ORIGINAL ARTICLE

Treatment outcomes and prognostic factors, including human papillomavirus, for sinonasal undifferentiated carcinoma: A retrospective review Stacey T. Gray, MD,1,2 Marc W. Herr, MD,1,2* Rosh K. V. Sethi, BS,3 Gillian Diercks, MD,1 Linda Lee, MD,1 William Curry, MD,2,4 Annie Chan, MD,2,5 John Clark, MD,2,6 Eric H. Holbrook, MD,1,2 James Rocco, MD, PhD,1,2 Peter M. Sadow, MD, PhD,1,7 Derrick T. Lin, MD1,2 1

Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts, 2Massachusetts Eye and Ear Infirmary/Massachusetts General Hospital Cranial Base Center, Boston, Massachusetts, 3Harvard Medical School, Boston, Massachusetts, 4Department of Neurosurgery, Massachusetts General Hospital, Boston, Massachusetts, 5 Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts, 6Department of Medical Oncology, Massachusetts General Hospital, Boston, Massachusetts, 7Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts.

Accepted 8 January 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23606

ABSTRACT: Background. Sinonasal undifferentiated carcinoma (SNUC) is a high-grade, aggressive neoplasm. Low incidence and poor outcomes make identification of prognostic factors and treatment standardization difficult. Similarly, little is known regarding the association of human papillomavirus (HPV) with SNUC. Methods. A retrospective review was conducted. Extracted information included treatment received, tumor recurrence, patient survival, p16 expression, and HPV status. The Kaplan–Meier method was used to estimate overall survival (OS) and disease-free survival (DFS). Survival trends were compared using the log-rank test. Results. Nineteen patients received multimodality treatment for SNUC. Five-year OS and DFS rates were 45.2% and 50.7%, respectively, with

INTRODUCTION 1

Originally described by Frierson et al in 1986, sinonasal undifferentiated carcinoma (SNUC) is a high-grade, aggressive neoplasm arising in the nasal cavity or paranasal sinuses. It is also an uncommon tumor, with a recent meta-analysis reporting only 167 documented cases since 1986.2 The median age at presentation is in the fifth decade, but there is a wide range (12–84 years).2,3 Patients are more likely to be men by a 2:1 ratio.2,4,5 SNUC is usually locally advanced upon presentation and, thus, orbital involvement, skull base invasion, and intracranial extension are common.2,3,5,6 Typical symptoms at presentation include bloody rhinorrhea, nasal obstruction, epistaxis, headaches, cranial neuropathies, visual changes, and facial pain.7 Approximately 10% to 30% of patients present with cervical lymph node involvement.8–11 Hematogenous spread is uncommon, but metastatic disease to the lung, bone, brain, and liver may occur.3,5 Prognosis

*Corresponding author: M. W. Herr, Department of Otology and Laryngology, Division of Head and Neck Surgery, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA 02114. E-mail: [email protected] Peter M. Sadow and Derrick T. Lin contributed equally to this work as senior authors.

no significant difference between treatment types. Tumors from 11 patients were p16-positive and 9 of these were also HPV-positive. Kaplan–Meier analysis demonstrated improved survival. Conclusion. Our series demonstrates a higher prevalence of HPV in SNUC than previously reported. HPV-positive SNUCs may benefit from improved survival and should be investigated further in future studies. C 2014 Wiley Periodicals, Inc. Head Neck 00: 000–000, 2014 V

KEY WORDS: sinonasal undifferentiated carcinoma, p16, human papillomavirus, sinonasal malignancy, treatment

for this disease is poor, and although a recent series reports a 5-year overall survival (OS) rate of 74%,12 the majority of studies describe survival rates ranging from 20% to 63%.2–4,7,9,10,13,14 Although its origins remain uncertain, SNUC is thought to develop from the Schneiderian epithelium, a specialized mucosal epithelium lining the sinonasal tract.15 Histologic features of SNUC are diverse and include sheets and nests of polygonal, pleomorphic cells with a high nuclear-to-cytoplasmic ratio, increased and atypical mitotic figures, coarse chromatin, and dense, hyperchromatic cytoplasm.16 Any number of these descriptive histologic features are shared with other malignancies of the sinonasal tract, including esthesioneuroblastoma, smallcell undifferentiated neuroendocrine carcinoma, nuclear protein in testis midline carcinoma, malignant mucosal melanoma, Ewing sarcoma/peripheral neuroectodermal tumor, rhabdomyosarcoma, and some hematopoietic malignancies. Therefore, additional immunohistochemical and molecular studies are often required to definitively establish the diagnosis of SNUC. Because of its low incidence, there is currently no consensus for the standard treatment of SNUC. However, given the aggressive nature of this malignancy and poor survival outcomes, the available literature does uniformly advocate a multimodality approach.2,3,7–9,12–14,17 Various HEAD & NECK—DOI 10.1002/HED

