asian Lung right lower lobe lobectomy 1.4 M locoregionally advanced nasopharyngeal carcinoma status-post chemoradiation in 2019 with relapse to lung in 2020 cT1N2 Stage III AWD 29
8 Positive, weak 49 M Asian Retroperitoneum 1 M rT4N3bM0 right-sided, EBV-associated NPC in 2017 status-post chemoradiation with relapse in thoracic and abdominal lymph nodes in 2020 now on chemotherapy rcT4N3bM0 AWD 44
9 Negative, endothelial cells 70 F Asian Right nasopharvnx 3.8 P locally advanced EBV-mediated NPC with bilateral enlarged cervical and retropharyngeal lymph nodes on chemoradiation cT3N2 Stage III AWD 6
EBV negative undifferentiated NPC (n=1)
1 Equivocal 59 F Caucasian Left sphenoid and nasopharvnx NA R EBV-negative and HPV-negative NPC status-post chemoradiation in 2018 with relapse in 2020 and now on chemotherapy T4N2M0 AWD 40
HPV-related sinonasal carcinoma (n=11)
1 Negative, endothelial cells 72 F Caucasian Nasopharvnx 3.8 P locally advanced HPV positive left nasopharyngeal carcinoma status-post chemoradiation cT2N0 NED 10
2 Negative, germinal centers and peritumoral lvmphocvtes 47 F Caucasian Nasopharvnx 5.3 P Nasopharyngeal carcinoma, HPV 45 +, on chemoradiation with excellent response cT3, cN2, cM0, stage IVa AWD 8
3 Negative, endothelial cells, germinal centers 69 M Caucasian Nasopharvnx NA P status-post chemoradiation in 2019; episode of phaiyngeal and a phlegmon of the posterior pharynx resolved with antibiotics cT4cN2cM0, Stage IV NED 23
4 Negative 67 F Caucasian Nasopharvnx NA P history of DLBCL, nasopharyngeal carcinoma status-post chemotherapy T3N2M0 NED 54
5 Negative 70 M Caucasian Nasopharvnx NA P HPV-associated, nasopharyngeal cancer with skull base involvement status-post chemoradiation in 2019 complicated by neuropathic symptoms secondary to radiation cT4N0M0 Stage IVA NED 25
6 Negative, staining in endothelial cells 67 F Caucasian left sinonasal tract NA P HPV-related multiphenotypic sinonasal carcinoma (HPV-56) s/p resection and radiation NA NED 6
7 Positive, focal 53 M Caucasian Nasopharvnx NA P locoregionally advanced HPV-associated nasopharyngeal squamous cell carcinoma status-post chemoradiation;Complication: chemotherapy-induced polyneuropathy Stage III (cT3, cN2, cM0 NED 13
8 Negative, staining in endothelial cells 62 F Caucasian left nasopharvnx NA P nasopharyngeal HPV+ squamous cell carcinoma status-post chemoradiation; residual neuropathy and fatigue from chemoradiation Stage III (cT4cN2cMX) NED 18
9 Negative, staining in endothelial cells 73 M Caucasian and Hispanic left nasal cavitv 3.2 R recurrent HPV related squamous cell carcinoma of the nasal septum status-post total rhinectomy and right neck dissection, chemoradiation rpT4aN3b NED as ofNov 2020 60
10 Negative, staining in endothelial cells 68 M Caucasian left nasal septum 2 P Schneiderian carcinoma and inverting papilloma status-post resection with negative margins, and adjuvant radiation pT2NX NED as of Dec 2020 23
11 Negative (<1% rare tumor cells) 61 M Caucasian left nasal cavity 5.1 P histoty of sinonasal squamous cell carcinoma (Schneiderian) with endophytic growth, non-keratinizing, arising from inverted sinonasal papilloma. NA NA 8
HPV-positive oropharyngeal carcinoma (n=10)
1 Negative, staining in endothelial cells, germinal centers and peritumoral lvmphocvtes 60 M Caucasian left inferior tonsil 0.4 P HPV-positive SCC of the left tonsil status-post resection, neck dissection in 2018 with relapse in 2019 status-post chemoradiation pT1N1 NED 31
2 Negative 67 M Caucasian Right base of tongue, right neck dissection 2.2 P HPV mediated squamous cell carcinoma of the base of tongue status-post resection and right neck dissection with close margin status-post radiation pT2N1 NED 4
3 Negative, staining in germinal centers and squamous crypt 65 M Caucasian Left base of tongue 3.1 P left tongue base HPV-associated SCC status-post chemoradiation (last treatment April 2021 complicated by mucositis, no further treatment planned) NA AWD 4
