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Background/Aim: The advent of immune checkpoint inhibitor (ICI) treatment has transformed the treatment of recurrent or metastatic head and neck cancer; however, nasopharyngeal carcinoma (NPC) has not been included in major phase III trials. The clinical outcomes of ICI for NPC in real-world practice remain to be fully elucidated. Patients and Methods: We retrospectively reviewed 23 patients with recurrent or metastatic NPC treated with nivolumab or pembrolizumab at 6 institutions from April 2017 to July 2021 and investigated the correlation of clinicopathological factors and immune-related adverse events with the effects of ICI therapy and the prognosis. Results: The objective response rate was 39.1% and the disease control rate was 78.3%. The median progression-free survival was 16.8 months and overall survival has not been reached. As with other treatment procedures, the efficacy and the prognosis tended to be better in EBER-positive cases than in EBER-negative cases. The rate of significant immune-related adverse events that necessitated discontinuation of treatment was only 4.3%. Conclusion: ICI monotherapy (e.g., nivolumab and pembrolizumab) was effective and tolerable for NPC in a real-world setting.

The efficacy of programmed cell death 1 (PD-1) inhibitor therapy for patients with recurrent and/or metastatic salivary gland carcinoma (R/M SGC) remains unclear.We retrospectively analyzed 36 patients with R/M SGC treated with PD-1 inhibitor. The expression of programmed cell death ligand 1 (PD-L1) and mismatch repair (MMR) proteins was also analyzed.The objective response rate (ORR) was 11.1%. The histopathological subtypes of patients who achieved complete response or partial response were salivary duct carcinoma (SDC) in three patients and poorly differentiated carcinoma in one patient, all of whom showed a positive PD-L1 expression. The expression of MMR proteins was not associated with the efficacy of PD-1 inhibitors.Although the efficacy of PD-1 inhibitor therapy in R/M SGC is limited, certain patients may respond and achieve long-term disease control. There is a potential therapeutic effect in SDC patients with positive PD-L1 expression.

Background/Aim: Surgery remains the standard treatment for salivary gland carcinoma (SGC). Our study investigated the association between epidermal growth factor receptor (EGFR) status in recurrent/metastatic SGC and the effectiveness of treatment with cisplatin/carboplatin and 5-fluorouracil plus cetuximab (EXTREME). Patients and Methods: We retrospectively collected 19 SGCs from patients treated with the EXTREME regimen. After analyzing EGFR expression and gene copy number gain, we evaluated the correlation between EGFR status and clinicopathological factors and prognosis. Results: EGFR overexpression was detected in 77.8% cases, but not statistically associated with clinicopathological factors or prognosis. EGFR gene copy number gain was detected in 16.7% cases, and statistically positively correlated with lymph node metastasis (p=0.0291). The best overall response was partial response in two cases, stable disease in 15, and progressive disease in one case. The EXTREME regimen was discontinued in all cases. Conclusion: Our results suggest that SGCs are positive for EGFR protein expression but the response rate to the EXTREME regimen was unremarkable.

Background/Aim: A long-term effect has been confirmed in clinical practice since the introduction of nivolumab for treating various malignant tumors. A similar phenomenon is speculated to occur in head and neck cancer; however, details remain unclear due to the lack of long-term reports. We aimed to investigate the five-year outcomes in long-term responders for over two years, and evaluate the optimal duration of therapy with nivolumab. Patients and Methods: In this retrospective observational study, we analyzed 203 cases of recurrent/metastatic head and neck squamous cell carcinoma (R/MHNSCC), including 33 long-term responders. Results: The median overall survival (OS), 5-year OS, median progression-free survival (PFS), and 5-year PFS values in the 203 cases were 13.1 months, 19.2%, 3.1 months, and 13.2%, respectively. Of the 33 long-term responders, 14 (42.4%) continued using nivolumab for more than 2 years. The remaining 19 patients (57.6%) discontinued nivolumab. The most common reason for discontinuation was severe immune-related adverse events (irAEs) (9 cases; 27.3%); in these 9 cases, the median disease-free survival was 33.2 (range=10.7-44.3) months. Nine patients (21.2%) were considered to have progressive disease (PD) after at least 2 years of administration, and 3 patients (9.1%) requested to discontinue treatment because a complete response (CR) was achieved. Conclusion: This study demonstrated the durable and long-term benefit of nivolumab in R/MHNSCC. In the future, we aim to accumulate real-world data for the establishment of criteria for completion of nivolumab treatment in long-term responders.