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combinations of surgery, radiation therapy, and chemotherapy have been reported. Nevertheless, several issues remain unclear, including the optimal order of treatment, the role of surgical debulking in unresectable cases, and the type of radiation that should be used. Similarly, with the exception of cervical lymph node involvement, significant prognostic factors have been difficult to ascertain.2,17 High-risk human papillomavirus (HPV) has a wellestablished association with head and neck carcinoma, most notably in the oropharynx, and the incidence of these HPV-related malignancies is increasing.18 Several recent series have demonstrated the presence of HPV in lesions of the sinonasal tract, including sinonasal papillomas, squamous cell carcinoma (SCC), small-cell undifferentiated neuroendocrine carcinoma, undifferentiated nasopharyngeal carcinoma, and SNUC.15,19–22 However, detection rates among all sinonasal tumors are widely disparate and only 2 cases of SNUC have been reported with positive HPV testing.21,22 Thus, in truth, little is known regarding the incidence or clinicopathologic behavior of HPV-associated SNUC. The purpose of this study was: (1) to review the experience and survival outcomes for patients with SNUC at the Massachusetts Eye and Ear Infirmary (MEEI)/Massachusetts General Hospital (MGH) Cranial Base Center treated with multimodality therapy; (2) to determine the prevalence of high-risk HPV in our population of patients with SNUC; and (3) to examine the prognostic factors and clinicopathologic behaviors associated with HPV-positive SNUC.

After approval from the Institutional Review Boards at MEEI and MGH, all patients diagnosed with SNUC between January 1995 and January 2013 were identified by a review of records. Each patient had been evaluated by a multidisciplinary team that included a radiation oncologist, medical oncologist, otolaryngologist, pathologist, and neurosurgeon. All patients were evaluated with an MRI of the brain and skull base and a fine-cut CT scan of the skull base. A treatment plan was formulated for each patient based on the clinical stage at presentation. All medical records including imaging reports, operative reports, and chemotherapy and radiotherapy records were reviewed retrospectively. Extracted information included patient demographics, symptoms at diagnosis, tumor stage and grade, treatment received, treatment complications, tumor recurrence, and patient survival. Tissue samples from all diagnostic biopsies and surgical specimens were evaluated by a single head and neck pathologist.

p16 Immunohistochemistry Adequate specimen was available in 14 patients to conduct immunohistochemical (IHC) evaluation for expression of the CDK-inhibitor p16, a biomarker of HPV E7 oncoprotein activity.23–28 The IHC technique has been previously described and begins with deparaffinized sections that are subjected to antigen retrieval using 10 mmol/L citrate buffer (92 C for 30 minutes).23 A primary mouse monoclonal antibody against p16 (MTM Laboratories) was visualized with the Leica Bond III autostainer. The p16 expression was scored as positive if strong, HEAD & NECK—DOI 10.1002/HED

Human papillomavirus in situ hybridization All of the formalin-fixed, paraffin-embedded specimens were then evaluated for high-risk HPV DNA using the in situ hybridization (ISH) – catalyzed signal amplification method. The technique for ISH testing was performed using the Ventana Autostainer and has also been described previously.23,29 The Inform HPV III Family 16 Probe (human papillomavirus DNA probe) from Ventana contains a cocktail with affinities to high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66. The ISH assay was performed in accord with the manufacturer’s guidelines using the BenchMark automated slide staining system. HPV control slides consisted of formalin-fixed, paraffin-embedded sections containing 3 separate collections of cells on a slide (Ventana Medical Systems). These cells consisted of the CaSki cervical cancer cell line (containing 200–400 copies of HPV16 per cell); the HeLa cervical cancer cell line (containing 10– 50 copies of HPV18 per cell); and the C-33A cell line, which served as a negative control. Reagent negative control was set using negative control probes provided by Ventana Medical Systems.29 Evidence of nuclear staining (classified as either punctate or diffuse) defined an HPVpositive tumor.23,29

Human papillomavirus polymerase chain reaction

MATERIALS AND METHODS

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diffuse nuclear and cytoplasmic staining was present in 70% of the tumor specimen. Cervical carcinoma provided a positive control for p16 staining.

