Post-Treatment Neck Dissection of Tonsillar and Base of Tongue Squamous Cell Carcinoma in the Era of PET-CT, HPV, and p16.

Human-papillomavirus (HPV)-positive tonsillar and base of tongue carcinomas (TSCC/BOTSCC) are rising in incidence and treatments with radiotherapy, chemoradiotherapy (RT/CRT), and neck dissections (NDs) have several side effects. Therefore, an improved selection of patients needing salvage NDs would be beneficial. We examined the prevalence and localisations of viable tumour cells in neck lymph nodes in patients post-RT/CRT, identified by fluorodeoxyglucose positron-emission tomography with computer-tomography (FDG PET-CT), with a focus on HPV-associated tumours. Patients with 217 TSCC/BOTSCC with tumours assessed for HPV-DNA and p16INK4a undergoing FDG PET-CT 12 weeks after treatment and/or an ND were included. The FDG PET-CT data were compared with the findings in the pathology report after the ND. In total, 36/217 (17%) patients were selected for an ND due to positive findings in post-treatment FDG PET-CT. Of these, 35/36 were HPV-associated, 10/36 (28%) had viable tumour cells in the pathology reports of the neck specimen, and 8/10 (80%) were consistent with the FDG PET-CT findings, while 2/36 (5%) were missed by FDG PET-CT. We conclude that FDG PET-CT 12 weeks after RT/CRT is useful, but not completely reliable for finding all the metastases of HPV-associated TSCC/BOTSCC. Nonetheless, our data indicate that an ND could be more selectively guided by FDG PET-CT.

Long-Term Survival and Recurrence in Oropharyngeal Squamous Cell Carcinoma in Relation to Subsites, HPV, and p16-Status.

Long-term survival data in relation to sub-sites, human papillomavirus (HPV), and p16INK4a (p16) for patients with oropharyngeal squamous cell carcinoma (OPSCC) is still sparse. Furthermore, reports have indicated atypical and late recurrences for patients with HPV and p16 positive OPSCC. Therefore, we assessed long-term survival and recurrence in relation to oropharyngeal subsite and HPV/p16 status. A total of 529 patients with OPSCC, diagnosed in the period 2000-2010, with known HPVDNA and p16-status, were included. HPV/p16 status and sub-sites were correlated to disease-free and overall survival (DFS and OS respectively). The overexpression of p16 (p16+) is associated with significantly better long-term OS and DFS in tonsillar and base of tongue carcinomas (TSCC/BOTSCC), but not in patients with other OPSCC. Patients with HPVDNA+/p16+ TSCC/BOTSCC presented better OS and DFS compared to those with HPVDNA-/p16- tumors, while those with HPVDNA-/p16+ cancer had an intermediate survival. Late recurrences were rare, and significantly more frequent in patients with p16- tumors, while the prognosis after relapse was poor independent of HPVDNA+/-/p16+/- status. In conclusion, patients with p16+ OPSCC do not have more late recurrences than p16-, and a clear prognostic value of p16+ was only observed in TSCC/BOTSCC. Finally, the combination of HPVDNA and p16 provided superior prognostic information compared to p16 alone in TSCC/BOTSCC.

Psoriasin expression is associated with survival in patients with human papillomavirus-positive base of tongue squamous cell carcinoma.

Patients with human papillomavirus-positive (HPV+) base of tongue squamous cell carcinomas (BOTSCC) have an improved survival compared with patients with HPV-negative BOTSCC and it has been suggested that treatment should be tailored. Before individualized treatment can be introduced, additional prognostic markers are required. A prognostic role of psoriasin has previously been demonstrated outside BOTSCC. Therefore, the present study aimed to examine psoriasin in BOTSCC, with focus on HPV+ BOTSCC, in relation to prognosis. A total of 72 BOTSCC samples were stained for psoriasin by immunohistochemistry, and the association between expression and clinical outcomes was analyzed. Patients with low psoriasin expression exhibited significantly improved overall survival (OS; P=0.001) and disease-free survival (DFS; P=0.007), which also was observed in patients with HPV+ BOTSCC (OS, P<0.001; DFS, P=0.02). Furthermore, psoriasin was a significant prognostic factor in univariable and multivariable analyses. In conclusion, psoriasin could be used as a prognostic marker in HPV+ BOTSCC.

Tumour inflammation signature and expression of S100A12 and HLA class I improve survival in HPV-negative hypopharyngeal cancer.