Although immune checkpoint inhibitors (ICIs) have emerged as new therapeutic options for refractory cancer, they are only effective in select patients. Tumor antigen-pulsed dendritic cell (DC) vaccine therapy activates tumor-specific cytotoxic T lymphocytes, making it an important immunotherapeutic strategy. Salivary ductal carcinoma (SDC) carries a poor prognosis, including poor long-term survival after metastasis or recurrence. In this study, we reported a case of refractory metastatic SDC that was treated with a tumor lysate-pulsed DC vaccine followed by a single injection of low-dose nivolumab, and a durable complete response was achieved. We retrospectively analyzed the immunological factors that contributed to these long-lasting clinical effects. First, we performed neoantigen analysis using resected metastatic tumor specimens obtained before treatment. We found that the tumor had 256 non-synonymous mutations and 669 class I high-affinity binding neoantigen peptides. Using synthetic neoantigen peptides and ELISpot analysis, we found that peripheral blood mononuclear leukocytes cryopreserved before treatment contained pre-existing neoantigen-specific T cells, and the cells obtained after treatment exhibited greater reactivity to neoantigens than those obtained before treatment. Our results collectively suggest that the rapid and long-lasting effect of this combination therapy in our patient may have resulted from the presence of pre-existing neoantigen-specific T cells and stimulation and expansion of those cells following tumor lysate-pulsed DC vaccine and ICI therapy.

This study investigated the effectiveness of pembrolizumab with or without chemotherapy on advanced-stage head and neck cancer (HNC), including nasopharyngeal, sinonasal cavity and external auditory canal cancer, in a real-world setting.We retrospectively collected data from 97 HNC patients who were treated with pembrolizumab alone (n=60) or with chemotherapy (n=37), and we investigated the association between clinicopathological findings and treatment response or prognosis.Patients treated with pembrolizumab and chemotherapy had a 1-year overall survival (OS) of 72.8%, objective response rate (ORR) of 48.6%, and serious (≥G3) adverse events (AEs) of 29.7%. Patients treated with pembrolizumab alone had a 1-year OS of 51.9%, ORR of 21.7%, and ≥G3 AEs of 6.7%. Both the ORR and disease control rate (DCR) in the pembrolizumab with chemotherapy group were significantly better than those in the pembrolizumab group (p=0.074 and p=0.00101, respectively). Among patients with distant metastasis, patients on pembrolizumab with chemotherapy achieved significantly better OS than pembrolizumab alone (p=0.0039). Among patients in the pembrolizumab group, both AE-positive and better performance status were associated with longer OS (p=0.011 and p=0.0037, respectively).Our real-world experience reinforces the durability and effectiveness of pembrolizumab for HNC patients. Additionally, our results suggest that pembrolizumab with chemotherapy might be recommended for patients with distant metastasis and no prior treatment. Further studies are needed to determine the optimal treatment strategy for HNC.

Sinonasal squamous cell carcinoma (SNSCC) is sometimes associated with high-risk human papillomavirus (HR-HPV) infection and inverted sinonasal papilloma or oncocytic sinonasal papilloma. Frequent mutations of EGFR and KRAS are reported in inverted sinonasal papilloma-related sinonasal squamous cell carcinoma (ISP-SCC) and oncocytic sinonasal papilloma-related SNSCC, respectively. Here, we attempted to determine the prevalence and the prognostic significances of these alterations in SNSCC. We retrospectively collected 146 SNSCCs, including 14 ISP-SCCs, and comprehensively analyzed the HR-HPV infection by human papillomavirus (HPV)-RNA in situ hybridization, EGFR gene copy number gain (CNG) by chromogenic in situ hybridization, and gene mutations in EGFR and KRAS by Sanger sequencing. HR-HPV was detected in 11 cases (7.5%), whereas all 14 ISP-SCCs were negative. EGFR mutations were present in 21 (14.7%) of 143 SNSCCs, including 13/14 (92.9%) ISP-SCCs and 8/129 (6.2%) non-ISP-SCCs (P