4 Negative 50 M Hispanic left tonsil 4.6 P cT1N1 HPV+ squamous cell carcinoma of left tonsil with prominent retropharyngeal node metastasis - plan for chemo radiation at outside institution cT1N1 AWD 6
5 Negative 50 M Caucasian Right base of tongue, right neck dissection 0.6 P Right base of tongue HPV-SCC metastatic to lymph node status-post resection and adjuvant radiation pT1N1 NED 6
6 Negative 53 M Caucasian Left base of tongue 1.7 P HPV+ left base of tongue oropharyngeal SCC status-post resection and chemoradiation pT1N1M0 NED 6
7 Negative 76 F Caucasian Left base of tongue, left neck dissection 2.9 P HPV-mediated oropharyngeal cancer cT2N1 NED 6
8 Negative, staining in intratumcral lymphocytes 76 M Caucasian bilateral base of tongue 3.4 R histoty of DLBCL status-post chemotherapy, T1N2aM0 moderately differentiated invasive SCC of base of tongue in 2011 with recurrence in Nov 2020 status-post chemoradiation yrpT2NX NED 120
9 Negative, staining in peritumoral lymphocytes and germinal centers 54 M Caucasian left tonsil and left neck dissection 1.5 P HPV-related SCC of tonsil with left cervical LN metastasis,status-post surgery, currently on chemoradiation pT1N1 NED 6
10 Negative, staining in peritumoral lymphocytes and germinal centers 50 M Caucasian left tonsil and left neck dissection 3.3 P T2N1 left tonsil HPV-mediated squamous cell carcinoma, 12 pack year distant smoking history, status post resection and radiation pT2N1 NED 6
Virus-negative keratinizing SCC oral cavity and tongue (n=2)
1 Negative 77 F Caucasian Right palate 1.5 P Outside biopsy showed moderate to severe dysplasia extending to tissue edges pT1NX NED 1
2 Negative 77 M Caucasian Right base of tongue 3.7 R history of right lateral tongue - moderately differentiated invasive keratinizing squamous cell carcinoma in 2010 status-post resection and adjuvant radiation, separate primary on left lateral tongue (pT1N0) status-post resection, left neck dissection and adjuvant radiation; now presenting with ulcerated lesion at right base of tongue recurrence, now on chemotherapy pT20N0MX of original primary AWD 12
HPV negative sinonasal carcinoma (n=9)
1 Negative (Rare tumor cells, <1%) 76 M Caucasian left sinonasal tract 5 P Presented with nasal congestion and found to have a left sinonasal mass pT1NX NED 8
2 Negative 75 F Caucasian right sinus NA P Sinonasal SCC with surrounding destructive changes status-post chemoradiation pT4N0 AWD 9
3 Negative 64 F Caucasian right sinonasal tract NA P right nasal cavity maxillary sinus squamous cell carcinoma in the background of inverted papilloma status-post excision and radiation NA NED 10
4 Negative 62 M Caucasian right sinonasal tract 4 cm in 2016 R histoiy of sinonasal SCC status-post resection and chemoradiation with recurrence pT4aNX of primary NED 57
5 Negative 69 M Caucasian left sinonasal tract NA P sinonasal SCC arising from inverted papilloma of ethmoid sinus status-post resection Stage 1 per note NED 11
6 Negative 69 F NA left sinonasal tract 2.8 P Squamous cell carcinoma, Schneiderian type, post resection and radiation pT1NX NED 17
7 Negative 93 F Caucasian right sinonasal tract 6.1 P T4BN0M0 right maxillary sinus SCC with invasion of orbital cavity and blindness, unresectable and declined chemoradiation T4BN0M0 AWD in 2019; lost to follow-up 1
8 Negative 64 M Caucasian left sinonasal tract NA P multifocal carcinoma sinonasal schneiderian type carcinoma arising in association with inverted papilloma with variable dysplasia status-post radiation and resection NA NED 21
9 Negative 61 F Caucasian left sinonasal tract NA P schneiderian carcinoma from left inferior turbinate status-post resection NA NED 26
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Abbreviation: NA - not available, M-Male, F-female, P-primary, M-metastasis, R-recurrence, NED-no evidence of disease, AWD-alive with disease, DOD - dead of disease, EBV-Epstein-Barr virus, HPV-human papilloma virus, SSTR2-somatostain receptor 2