Hypopharyngeal squamous cell carcinoma (HPSCC) has a very poor prognosis. Local surgery may increase survival, but is often avoided due to significant post-op co-morbidities. Since prognostic markers are lacking, the aim was to find predictive biomarkers that identify patients whose response to oncological treatment is poor and who may benefit from primary surgery to increase survival. Pretreatment biopsies from 23 HPSCC patients, 3 human papillomavirus (HPV) positive and 20 HPV-negative, were analyzed for expression of 750 mRNAs using the Nanostring nCounter IO360 panel in relation to 3-year survival. Validation was performed through immunohistochemistry (IHC) for HLA class I and S100A12 in 74 HPV-negative HPSCC samples. Clustering identified a subset of HPV-negative HPSCC with favorable prognosis and a gene expression signature overexpressing calgranulins and immune genes, distinct from that of HPV-positive HPSCC. Enrichment analysis showed immune signaling, including the tumor inflammation signature, to be enriched in surviving patients. IHC validation confirmed high S100A12 and HLA class I expression to correlate with survival in HPV-negative HPSCC. This shows that immune activity is strongly related to survival in HPV-negative HPSCC. Enrichment of the tumor inflammation signature indicates a potential benefit of immunotherapy. Low expression of both HLA class I and S100A12 could be used to select patients for local surgery.

Survival of patients with oropharyngeal squamous cell carcinomas (OPSCC) in relation to TNM 8 - Risk of incorrect downstaging of HPV-mediated non-tonsillar, non-base of tongue carcinomas.

BACKGROUND: TNM-8 staging separates oropharyngeal squamous cell carcinomas (OPSCC) into human papillomavirus (HPV)-mediated and -unrelated OPSCC based on p16INK4a overexpression (p16+), as surrogate marker for HPV. However, OPSCC is histologically and clinically heterogenous including tonsillar and base of tongue squamous cell carcinomas (TSCC and BOTSCC respectively), and carcinomas of soft palate and walls (otherOPSCC). The significance of HPV is established in TSCC/BOTSCC, while its role in otherOPSCC is unclear, which is not considered in TNM-8. Here, p16+ was therefore evaluated in relation to overall survival (OS) and tumor stage per OPSCC subsite. PATIENTS AND METHODS: All 932 patients, treated with curative intent in Stockholm 2000-2016 with OPSCC, previously analyzed for p16 expression, were included. Clinical data, including stage and OS, was collected retrospectively. RESULTS: Patients with p16+ otherOPSCC had significantly poorer OS compared to patients with p16+ TSCC/BOTSCC (p = 0.005) and their survival was similar to that of patients with p16-otherOPSCC/TSCC/BOTSCC. Moreover, patients with TNM-8 stage I-II and p16+ otherOPSCC had a significant poorer OS compared to patients with p16+ TSCC/BOTSCC and similar stage (p = 0.02). Lastly, patients with otherOPSCC and low TNM-7 stage had a significant better OS, as compared to those with a high stage (p = 0.019) while no hazard discrimination was observed with TNM-7 in TSCC/BOTSCC. CONCLUSION: Our results indicate a risk of misclassification of patients with otherOPSCC and low TNM-8 stage. We suggest that p16 should only be evaluated in TSCC/BOTSCC and that patients with otherOPSCC should all be staged as patients with HPV-unrelated (p16-) OPSCC.

Immune related proteins and tumor infiltrating CD8+ lymphocytes in hypopharyngeal cancer in relation to human papillomavirus (HPV) and clinical outcome.

BACKGROUND: Hypopharyngeal cancer (HPSCC) shows a poor clinical outcome, while HPSCC, caused by human papillomavirus (HPV), presents a better outcome. Here, HPCC, immune proteins, and tumor infiltrating CD8+ lymphocytes (CD8+ TILs) were evaluated in relation to HPV and outcome. METHODS: Fresh frozen tissue from four HPV-positive HPSCC, 39 HPV-negative HPSCC, and normal samples were analyzed for protein expression by the Proseek immuno-oncology immunoassay. CD8+ TIL numbers evaluated by immunohistochemistry on 144 formalin-fixed biopsies were analyzed in relation to clinical outcome. RESULTS: Proteins differing between HPV-positive and negative HPSCC included CD8A, PD-L1, Fas ligand, and chemokines. High CD8+ TIL numbers were correlated to improve clinical outcome in HPV-negative HPSCC. CONCLUSIONS: High expression of immune proteins in HPV-positive HPSCC may explain the better clinical outcome. CD8+ TILs are of relevance for outcome of HPV-negative HPSCC, while tumors with high immune activity but poor patient survival suggest a role for immune therapy.