External auditory canal squamous cell carcinoma (EACSCC) is an extremely rare and aggressive malignancy. Due to its rarity, the molecular and genetic characteristics of EACSCC have not yet been elucidated. To reveal the genetic alterations of EACSCC, we performed whole exome sequencing (WES) on 11 primary tumors, 1 relapsed tumor and 10 noncancerous tissues from 10 patients with EACSCC, including 1 with a rare case of synchronous bilateral EACSCC of both ears. WES of the primary tumor samples showed that the most frequently mutated gene is TP53 (63.6%). In addition, recurrent mutations in CDKN2A, NOTCH1, NOTCH2, FAT1 and FAT3 were detected in multiple samples. The mutational signature analysis of primary tumors indicated that the mutational processes associated with the activation of apolipoprotein B mRNA‐editing enzyme catalytic polypeptide‐like (APOBEC) deaminases are the most common in EACSCC, suggesting its similarity to SCC from other primary sites. Analysis of arm‐level copy number alterations detected notable amplification of chromosomes 3q, 5p and 8q as well as deletion of 3p across multiple samples. Focal chromosomal aberrations included amplifications of 5p15.33 (ZDHHC11B) and 7p14.1 (TARP) as well as deletion of 9p21.3 (CDKN2A/B). The protein expression levels of ZDHHC11B and TARP in EACSCC tissues were validated by immunohistochemistry. Moreover, WES of the primary and relapsed tumors from a case of synchronous bilateral EACSCC showed the intrapatient genetic heterogeneity of EACSCC. In summary, this study provides the first evidence for genetic alterations of EACSCC. Our findings suggest that EACSCC mostly resembles other SCC., As the genetic characteristics of external auditory canal squamous cell carcinoma (EACSCC) are not yet elucidated due to its rarity, we performed whole exome sequencing and copy number analysis in EACSCC samples. The genetic alterations of EACSCC mostly resemble other SCC, although the novel amplified loci that harbor oncogenes were found.

Summary Background As the base of the tongue (BOT) plays essential roles in speech and swallowing, surgical resection of BOT cancer is typically avoided. Moreover, standard reconstructive procedures for larynx-preserving BOT defects have not yet been established. We performed immediate flap reconstruction after wide resection of BOT cancer with laryngeal preservation. Herein, the functional and oncological results of our strategy were analysed. Methods We retrospectively evaluated patients who underwent extended BOT resection (including the oral tongue, upper/lateral oropharyngeal wall, epiglottis and false vocal cord) with laryngeal preservation between April 2006 and April 2016. We classified defects involving the oral tongue or upper/lateral oropharyngeal wall as the lateral extension type and those involving the epiglottis or false vocal cord as the laryngeal extension type. Lateral extension-type defects were closed primarily and filled with a deepithelialised skin or muscle flap. Laryngeal extension-type defects were reconstructed using a bulky skin flap plus hyo-thyroid-pexy to create a neo-epiglottis. Postoperative functional and oncologic outcomes were assessed. Results We enrolled 18 patients with extended BOT defects. Of them, 11 had a history of irradiation. The tracheal cannula was removed in all cases, although laryngeal extension defects were associated with a longer duration to removal. All patients achieved complete oral intake and retained intelligible speech, with preservation of laryngeal function. There was no local recurrence, and the 5-year overall survival was 88.9%. Conclusions Following wide BOT resection, reconstruction with laryngeal preservation is feasible even in cases involving irradiated tumours with laryngeal extension.

Background/Aim: The inflammation-based prognostic score (IBPS) has attracted attention recently as a prognostic biomarker for head and neck cancer patients. However, as the IBPS often changes after anticancer drug therapy, its independent prognostic value remains controversial. We aimed to investigate the relationship between the IBPS and prognosis in recurrent and/or metastatic head and neck squamous cell carcinoma (RMHNSCC) treated with nivolumab, and investigate changes in the IBPS before and after nivolumab treatment. Patients and Methods: Total of 164 patients with RMHNSCC received nivolumab therapy were retrospectively analyzed. Results: Univariate analysis among the 164 patients revealed that the performance status (PS), immune-related adverse event (irAE) status, pre- and post-therapy Glasgow Prognostic Score (GPS), neutrophil-to-lymphocyte ratio (NLR), C-reactive protein-to-albumin ratio (CAR), platelet-to-lymphocyte ratio (PLR), and post-eosinophil count, were all significant predictors of overall survival (OS) (p

Pembrolizumab and chemotherapy (chemoimmunotherapy) were administered to 2 head and neck squamous cell carcinoma (HNSCC) patients with extremely advanced local tumors and distant metastases with palliative intent. However, they demonstrated strikingly good responses and achieved remission. Expanded application of induction chemoimmunotherapy may be useful for locally advanced HNSCC.