Among the undifferentiated NPC (n=10), 90% showed prominent multifocal to diffuse SSTR2 staining, including 8 of 9 EBV-positive NPC and the single EBV-negative NPC (Figure 1). In the rest of the cohort, only one HPV-positive sinonasal carcinoma (1/11, 9%) showed only focal SSTR2 staining with moderate intensity, whereas the remaining HPV-postive sinonasal carcinomas, HPV-positive OPSCC, HPV-negative sinonasal carcinomas and virus-negative oral cavity and tongue SCC showed no significant staining (occasional, rare cells) and were considered negative. Finally, in normal palatine tonsil and adenoid tissue, and in negative tumor cases, SSTR2 expression was localized to germinal centers, endothelial cells, and lymphoreticular crypt epithelium (Figure 4). Overall, the sensitivity, specificity, positive predictive value and negative predictive value of SSTR2 for non-keratinizing/undifferentiated NPC (either EBV-positive or EBV-negative) are 90%, 97%, 90%, and 97%, respectively (Table 2).

Figure 1.

Figure 1.

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Staining profile of EBV-positive nasopharyngeal carcinoma. Representative H&E sections of EBVNPC (A), p40 IHC (B), EBER chromogenic in situ hybridization (ISH) and IHC for SSTR2. EBVNPC shows diffuse nuclear p40 expression, positive EBER ISH and strong, membranous SSTR2 expression. Images are 100x magnification.

Figure 4.

Figure 4.

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Staining profile of the sole HPV-positive sinonasal carcinoma (H&E, A). SSTR2 expression (B) is focal and moderate in intensity. The tumor is diffusely positive for p16 (C) and is positive for high-risk HPV 16/18 by chromogenic in situ hybridization (D). The remaining HPV-positive sinonasal carcinoma cases (n=9) are negative for SSTR2 expression. Images are 100x magnification.

Table 2.

Performance characteristics for SSTR2 in various virus-positive and negative head and neck squamous cell carcinomas

Tumor type (n=number of patients) SSTR2 Positive (%)
EBV-positive NPC (n=9) 8/9 (89%)
EBV-negative undifferentiated/non-keratinizing NPC (n=1) 1/1 (100%)
HPV-positive sinonasal carcinoma (n=11) 1/11 (9%)
HPV-negative sinonasal carcinoma (n=9) 0/9 (0%)
HPV-positive oropharyngeal carcinoma (n=10) 0/10 (0%)
Virus-negative SCC of oral cavity and tongue (n=2) 0/2 (0%)
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Abbreviation: NPC-nasopharyngeal carcinoma, SCC-squamous cell carcinoma, EBV-Epstein-Barr Virus, HPV-human papilloma virus, SSTR2-somatostain receptor 2

Discussion

HNSCC is a heterogeneous group of diseases with various histologic patterns depending upon the cell of origin associated with an anatomic site as well as the inciting etiologic agent (1, 2). Accordingly, classification of the site-specific HNSCC with additional ancillary testing is necessary to ensure appropriate treatment regimens and prognostication. EBV-positive and HPV-positive HNSCC account for the majority of virally-mediated HNSCC. As expected, EBVNPC were more prevalent in patients with Asian descent. In our cohort, EBVNPC and HPV-positive malignancies trended toward younger patients presenting at advanced stage. Management of these disease processes requires multidisciplinary discussion and a multimodal approach that relies on surgery and chemoradiation. Long term prognosis for these disease processes is still grim, despite incremental improvements in therapeutics, so there is a continuing need for identifying potential biomarkers for therapeutic, prognostic or imaging purposes (1, 2).