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Tumors that were discordantly p16-positive by IHC but demonstrated negative or equivocal results for high-risk HPV DNA testing by ISH were further evaluated by restriction fragment length polymorphism-based polymerase chain reaction (RFLP-PCR). PCR analysis was performed using a standard protocol, which has been previously described and has been a clinical standard at our institution for 20 years.30,31 This method can identify most known high-risk HPV subtypes, such as type 16, 18, 31, and 33. Determination of the specific subtype was based on comparison with known HPV standards and established restriction maps of the amplified regions.30,31

Statistical analysis Patient demographics and treatment methods were compared based on p16 and HPV status using the chi-square test for proportions and the t test for means. The Kaplan– Meier method was used to estimate OS, with an endpoint of death from any cause, and disease-free survival (DFS), with an endpoint of disease recurrence. Survival trends were compared by patient age (50 vs >50), sex, p16 status, HPV status, and treatment modality using the logrank test. Associations with p < .05 were considered significant. All analyses were performed using STATA version 12 (StataCorp LP, College Station, TX).

RESULTS Patient and tumor characteristics Nineteen patients with SNUC were treated at MEEI and MGH from 1995 to 2012. There were 15 men and

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HPV

TABLE 1. Patient demographics, tumor classification, p16 and human papillomavirus status, treatment, and outcomes. Patient no.

Age, y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

52 42 42 88 35 64 55 64 64 41 43 53 70 19 46 74 57 34 40

Sex

M M F M F M M M M F M F M M M M M M M

TNM classification

T4bN0 T4aN0 T4bN0 T4bN1 T4bN0 T4bN0M1 T4bN0 T4bN0 T4bN0 T4aN2a T4aN0 T4bN0 T4bN0 T4bN0 T4bN0 T4aN0 T4bN1 T4bN0 T4bN0

p16

Neg Pos N/A Pos Pos N/A N/A Pos Pos Pos N/A Pos Neg Neg N/A Pos Pos Pos Pos

HPV

Treatment

Status

Survival, mo

Neg Neg N/A Pos Pos N/A N/A Pos Pos Pos N/A Pos Neg Neg N/A Pos Pos Neg Pos

C

DOD NED DOD DOD NED DOD NED NED DOD NED AWD DOD DOD DOD NED NED NED NED NED

6 94 30 2 60 23 58 35 12 34 32 16 3 21 15 12 9 6 3

Chemo 1 XRT CFR 1 ChemoI,C 1 XRT Sub 1 ChemoC 1 XRT Sub 1 ChemoC 1 XRT Sub 1 ChemoC 1 XRT ChemoC 1 XRT ChemoI,C 1 XRT CFR 1 ChemoC 1 XRT CFR 1 ChemoC 1 XRT ChemoI,C 1 XRT ChemoI,C 1 XRT CFR 1 ChemoC 1 XRT Sub 1 ChemoI,C 1 XRT CFR 1 ChemoC 1 XRT ChemoC 1 XRT CFR 1 ChemoC 1 XRT ChemoC 1 XRT CFR 1 ChemoC 1 XRT CFR 1 ChemoC 1 XRT

Abbreviations: HPV, human papillomavirus; Neg, negative; ChemoC, concurrent chemotherapy; XRT, proton beam radiation; DOD, died of disease; Pos, positive; CFR, craniofacial resection; ChemoI,C, induction and concurrent chemotherapy; NED, no evidence of disease; N/A, available specimen inadequate for testing; Sub, subtotal resection; AWD, alive with disease.

4 women in the cohort with a mean age of 52 years (range, 19–88 years; Table 1). Tumors were staged in accordance with the American Joint Committee on Cancer staging for malignancies of the paranasal sinuses.32 All patients in our population presented with locally advanced (T4) disease. Fourteen patients (74%) exhibited invasion of the dura and 4 patients (21%) had cavernous sinus involvement evident on preoperative imaging. Tumor invasion of the orbit(s) or orbital apex was seen in 12 patients (63%). Three patients (16%) had regional metastases to the cervical lymph nodes at the time of presentation. One patient (5%) presented with distant metastases (M1) to the liver and spine (Table 1).

Tumor histology Histologic evaluation demonstrated tumors that were largely present in the submucosal tissues of the nasal cavity and sinuses and could readily be seen infiltrating the bone. Typically, no in situ or well-differentiated component was seen. Classic SNUC morphology is demonstrated in Figure 1. Lesions demonstrated a nested (Figure 1A) or diffuse, sheet-like (Figure 1B) architecture with pleomorphic to rounded cells. Additional histologic features included a high nuclear:cytoplasmic ratio, dense cytoplasm with scattered clear (cytoplasmic) cells, course chromatin with a prominent nucleolus, brisk mitotic activity, and necrosis or apoptosis. A panel of immunostains and molecular testing was also utilized to support the diagnosis of SNUC and discount other entities. Tumor cells uniformly stained for cytokeratin, confirming the lesion as a carcinoma (Figure 1D). Tumors were negative for chromogranin, synaptophysin, S100, HMB45, CD 99, vimentin, desmin, neuronspecific enolase, and p63. Testing for Epstein–Barr virus was routinely performed and was uniformly negative. Immunostaining for p16 and ISH for high-risk HPV16 family isoforms is demonstrated in Figure 2. Positive ISH

analysis produced variable nuclear staining, with dark blue/black dot-like staining indicating expression of highrisk HPV (Figure 2C).