Human papillomavirus (HPV) is absent in branchial cleft cysts of the neck distinguishing them from HPV positive cystic metastasis.

BACKGROUND: Distinguishing branchial cleft cysts (BCCs) from cystic metastases of a human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (OPSCC) is challenging. Fine needle aspirates (FNAs) from cystic metastasis may be non-representative, while reactive squamous cells from BCC can be atypic. Based on cytology and with the support of HPV DNA positivity many centers treat cystic metastasis oncological and thus patients are spared neck dissection. To do so safely, one must investigate whether HPV DNA and p16INK4a overexpression is found exclusively in cystic metastases and not in BCC. PATIENTS AND METHODS: DNA was extracted from formalin fixed paraffin embedded (FFPE) surgically resected BCCs from 112 patients diagnosed 2007-2015 at Karolinska University Hospital and amplified by PCR. A multiplex bead-based assay used to detect 27 HPV-types and p16INK4a expression was analyzed by immunohistochemistry (IHC). RESULTS: All 112 BCCs were HPV DNA negative, and of 105 BCCs possible to evaluate for p16INK4a, none overexpressed p16INK4a. CONCLUSIONS: HPV DNA and p16INK4a overexpression were absent in BCCs. Lack of HPV DNA and p16 protein overexpression in BCCs is helpful to discriminate benign BCCs from HPV+ OPSCC metastasis. HPV testing definitely has a role in the diagnostics of cystic masses of the neck.

Validation of Human Papillomavirus as a Favourable Prognostic Marker and Analysis of CD8+ Tumour-infiltrating Lymphocytes and Other Biomarkers in Cancer of Unknown Primary in the Head and Neck Region.

BACKGROUND: Human papillomavirus (HPV) is a favourable prognostic factor in oropharyngeal cancer. Moreover, we and others reported that HPV-positive cancer of unknown primary in the head and neck region (HNCUP) has better outcome than HPV-negative HNCUP. However, not all studies concord. Here, our previous finding was investigated in a new cohort and additional biomarkers were analyzed. MATERIALS AND METHODS: A total of 19 HNCUPs diagnosed 2008-2013 were analyzed for HPV DNA by polymerase chain reaction assay (PCR) and p16 by immunohistochemistry (IHC). Thereafter, 69 HNCUPs diagnosed between 2000-2013 were analyzed for HPV16 mRNA by PCR (if HPV16DNA-positive) and cluster of differentiation 8 positive (CD8+) tumour-infiltrating lymphocytes (TILs) and human leukocyte antigen (HLA) class I-expression using IHC. RESULTS: HPV DNA, alone and in combination with p16 overexpression, was validated as a favourable prognostic factor in HNCUP. HPV16 mRNA was present in most HPV16 DNA-positive cases, confirming HPV-driven carcinogenesis in HNCUP. High CD8+ TIL counts indicated favourable prognosis. CONCLUSION: HPV status is useful for the management of patients with HNCUP and the role of CD8+ TILs should be further explored.

Human papillomavirus DNA detection in fine-needle aspirates as indicator of human papillomavirus-positive oropharyngeal squamous cell carcinoma: A prospective study.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (SCC) has a better outcome than most head neck squamous cell carcinomas (HNSCCs) and an HPV-positive lymph node metastasis likely has an HPV-positive oropharyngeal SCC origin. Determining HPV-status in cervical lymph nodes by fine-needle aspiration cytology (FNAC) may be useful for diagnosis. METHODS: FNACs from 66 patients with neck masses were prospectively examined for HPV DNA and HPV16 mRNA by a polymerase chain reaction (PCR)-based assay, and the data correlated to diagnosis and HPV-status obtained from histopathological specimens. RESULTS: Aspirates from 17 of 66 patients, later diagnosed with HPV-positive oropharyngeal SCC, were HPV16 DNA-positive. HPV16 mRNA was detected in all cases with extractable RNA. All remaining FNACs, including 18 branchial cleft cysts, were HPV DNA-negative. HPV DNA status in the aspirates showed perfect concordance with corresponding biopsies. CONCLUSION: HPV16 DNA detection in fine-needle aspirations from neck masses is reliable and HPV16 DNA in a metastasis is a strong indicator of an HPV-positive oropharyngeal SCC. © 2016 Wiley Periodicals, Inc. Head Neck 39: 419-426, 2017.