SSTR2 is one such recent biomarker that gained attention for HNSCC, particularly EBVNPC. A recent study from Lechner et al. performed detailed immunohistochemical and molecular examination of 402 primary, recurrent and metastatic EBVNPC (6). The group demonstrated that EBV-infected cells express LMP1 that activates NFKB signaling and drives SSTR2 expression. In their study, SSTR2 IHC had a sensitivity of 81% with expression notably in EBVNPC and in NPC with a non-keratinizing/undifferentiated histology. Our findings are in keeping with their study, where 89% of our EBVNPC and our single EBV-negative undifferentiated NPC demonstrated moderate to strong SSTR2 IHC expression. While we do not have molecular characterization of our single EBV-negative undifferentiated NPC, one potential explanation for the SSTR2 expression in this case is that EBV-negative undifferentiated NPC may harbor NFKB pathway mutations mutually exclusive from the EBV-positive NPC (3–6) and that both mechanisms activate the NFKB signaling pathway leading to downstream SSTR2 expression.

A unique aspect to our study is that we explored whether HPV-positive head and neck malignancies may demonstrate SSTR2 expression. HPV is known for its canonical role in inactivating p53 and Rb via the E6 and E7 proteins, respectively, to drive tumor progression. However, studies have shown that HPV can also activate NFKB signaling in various organ systems (20). Moreover, recent studies have shown that despite unique viral drivers, EBVNPC and HPV-positive HNSCC can share molecular signatures, including alterations in the NFKB pathway (3–5). In our study, only one case of HPV-positive sinonasal carcinoma showed focal moderate SSTR2 staining with neither of the remaining HPV-positive HNSCC, including OPSCC and HPV-positive sinonasal carcinomas, nor the virus-negative HNSCC demonstrating any significant SSTR2 expression. Thus, by comparison to non-keratinizing/undifferentiated NPC (EBV and non-EBV), HPV-positive HNSCC do not appear to rely on SSTR2 as a primary mechanism.

Given that only a single HPV-positive HNSCC showed significant SSTR2 expression, there are several potential explanations. First, EBV and HPV infect unique cell types and anatomic sites, predominantly the fossa of Rosenmüeller and the sinonasal tract or oropharynx, respectively. The surrounding milieu and the cell of origin likely harbor unique genetic and epigenetic expression signatures that may impact the overall induction of SSTR2 expression in response to viral infection. However, it is important to note that in the study by Lechner et al., they demonstrated SSTR2 expression in other non-head and neck EBV-positive tumors, including pulmonary lymphoepithelial carcinoma and EBV-positive Hodgkin lymphoma, with SSTR2 expression restricted to the EBV infected cells (6). This would suggest that the EBV-NFKB-SSTR2 pathway may not necessarily be restricted to certain cell types, but rather appears to be an EBV-specific phenomenon. Conversely, the same study showed that a percentage of EBV-negative NPC demonstrated SSTR2 expression; thus, this interaction between EBV, NFKB and SSTR2 is complex and likely multifactorial (6).

Second, there are a variety of low-risk and high-risk HPV subtypes and sublineages which may impact the NFKB and p53-Rb pathways in distinct ways. In our cohort, most of our HPV-positive HNSCC were driven by HPV16/18; however, we had one HPV-positive sinonasal carcinoma that arose in a background of low-risk HPV and a case of multiphenotypic sinonasal carcinoma which is typically mediated through HPV33 and HPV57 (Reference: WHO Head and Neck 2017). While we did not determine the sublineage of the HNSCC driven by HPV16/18, as the frequency of SSTR2 expression in HPV-positive HNSCC is very low, a unique HPV sublineage driving SSTR2 expression seems unlikely. Thus, while HPV-positive HNSCC share molecular signatures with EBVNPC, these shared features alone do not seem to be sufficient to drive SSTR2 expression in HPV-positive HNSCC and likely requires interaction between multiple factors.