p16 and human papillomavirus status Pathologic specimens were available for p16 and HPV testing in 14 patients. Definitive IHC, ISH, or RFLP-PCR was not possible on the existing specimens from the 5 remaining patients. Nine patients demonstrated diffuse nuclear and cytoplasmic positivity for p16 with the associated expression of high-risk HPV subtypes (HPV16 family) by ISH or RFLP-PCR (Table 1). The tumors from 2 patients were positive for overexpression of p16, but showed no evidence of HPV DNA on ISH or PCR analysis. The remaining 3 patients were negative for p16 and HPV. There were no significant differences in patient demographics or treatment method when comparing patients by p16 or HPV status.

Treatment Eight patients underwent open craniofacial resection followed by adjuvant chemoradiation (CFR 1 chemo 1 XRT). Five of these patients (62.5%) are currently alive with no evidence of disease (NED) and 3 patients (37.5%) have died of disease (DOD; Table 1). Four patients were treated with subtotal resection for control of symptoms and to facilitate concurrent chemoradiation therapy (sub 1 chemo 1 XRT). One patient died before the start of chemoradiation. A second patient died of his disease in the midst of chemoradiation therapy. The final 2 patients completed treatment: 1 of these patients (25%) is currently alive with NED, whereas the other DOD 30 months from diagnosis (Table 1). The remaining 7 patients were treated primarily with proton beam radiation and chemotherapy (chemoC 1 XRT). Three patients received induction chemotherapy with cisplatin and HEAD & NECK—DOI 10.1002/HED

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FIGURE 1. Sinonasal undifferentiated carcinoma (SNUC) morphology (hematoxylin-eosin stain). Tumor shows a nested (A; original magnification 3100) or diffuse, sheet-like (B; original magnification 3100) pattern of infiltration. As seen at high power (C; original magnification 3600), cells are characterized by a rounded to pleomorphic appearance, with dense cytoplasm, coarse nuclear chromatin with prominent nucleoli, numerous mitotic figures (black arrows), apoptotic bodies (arrowheads), and crush artifact (white arrows). The only uniformly positive immunostain is cytokeratin; shown here, cytokeratin cocktail AE1/3/CAM5.2 (D; original magnification 3400).

etoposide before definitive treatment (chemoI,C 1 XRT). Currently, 4 patients (57%) within this group are alive with NED and 1 patient (14%) is alive with disease. Two patients (29%) DOD at 6 and 23 months (Table 1). As described above, all patients in our series received proton beam radiation (either adjuvant or primary) to the primary site with median dose of 70 cobalt-gray equivalents (CGE; range 50–76 CGE). All patients were also treated with bilateral elective neck irradiation—60 CGE of proton beam radiation to the upper neck and 50 Gy of external beam photons to the lower neck. The technique for planning and delivery of proton beam therapy at MGH has been described elsewhere.33 There were no breaks in treatment because of acute radiation toxicities. Of the 19 patients in the study cohort, 10 were managed with concurrent chemotherapy using our standard regimen of cisplatin or carboplatin and etoposide. The remaining 9 patients were treated at the discretion of the medical oncologist with the various regimens of platinum-based monotherapy, carboplatin and paclitaxel, or methotrexate, cisplatin, and bleomycin. Across all treatment groups, a total of 5 patients received induction chemotherapy. 4

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Outcomes Local, regional, and distant control. Six patients had recurrences an average of 11.7 months (range, 7–16 months) from diagnosis. Of these cases, 4 patients had initially undergone craniofacial resection followed by adjuvant chemoradiation and recurred locally, most commonly at the dura (Table 2). One patient who was treated with subtotal resection followed by chemoradiation (patient 3), initially presented with recurrent disease in the maxillary sinus and cervical lymph nodes 15 months after diagnosis. Seven months after maxillectomy, neck dissection, and adjuvant chemoradiation, she developed distant metastases in the liver and spine and eventually died of her disease (Table 2). One patient who was treated with chemotherapy and radiation therapy alone (patient 11) developed recurrence in the dura, spine, and mediastinal lymph nodes 16 months after diagnosis. After failing to respond to radiation therapy and chemotherapy, he transitioned to an alternate, experimental chemotherapeutic protocol and was alive with disease at his most recent follow-up (Table 2).