The prevalence and prognostic value of human papillomavirus (HPV) infection and epidermal growth factor receptor (EGFR) alteration in sinonasal squamous cell carcinoma (SNSCC) are not known. The reliability of p16 overexpression as a surrogate for HPV infection in SNSCC is also unclear. We investigated the prognostic and diagnostic significances of HPV infection, EGFR alteration, and p16 expression in SNSCC. We analyzed high-risk HPV infection by HPV-RNA in situ hybridization and EGFR gene copy number gain (CNG) by chromogenic in situ hybridization and by determining the protein expressions of p16, Rb, and EGFR by immunohistochemistry in 101 SNSCC cases. HPV infection (n=9, 8.9%) and p16 overexpression (n=15, 14.9%) were associated with better overall survival (P=0.0042 and 0.005, respectively). The HPV cases were located predominantly at the nasal cavity with nonkeratinizing histology and partial loss of Rb. Notably, 40% (6/15) of p16 SNSCCs were HPV. Two of these cases showed complete loss of Rb expression by immunohistochemistry, suggesting a reason for the above discrepancy. EGFR CNG, detected in 30.5% of the SNSCCs, was correlated with EGFR protein overexpression (P=0.0001). HPV infection and EGFR CNG were mutually exclusive. The HPV/EGFR CNG group had significantly better overall survival than the HPV/EGFR CNG and HPV/EGFR CNG groups (P=0.0471 and 0.0343, respectively). Our results suggest that HPV infection is a favorable prognostic marker in SNSCC, but p16 is not a perfect surrogate marker; the Rb expression pattern may improve the diagnostic accuracy. The molecular subclassification of SNSCCs based on HPV infection and EGFR copy number status might provide important information for therapeutic strategies.

Background: Biomarkers predicting therapeutic response to immunotherapy have been widely explored via monitoring the liquid and tissue-derived components. Increasing treatment options for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) mandates prediction of the therapeutic response of anti-PD-1 antibody alone as well as optimization of the treatment sequence. In view of improving biomarkers predicting the efficacy of immunotherapy for R/M HNSCC, we hypothesized that biomarkers can be personalized depending on clinicopathological backgrounds and treatment sequence. Methods: In this study, we retrospectively included formalin-fixed paraffin-embedded (FFPE) samples, peripheral blood cell counts at treatment, clinicopathological information, and outcome data for patients with R/M HNSCC receiving nivolumab across 22 institutions in Japan (N = 100). FFPE samples were subjected to 14-marker multiplex immunohistochemistry (IHC) and image cytometry analysis (Tsujikawa T et al. Cell Reports, 2017) to quantitatively evaluate CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, CD66b+ granulocytes, mast cells, programmed death ligand 1 (PD-L1) and PD-1 expression in a single slide. Intratumoral and circulating immune cell frequencies were comparatively analyzed between responders (CR, n = 14; PR, n = 39) and non-responders (SD, n = 2; PD, n = 45). Results: Of 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-L1 expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. Next, focusing on the history of prior therapy, stratified analysis revealed that the frequency of NK cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Furthermore, stratified analysis by patient age revealed that nivolumab response was significantly associated with high CPS and lymphoid-inflamed profiles based on cell densities of nine immune cell lineages in the group aged 65 years or older, but not in the group under 65 years of age. On the contrary, the neutrophil/lymphocyte ratios (NLR) in peripheral blood counts at nivolumab treatment were significantly lower in responders (mean 4.96) than those in non-responders (mean 10.46) in the group under 65 years of age, but not in those over 65 years of age (7.41 versus 8.47). Conclusions: Using peripheral blood data and tumor tissue profiling stratified by patient age and prior treatment might provide better predictive biomarkers in nivolumab response to HNSCC. Further preclinical and clinical studies elucidating immune mechanisms in different patient backgrounds will be warranted. Citation Format: Takahiro Tsujikawa, Kazuchika Ohno, Sumiyo Saburi, Junichi Mitsuda, Kanako Yoshimura, Alisa Kimura, Hiroki Morimoto, Gaku Ohmura, Akihito Arai, Hiroshi Ogi, Saya Shibata, Yosuke Ariizumi, Akihisa Tasaki, Ryosuke Takahashi, Yumiko Tateishi, Hiroaki Kawabe, Sadakatsu Ikeda, Kei-ichi Morita, Tatsuhiko Tsunoda, Takumi Akashi, Morito Kurata, Issei Imoto, Yasushi Shimizu, Akihito Watanabe, Yukinori Asada, Ryuichi Hayashi, Yuki Saito, Hiroyuki Ozawa, Kiyoaki Tsukahara, Nobuhiko Oridate, Arata Horii, Takashi Maruo, Nobuhiro Hanai, Hidenori Inohara, Hiroshi Iwai, Takashi Fujii, Ken-ichi Nibu, Shigemichi Iwae, Tsutomu Ueda, Ryuji Yasumatsu, Hirohito Umeno, Muneyuki Masuda, Kyoko Itoh, Shigeru Hirano, Takahiro Asakage. Tumor immune characterization identifies age-stratified biomarkers for nivolumab in patients with head and neck squamous cell carcinoma: A nationwide collaborative study in Japan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5210.