There are several clinical implications from both our study and from Lechner et al (6). First, patients with SSTR2-expressing EBV and non-EBV NPC could be staged with 68-Ga-DOTA. Few studies had previously noted this correlation of EBVNPC demonstrating 68-Ga-DOTA uptake; however, the precise mechanistic link for this observation had not been established until recently (6). Lechner et al. also demonstrated a positive correlation between SSTR2 expression and 68-Ga-DOTA signal intensity. While our study did not explore this aspect, we anticipate that these SSTR2-expressing EBVNPC are likely to show 68-Ga-DOTA uptake. With this imaging approach, 68-Ga-DOTA could potentially be used for not only staging and localizing disease burden, but also to assess for recurrence in conjunction with plasma EBV levels.

Second, there are important treatment implications. While a large proportion of EBV-positive and negative NPC are responsive to chemoradiation, patients with advanced/metastatic disease have poor outcomes with limited therapeutic options. The identification of SSTR2 expression on NPC (EBV positive and negative) may enable targeting with SSTR2-modulating therapeutics. Lechner et al. demonstrated efficacy of a SSTR2-targeting cytotoxic drug, known as PEN221, towards EBV-driven NPC in vitro and in vivo in animal xenograft models (6). While a recent clinical trial examined the efficacy of this drug in the context of pulmonary neuroendocrine tumor (Clinical trial #NCT02936323), PEN221 may have therapeutic potential for treating advanced/metastatic undifferentiated EBV-positive and negative NPC.

Beyond the potential for treatment, SSTR2 expression may have significant prognostic implication for NPC patients. In their study, Lechner et al. demonstrated that SSTR2 expression in NPC was an independent prognostic marker for overall improved survival, regardless of EBV status (6). The precise mechanism dictating this improved survival, whether directly or indirectly linked to SSTR2, is not known. Our cohort size is limited and therefore not powered enough to provide a definitive answer on this question. Cracolici et al. did not identify a survival benefit for olfactory neuroblastomas expressing SSTR2; however, this may reflect either the unique intrinsic tumor properties (i.e. neuroendocrine differentiation) or, as suggested by the authors in that study, their cohort may be insufficiently powered (8). Nevertheless, this would be an important clinicopathologic parameter to examine in future studies with larger cohorts.

Based on our study and Lechner et al., SSTR2 may represent a useful diagnostic surrogate marker for EBVNPC (6). Given the retention of SSTR2 expression in the primary, recurrent and metastatic tumor, SSTR2 IHC might be helpful particularly in lymph node metastases where the primary is unknown. If SSTR2 IHC shows moderate to strong and extensive staining, then it would be worthwhile to assess for EBV with either in situ hybridization or plasma EBV levels. However, it is important to remember that among head and neck malignancies, SSTR2 expression is not exclusive to EBV-infected tumors. Merkel cell carcinoma, where Merkel cell polyoma virus is considered an important driver in a moderate proportion of cases, also shows increased SSTR2 expression (21), although whether this is related to viral-mediated NFKB activation or simply a downstream effect of neuroendocrine differentiation of the tumor is unclear. Other non-viral neuroendocrine tumors have also been shown to express SSTR2, including meningiomas and olfactory neuroblastoma (8). Cracolici et al. showed that SSTR2 is promising in separating olfactory neuroblastomas from other histologic mimics with sinonasal neuroendocrine carcinoma creating the only potential diagnostic dilemma with focal and/or weak SSTR2 staining (8). High-grade (Hyams III-IV) olfactory neuroblastomas retain their high SSTR2 expression and while these could potentially show overlap with EBVNPC or NPC with undifferentiated histology, EBV testing and correlation with clinicopathologic and radiologic findings in conjunction with additional immunohistochemistry will be able to separate these entities.

Of note, a potential caveat was noted in our cohort of tumors that were negative with SSTR2 staining. We observed consistent moderate SSTR2 staining in squamous crypt epithelium in tonsillar tissue, and in germinal centers, peritumoral lymphocytes (regardless of viral or non-viral HNSCC) and endothelial cells, regardless of site. Similar staining patterns were seen in non-neoplastic tonsillar and adenoid tissue. SSTR2 expression has been reported in lymphoid cells (10), but not in the other normal cell types seen in our study (22); however, whether this reflects antibody clone (Abcam clone used in our study and Sigma-Aldrich clone was used in the Human Protein Atlas reference) specific differences remains to be seen. Regardless, the biologic significance of SSTR2 staining in these non-tumoral cell types remains unclear. As such, in limited biopsy samples, caution should be taken to not overinterpret SSTR2 expression in these non-tumoral cells, especially when these non-neoplastic cells are intermingled with the tumor cells.