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UNDIFFERENTIATED CARCINOMA AND

HPV

FIGURE 2. Sinonasal undifferentiated carcinoma (SNUC) and human papillomavirus (HPV). Case 1 (top row) and case 2 (bottom row) demonstrate the variable tumor morphology (A–D; hematoxylin-eosin stain, original magnification 3400), with positive (nuclear and cytoplasmic) staining for p16 (B; original magnification 3400) corresponding to the blue dot-like positivity for HPV (arrows) by in situ hybridization (C; original magnification 3400). Conversely, the SNUC negative for p16 (E; original magnification 3400) is also negative for HPV16 (F; original magnification 3400).

Survival rates Overall, the mean follow-up time was 25 months (range, 2–94 months). Kaplan–Meier curves were calculated for the entire population and revealed a 5-year OS rate of 45.2% (95% confidence interval [CI], 18.4–68.9) and 5-year DFS rate of 50.7% (95% CI, 20.6–74.6). When stratified by age, 5-year OS was greater for patients aged 50 and younger (63.5%; 95% CI, 14.7– 89.8) as compared to those older than 50 (31.1%; 95% CI, 6.2–61.2). This difference approached statistical significance (log-rank p 5 .057). There was no significant difference in DFS by age (log-rank p 5 .571). OS and DFS were also stratified by treatment type: CFR 1 chemo 1 XRT versus subtotal resection 1 chemo 1 XRT versus chemo 1 XRT. Five-year OS rates were 45.5% (95% CI, 8.1–78.1), 25.0% (95% CI, 0.9–66.5), and 65.8% (95% CI, 17.8–90.4), respectively. Five-year DFS rates were 30.6% (95% CI, 2.9–67.3), 50.0% (95% CI, 0.6–91.0), and 75.0% (95% CI, 12.8–9.1), respectively. Comparison using the log-rank test demonstrated no statistical difference in OS (log-rank p 5 .417) between the treatment types. There was a trend toward improved DFS with primary chemoradiation (chemo 1

XRT) compared with the other treatment regimens; however, the difference was not significant (log-rank p 5 .079).

Survival – p16 and human papillomavirus status Kaplan–Meier analysis was also performed for the subset of 14 patients with p16 and HPV data. Patients who were positive for overexpression of p16 demonstrated a significant improvement in OS: 61.9% (95% CI, 21.6–86.1) compared to 0% in patients who were p16-negative (log-rank p 5 .048; Figure 3). There was also a trend toward improved DFS in p16-positive patients, although this did not reach statistical significance (log-rank p 5 .094; Figure 4). Evaluation of survival based upon HPV status revealed a nonsignificant trend toward improved OS in HPV-positive patients. Five-year OS rates were 55.7% (95% CI, 15.2–83.4) for HPV-positive patients and 27.8% (95% CI, 1.1–69.5) for HPV-negative patients (log-rank p 5 .432). There was no significant difference in DFS: 5-year DFS rates were 48.2% (95% CI, 9.0–80.1) for HPV-positive patients and 71.4% (95% CI, 9.0–95.4) for HPV-negative patients (log-rank p 5 .869). HEAD & NECK—DOI 10.1002/HED

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TABLE 2. Tumor recurrence, treatment, and outcomes.

Patient

TNM

Time to recurrence, mo

Location of recurrence

CFR 1 Chemotherapy and proton beam radiotherapy 8 T4bN0 11 Dura (frontal) 9 T4bN0 7 Dura (parietal) 12 T4bN0 13 Skull base, dura, bilateral orbital apex 14 T4bN0 8 Dura (anterior falx) Subtotal resection 1 chemotherapy and proton beam radiotherapy 3 T4bN0 15; 22 Maxillary sinus and neck, liver, spine Chemotherapy and proton beam radiotherapy only 11 T4aN0 16 Dura, spine, mediastinal LNs

Treatment

Status

Survival, mo

Rsxn and postoperative XRT (60 Gy - proton)/cisplatin Palliative chemotherapy (temodar) None

NED DOD DOD

35 12 16

Rsxn of tumor and sagittal sinus -> 70 Gy (proton) 1 carbo/paclitaxel

DOD

21

Inferior maxillectomy and ND -> Close margins -> XRT 1 carbo/paclitaxel -> palliative chemo XRT

DOD

30

XRT 1 protocol 10–262 with BYL19 -> NR -> paclitaxel/GDC0068 protocol

AWD

32

Abbreviations: CFR, craniofacial resection; Rsxn, Resection; XRT, radiotherapy; NED, no evidence of disease; DOD, died of disease; carbo, carboplatin; ND, neck dissection; chemo, chemotherapy; LNs, lymph nodes; NR, no response; AWD, alive with disease.