The antitumor efficacies of immune checkpoint inhibitors (ICIs) and the usefulness of potential predictive markers such as programmed death-ligand 1 (PD-L1) expression, density of tumor-infiltrating lymphocytes (TILs) and microsatellite instability (MSI) in sinonasal squamous cell carcinoma (SNSCC) have not been fully elucidated. We retrospectively analyzed 131 SNSCCs with immunohistochemistry for PD-L1 expression, TIL subpopulations and loss of mismatch repair (MMR) proteins as a surrogate for MSI-high. We also comprehensively evaluated the mutual relationships among these immuno-markers, high-risk human papillomavirus (HPV) infection, epidermal growth factor receptor (EGFR) gene status, and KRAS mutation. PD-L1 expression (tumor proportion score ≥ 1%) was detected in 60 (45.8%) SNSCC cases and was significantly associated with worse overall survival (OS) (p = 0.0240). High density of cluster of differentiation 8 (CD8)-positive TILs was significantly associated with better progression-free survival (PFS) (p = 0.0368), and high density of forkhead box protein P3-positive TILs was significantly associated with better PFS and OS (p = 0.0007 and 0.0143, respectively). With respect to the combination of CD8 + TIL and PD-L1 expression, the high-CD8/PD-L1-negative group showed the most favorable prognosis, whereas the low-CD8/PD-L1-positive group showed the worst prognosis. MMR loss was detected in 3 (2.3%) of the 131 cases. HPV infection (6.1%), EGFR mutation (14.5%), EGFR copy number gain (26%), and MMR loss were essentially mutually exclusive; patients in these molecular groups showed significant differences in prognosis but not in the degree of PD-L1 expression or TILs. Among the nine ICI-treated patients, three (33.3%) were responders, and the EGFR-wild type cases (n = 7) showed better clinical responses to an ICI compared to the EGFR-mutant cases (n = 2). Among the patients with residual/recurrent EGFR-wild type tumors (n = 43), ICI treatment significantly improved OS (p = 0.0281). The results suggest that the evaluation of immuno-markers and molecular subclassification may be helpful for prognostic prediction and selecting an individualized therapeutic strategy for patients with SNSCC.

Significant immune-related adverse events (irAEs) requiring therapy discontinuation sometimes occur. The influence of discontinuation on disease control after an irAE is unclear.The aim of this study was to investigate whether or not patients continued to show a response or durable disease control even after stopping therapy following an irAE.The response after nivolumab monotherapy discontinuation was examined for 14 patients in whom therapy was stopped without progression.The best response was CR in 5 (36%) patients, PR in 8 (57%) patients and SD in 1 (7%) patient. The estimated 1-year overall and progression-free survival rates were 92.9% and 78.6%, respectively. The best response during nivolumab therapy in patients who developed PD was CR in 0 of 5 patients (0%), PR in 3 of 8 patients (38%) and SD in 1 patient (100%). Patients obtaining CR tended to have a lower risk of PD than those with PR or SD.Patients with CR status may continue to show a response or durable disease control even after stopping therapy due to an irAE.