An important question that needs to be considered is the selection of cases for SSTR2 testing to ensure the highest yield and the most benefit for patients. Based on our findings in this study and in the prior work by Lechner et al., we propose that all EBVNPC should be tested with SSTR2 IHC, due to the potential for SSTR2 targeted therapies, such as FEN-221, imaging with 68-Ga-DOTA, and for enrollment into clinical trials (6). Given that in our cohort and in Lechner et al., either most or all of the EBVNPC showed multifocal to diffuse SSTR2 expression in both the primary, recurrent, and/or metastatic NPC, the pre-test probability in any of these tissue sample types is high. In addition, the SSTR2 IHC has rapid turnaround time and the membranous/cytoplasmic staining is relatively straightforward to interpret in tumor cells when caution is taken to exclude the background SSTR2 staining of non-neoplastic cells.

Among HPV-positive sinonasal HNSCC, OPSCC, and virus-negative HNSCC, only one HPV-positive sinonasal HNSCC showed focal positive SSTR2 staining with moderate intensity. In addition, one HPV-positive sinonasal HNSCC and one HPV-negative sinonasal HNSCC showed SSTR2 expression in a rare tumor cells (<1%); however, we considered this extent of staining insufficient to be deemed positive. Given the very low frequency of SSTR2 staining in HPV-positive sinonasal carcinoma, OPSCC and virus-negative HNSCC, the return on upfront SSTR2 IHC testing on these tumor types would not be cost-effective.

In summary, we demonstrate that among the tested HNSCC in our cohort, EBVNPC and an undifferentiated EBV negative NPC demonstrated multifocal to diffuse strong SSTR2 expression. A potential limitation of this study is the retrospective nature and the limited number of evaluated cases; however, SSTR2 expression was strongly enriched in EBV-positive NPC over HPV-positive head and neck malignancies and virus-negative SCC. Ultimately, SSTR2 is a robust surrogate biomarker for primary NPC and given the potential for prognostication, imaging, and therapeutic modalities, we recommend performing SSTR2 immunohistochemistry on all potential NPC cases.

Figure 2.

Figure 2.

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SSTR2 expression in HPV-positive sinonasal squamous cell and oropharyngeal squamous cell carcinoma. Representative H&E sections of specified tumors are paired with corresponding SSTR2 IHC (sinonasal: A, B; OPSCC: C, D). No significant SSTR2 staining is seen in any of the representative cases. Images are 100x magnification.

Figure 3.

Figure 3.

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SSTR2 expression in HPV-negative sinonasal carcinoma and virus-negative keratinizing squamous cell carcinoma of oral cavity. Representative H&E sections of specified tumors are paired with corresponding SSTR2 IHC (sinonasal: A, B; oral cavity: C, D). No significant SSTR2 staining is seen in any of the representative cases. Images are 100x magnification.

Figure 5.

Figure 5.

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SSTR2 expression in non-neoplastic cells in SSTR2-negative tumors and normal palatine tonsil and adenoid tissue. Representative sections of benign tonsil (A-B) and adenoid tissue (C-D) stained with either H&E stain or SSTR2 IHC. SSTR2 expression is present in germinal centers and endothelial cells. Additionally, in tonsillar tissue, SSTR2 is expressed in squamous crypt epithelium. Tonsil tissue is 40x magnification and adenoid tissue is 100x magnification.

Acknowledgements

Funding to Dr. Sadow is provided by the National Cancer Institute of the National Institutes of Health (1P01CA240239-01). Drs. Viswanathan and Sadow were awarded an MGH Pathology Vickery-Colvin Award for this project.

Declaration section

Ethical approval: This research study was conducted retrospectively from data gathered for clinical purposes. This study was approved by the Massachusetts General Hospital IRB (2012P001024, PMS). Consent is waived due to the retrospective nature of this study.

Footnotes

Conflicts of Interest/Competing interests Disclosures: The authors do not have any conflicts of interest/competing interests to disclose.

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