Complications

had incisional breakdown and surgical wound infections after completion of radiation therapy (grade 3). A fourth patient required removal of her frontal bone flap after developing osteomyelitis 6 weeks after craniofacial resection (grade 3).

Treatment toxicity was evaluated based upon the Common Terminology Criteria for Adverse Events (version 4.0) of the National Cancer Institute. Eight of the 19 patients (42.1%) in our cohort experienced a total of 10 moderate to severe complications from all modalities of therapy. Three patients (15.8%) developed ocular complications including: mild, self-limiting diplopia (grade 2), chronic epiphora treated with endoscopic dacryocystorhinostomy (grade 3), and ipsilateral vision loss (grade 4). Two patients (10.5%) had a complication involving the central nervous system. One of the patients with a central nervous system complication developed peripheral neuropathy (grade 2), whereas another had recurrent seizures secondary to radiation-induced brain edema (grade 3). Four patients (21.1%) suffered from a total of 5 infectious or wound complications. One patient presented with facial cellulitis and a maxillary mucocele that required IV antibiotics and endoscopic drainage (grade 3). Two patients

The histopathologic diagnosis of SNUC can be difficult and an experienced pathologist is critical for accurate identification of the disease. Histologically, it is a highgrade malignancy that demonstrates varied growth, prominently including nested, lobular, and sheet-like patterns.1,15,16 The morphology is also variable, with medium to large cells that are round to oval, have hyperchromatic nuclei, and prominent nucleoli. Cytoplasmic content is variable with largely dense cytoplasm and scattered clear cells. As in other high-grade malignancies, there is typically a high nuclear to cytoplasm ratio, as well as marked increase in mitotic activity, tumor

FIGURE 3. Five-year overall survival (OS) Kaplan–Meier curves by p16 status. Five-year OS rates were 61.9% for p16-positive patients (11 cases) and 0% for p16-negative patients (3 cases), log-rank p 5 .048.

FIGURE 4. Five-year disease-free survival (DFS) Kaplan–Meier curves by p16 status. Five-year DFS rates were 56.6% for p16positive patients (11 cases) and 0% for p16-negative patients (3 cases), log rank p 5 .094.

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DISCUSSION

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necrosis, and apoptosis. Thus, IHC techniques are often required to distinguish SNUC from other tumors. Unlike other carcinomas, SNUC is consistently immunoreactive for cytokeratin markers of simple epithelia, including CK 8 (100% cases) as well as CK 7 and CK 19 (50% of cases).15,16,34 Reactivity for other markers, such as epithelial membrane antigen, neuron-specific enolase, and p53, is reported in less than half of the cases.11,15,16 Vimentin, muscle markers (desmin, myoglobin, actins), hematolymphoid markers (leukocyte common antigen, B and T-cell lineage) melanocytic cell markers (HMB-45, melan A), and CD99 are uniformly negative.15,16 The treatment of SNUC continues to be challenging as the limited number of patients precludes robust studies of therapeutic protocols. Survival outcomes are poor despite universal application of aggressive multimodality therapies, indicating that optimal management of these tumors remains elusive. Several institutions have reported their experience treating SNUC using a variety of single and combined-modality protocols, including surgery alone, radiation alone, surgery with adjuvant photon beam radiation and chemotherapy, preoperative radiation therapy followed by surgery, and induction chemotherapy followed by surgery or radiation therapy with or without adjuvant chemotherapy.4,7–10,12–14,17 Calculated 5-year OS rates range from 20% to 74%. Although small case numbers and variable treatment strategies limit definitive conclusions, the authors universally endorse aggressive, multimodality therapy.3,8–10,12,13,17 In 2002, Musy et al7 studied the outcomes of 20 patients treated at the University of Virginia from 1986 to 2000. Patients with limited intracranial involvement or intraorbital disease were treated with chemotherapy (cyclophosphamide, doxorubicin, and vincristine or cisplatin and etoposide) and photon beam radiation therapy followed by craniofacial resection. The remaining patients were treated with palliative radiotherapy or chemoradiation alone. The overall 2-year survival for the entire population was 47%. Although there was a trend toward improved survival in the surgical group, this was not statistically significant (p 5 .076). It is interesting to note, however, that the authors found residual tumors in 70% of the surgical specimens after primary chemoradiation. These findings suggest that surgery is an essential part of multimodality therapy in patients with SNUC.7 In 2008, Chen et al14 at the University of CaliforniaSan Francisco reviewed their results for 21 patients with SNUC, all of whom were treated with multimodality therapy. Seventeen patients underwent surgery followed by postoperative radiotherapy with or without adjuvant chemotherapy. Two patients were treated with neoadjuvant chemoradiation followed by surgery and 2 patients received chemoradiation only. These strategies achieved a 5-year OS rate of 43%, with no difference in local control based on initial treatment type. However, the authors found that for the patients who underwent surgical intervention, the local control rate was 74% when gross total resection was achieved compared with local control rates of only 24% after subtotal resection.14 Utilizing similar combined-modality regimens, Al-Mamgani et al12 achieved 5-year OS and DFS rates of 74% and 64%, respectively, in their recent series of 21 patients.