Recent studies have suggested the benefit of salvage chemotherapy (SCT) after immune checkpoint inhibitor (ICI) treatment for recurrent and metastatic head and neck squamous cell carcinoma (R/M HNSCC). We retrospectively examined the outcome of SCT and the usefulness of the serum C-reactive protein level (CRP) and neutrophil-to-lymphocyte ratio (NLR) as prognostic biomarkers. Thirty-nine patients with R/M HNSCC were enrolled in this study. Twenty-five patients (64.1%) received combination chemotherapy of weekly paclitaxel and cetuximab (PC) as SCT, and 14 patients (35.9%) received S1, an oral fluoropyrimidine. In all patients, the response rate, disease control rate, median progression-free survival (PFS), and median overall survival (OS) were 45.2%, 85.7%, 6.5 months, and 13.5 months, respectively. No chemotherapy-related deaths were observed. These PC groups had low CRP (&lt, 1.2 mg/dL) or low NLR (&lt, 7.0) values at the time of SCT induction, which was significantly associated with an improved OS (p = 0.0440, p = 0.0354). A multivariate analysis also showed that a lower CRP value was significantly associated with a better OS (p = 0.0078). We clarified the usefulness of the PC and S1 regimens as SCT. In addition, SCT with the PC regimen showed a better prognosis with a lower CRP or NLR at induction than a higher CRP or NLR This is the first report on biomarkers of SCT in R/M HNSCC

Background Drug-induced interstitial lung disease (DI-IP) is one of the most serious adverse reactions associated with the use of anticancer drugs. DI-IP prevalence among molecular-targeting drugs and immune checkpoint inhibitors (ICIs) is relatively high in Japanese patients. To assess the risk of cetuximab and/or nivolumab-related IP is important. Patients and Methods The medical records of 138 patients with recurrent and/or metastatic head and neck squamous cell carcinoma treated with cetuximab-containing chemotherapy and/or nivolumab monotherapy were retrospectively reviewed. Results The incidence of DI-IP with R/M HNSCC was 7.2%. DI-IP occurred more frequently in patients treated with cetuximab-containing chemotherapy following nivolumab monotherapy than in patients with other regimens. However, tumor suppression was detected in all patients treated with cetuximab-containing chemotherapy following nivolumab monotherapy, and two achieved a complete response. Conclusions Although patients treated with cetuximab-containing chemotherapy following nivolumab showed dramatic efficacy, careful monitoring should be recommended.

MOB1 deficiency promotes rapid onset and progression of oral SCC (OSCC) via YAP1 activation., Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common group of cancers in the world, and patients have a poor prognosis. Here, we present data indicating that YAP1 may be a strong driver of the onset and progression of oral SCC (OSCC), a major subtype of HNSCC. Mice with tongue-specific deletion of Mob1a/b and thus endogenous YAP1 hyperactivation underwent surprisingly rapid and highly reproducible tumorigenesis, developing tongue carcinoma in situ within 2 weeks and invasive SCC within 4 weeks. In humans, precancerous tongue dysplasia displays YAP1 activation correlating with reduced patient survival. Combinations of molecules mutated in OSCC may increase and sustain YAP1 activation to the point of oncogenicity. Strikingly, siRNA or pharmacological inhibition of YAP1 blocks murine OSCC onset in vitro and in vivo. Our work justifies targeting YAP1 as therapy for OSCC and perhaps HNSCC, and our mouse model represents a powerful tool for evaluating these agents.