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HPV

Although 6 patients did have a total of 10 serious complications from treatment, the incidence of late toxicity was improved by the introduction of intensity-modulated radiation therapy. Collective review of these series leads to several important conclusions. First, better outcomes are achieved when surgical resection involving complete tumor extirpation is incorporated into treatment. Second, combinedmodality treatment should be offered to all patients. The sequence of these modalities may be dictated by the extent of disease, performance status of the patient, and available treatment resources.12 These concepts were echoed by Reiersen et al2 in their recent meta-analysis of 167 patients. With a median follow-up of 15 months, the OS rate averaged 47.3%. Despite wide variability in treatment regimens, they found converging evidence to suggest that surgery with adjuvant radiation and/or chemotherapy produces the best outcomes.2 Additional analysis also found the presence of neck disease to be a poor prognostic sign. To date, our series is one of the largest to examine the outcomes after using multimodality therapy to treat all patients diagnosed with SNUC. Although 1 of 3 distinct therapeutic regimens was used for each case, every patient in our cohort received proton beam radiation (to the primary tumor and elective neck irradiation) as well as chemotherapy. Eight patients underwent up-front surgical extirpation of the tumor via open craniofacial resection. Subtotal resection was performed on another 4 patients in an effort to alleviate symptoms and decrease tumor volume before chemoradiation therapy. Despite uniformly aggressive multimodality management, calculated 5-year OS and DFS rates were 45.2% and 50.7%, respectively, similar to those reported in the literature. Analysis was limited by the small number of patients and inherently short follow-up times, thus, a statistical difference in survival was not observed between the 3 treatment groups. In our series, patients younger than age 50 did demonstrate improved survival; however, clinical applicability of this finding is tempered by the fact that universally aggressive management of SNUC across all age groups failed to produce satisfactory results.

p16 and human papillomavirus: Prevalence and prognostic implications In the past decade, high-risk HPV has become a wellestablished cause of head and neck carcinoma.35,36 Perhaps even more importantly, HPV status has a significant impact on patient profiles and clinical outcomes.37,38 Several reports have demonstrated that HPV-positive cancers of the head and neck tend to occur in younger patients without a history of tobacco or alcohol abuse,39 involve the oropharynx,40,41 and have better OS than HPV-negative tumors.15,18,36–38,42–44 The presence of HPV has also been documented in lesions of the sinonasal tract, including sinonasal papillomas (low risk HPV6/11), SCC, poorly differentiated neuroendocrine carcinoma (small cell carcinoma), nasopharyngeal carcinoma, and SNUC.15,19–22 In a review of 22 studies encompassing 322 cases of sinonasal SCC, Syrj€anen19 found that 70 cases (21.7%) were positive for HPV DNA with HPV16 and 18 being HEAD & NECK—DOI 10.1002/HED

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the most common subtypes. El-Mofty and Lu21 examined 39 cases of sinonasal carcinoma, including 10 patients with SNUC. Two cases of SNUC demonstrated positive reactivity for p16, although HPV DNA was detected in only 1 of these patients. Bishop et al22 at Johns Hopkins reviewed 161 cases of sinonasal carcinoma. Of the 16 patients in this cohort who were diagnosed with SNUC, 4 were positive for p16. HPV16 DNA was present in only 1 of these cases. Finally, Wadsworth et al15 analyzed 5 cases of SNUC and found strong diffuse positivity for p16 in every specimen. In spite of this, HPV DNA was not detected in a single instance. Thus, of the 31 cases of SNUC analyzed for HPV in the literature, a total of 11 cases (35.4%) were reactive for p16, and 2 cases (6.5%) revealed the presence of HPV DNA. Utilizing the scientific gold standard for detection, p16 and HPV testing was performed on the pathologic specimens of 14 patients in our cohort. Similar to Wadsworth et al15 who saw nearly universal reactivity to p16, 11 cases (78.6%) in our series were positive. Interestingly, HPV DNA was present in 9 patients (64.3%), a rate significantly higher than previously noted in the literature. We did not find any significant difference in patient demographics, including age, by p16 or HPV status. Kaplan–Meier analysis revealed a significant improvement in OS for patients whose tumors demonstrated p16 overexpression. There was also a nonsignificant benefit in DFS for p16-positve patients. Furthermore, although not statistically significant, there was a similar trend toward improved survival in patients with HPV-positive tumors. Although our small population precludes any definitive conclusions, these preliminary findings certainly suggest that p16 overexpression and HPV infection may play a role in the carcinogenic process of a subset of SNUCs.15 Furthermore, HPV-positive SNUCs may benefit from improved outcomes similar to HPV-positive oropharyngeal squamous cell carcinomas (SCCs). The mechanisms for these higher survival rates are not well understood; however, HPV-positive oropharyngeal SCCs are genetically distinct from HPV-negative cancers of the region.43,44 The former are more likely to possess functional, wild-type TP53, which may render the tumors more susceptible to radiation-induced apoptosis.43,45 They are also less likely to carry gene amplification at 11q13, a molecular marker that has been associated with more rapid and frequent recurrence of disease, as well as poor survival.43,45,46 Although no direct correlation has been made between survival and any specific mechanism, previous work with oropharyngeal SCC has demonstrated higher rates of response to radiation therapy and chemotherapy among patients with HPV-positive tumors.23,44 While this tendency was not clearly demonstrated in our population of p16 and HPV-positive patients with SNUC, their improved OS rates indicate that these tumors are, indeed, less aggressive biologically. Further investigation with larger patient populations and additional molecular testing is essential for more complete understanding of the clinicopathologic behavior of HPV-associated SNUCs and its impact on treatment outcomes. The relatively frequent discrepancy between p16 reactivity and HPV positivity in patients with SNUC is 8

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curious, especially in light of the fact that up to 97% of HPV-positive oropharyngeal SCCs show strong expression of p16INK4A.15,47 p16 is a sensitive marker for cells expressing the E7 oncoprotein, which is encoded by the E7 viral oncogene. However, it is not specific for HPV infection. Benign mucosa, as well as carcinomas unrelated to HPV infection, may stain strongly with p16.15 In fact, Schwerer et al47 detected p16 expression in normal respiratory epithelium, sinonasal papillomas, and adjacent mucosa. With this in mind, and considering the unique pathologic and clinical behavior of SNUC, cases of p16 overexpression in the absence of HPV DNA would not be unexpected. The role that each plays in the development of SNUC, either singly or in conjunction with each other, requires further study. Finally, the high prevalence of HPV in our patient population also bears further discussion. Reported HPV detection rates are notoriously disparate because of dissimilar assays and varied study populations.22 At our institution, we reflexively test all oropharyngeal cancers for HPV. However, additional p16 testing is conducted on a variety of other nonkeratinizing malignancies of the head and neck to screen for the possibility of HPVrelated abnormalities. This is followed by high-risk HPV16 family ISH testing, and in some cases, PCRbased follow-up for discrepant results. In our hands, the HPV ISH is largely specific, although it may lack sensitivity for low levels of HPV activity. The PCR-based assay, however, is quite sensitive because the amplicon is relatively comprehensive, covering both low-risk and high-risk HPV types. Thus, the high rate of HPVassociation in our series of patients with SNUC is likely a reflection of our robust testing techniques uncovering a previously underappreciated prevalence, or a rising incidence, of the virus in the population of patients with SNUC as a whole.

CONCLUSION Nineteen patients with SNUC were treated at MEEI and MGH from 1995 to 2012. A multimodality treatment approach was utilized with each patient receiving 1 of 3 therapeutic regimens: (1) open craniofacial resection followed by adjuvant chemoradiation; (2) subtotal resection for control of symptoms and to facilitate concurrent chemoradiation therapy; or (3) concurrent proton beam radiation and chemotherapy. Calculated 5-year OS was 42.5% and DFS was 50.7%, which is similar to that reported in the literature. There was no significant difference in OS or DFS between treatment types. When stratified by age, 5-year OS was greater for patients age 50 and younger as compared to those older than 50, and this difference approached statistical significance. Our series of patients demonstrates a much higher prevalence of HPV in SNUC than previously reported in the literature. Further analysis also suggests that, as with HPV-associated tumors in other subsites of the upper aerodigestive tract, p16 and HPV-positive SNUCs benefit from improved survival outcomes. Given the universally abysmal outcomes for patients with SNUC, this new evidence of increased HPV association may unlock additional treatment strategies and should be investigated further in future studies.

SINONASAL

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