Differential diagnosis among basal cell adenoma (BCA), basal cell adenocarcinoma (BCAC), adenoid cystic carcinoma (ACC) and pleomorphic adenoma (PA) of the salivary gland can be challenging due to their similar histological appearance. Although frequent nuclear β-catenin expression and CTNNB1 mutations have been reported in BCA, further details of the Wnt/β-catenin signal alterations are unclear. The aim of this study was to assess the diagnostic utility of Wnt/β-catenin signal alteration in BCA and morphological mimics. We performed immunohistochemical staining for β-catenin and mutation analysis for Wnt/β-catenin-related genes (CTNNB1, APC, AXIN1 and AXIN2) in BCA (n = 34), BCAC (n = 3), ACC (n = 67) and PA (n = 31). We also analyzed ACC-specific MYB and MYBL1 gene rearrangements by fluorescence in situ hybridization (FISH). Nuclear β-catenin expression (≥3%) was present in 32/34 cases (94.1%) of BCA, and the nuclear β-catenin labeling index was significantly higher than in other tumor types (p = 0.0001). In BCA, we found mutations in CTNNB1, APC and AXIN1 genes (41.1%, 2.9% and 8.8%, respectively). In BCAC, nuclear β-catenin expression with CTNNB1 mutation was present in 1/3 cases (33.3%). As for ACC, nuclear β-catenin expression was observed in 3/67 cases (4.4%), but all 3 cases harbored either MYB or MYBL1 gene rearrangement. The results suggest that nuclear β-catenin immunoreactivity with appropriate criteria may be helpful to distinguish BCA from histologically similar tumors. However, a minor subset of ACCs with nuclear β-catenin expression require careful diagnosis. In addition, Wnt/β-catenin signal alteration may play a role in the pathogenesis of BCA and BCAC.

Human papillomavirus (HPV)-related oropharyngeal small-cell carcinoma (OPSmCC) is a rare malignancy with aggressive behavior, whereas HPV-related oropharyngeal squamous-cell carcinoma (OPSqCC) displays a favorable prognosis. Notably, these two malignancies occasionally arise in an identical tumor. In this case study, we explored the molecular characteristics that distinguishes these two carcinomas using a rare case of HPV-related oropharyngeal carcinoma (OPC) with the combined histology of SmCC and SqCC. Immunohistochemical analysis and HPV-RNA in situ hybridization (ISH) suggested that both SmCC and SqCC were HPV-related malignancies. Targeted exome sequencing revealed that SmCC and SqCC had no significant difference in mutations of known driver genes. In contrast, RNA sequencing followed by bioinformatic analyses suggested that aberrant transcriptional programs may be responsible for the neuroendocrine differentiation of HPV-related OPC. Compared to SqCC, genes up-regulated in SmCC were functionally enriched in inflammatory and immune responses (e.g., arachidonic acid metabolism). We then developed a SmCC-like gene module (top 10 up-regulated genes) and found that OPC patients with high module activity showed poor prognosis in The Cancer Genome Atlas (TCGA) and GSE65858 cohort. Gene set enrichment analysis of the SmCC-like gene module suggested its link to MYC proto-oncogene in the TCGA data set. Taken together, these findings suggest that the SmCC-like gene module may contribute to acquisition of aggressive phenotypes and tumor heterogeneity of HPV-related OPC. The present case study is the first report of genetic and transcriptomic aberrations in HPV-related OPSmCC combined with SqCC.

Objectives: Small cell carcinomas in extrapulmonary sites (ESmCCs) are very rare. ESmCCs originating in the head and neck account for approximately 10% of all ESmCCs, and there are few reports about this disease. ESmCCs have an aggressive natural history characterized by widespread metastasis. The aim of this study was to investigate the characteristics and outcomes of patients with ESmCCs of the head and neck. Methods: The outcomes of 21 patients with ESmCCs of the head and neck treated between January 2001 and December 2015 at the authors’ hospital and associated facilities were reviewed. Results: There were 18 men and 3 women, and the median age was 74 years (range, 53-91 years). The tumor site was the larynx in 6 patients; the paranasal sinus in 5; the hypopharynx in 3; the oropharynx in 2; the nasopharynx in 2; and the oral cavity, salivary gland, and primary unknown in 1 patient each. The extent of the disease was staged as follows: stage I or II, 3 cases; stage III, 4 cases; stage IVA, 9 cases; stage IVB, 1 case; and stage IVC, 4 cases. The median observation time was 17 months (range, 1-103 months). Four patients (19%) had distant metastasis at initial treatment, and 13 patients (62%) developed distant metastasis within 3 years. Treatments were administered, including radical surgery (9 patients), radiation therapy (5 patients), chemoradiotherapy (7 patients), and chemotherapy (6 patients). The 1- and 3-year overall survival rates of patients were 56% and 37%, respectively. More than half of the patients died of distant metastasis. Conclusions: ESmCCs of the head and neck have a poor prognosis, similar to those of carcinomas in many other sites. Control of distant metastasis would contribute to improving the prognosis of ESmCCs of the head and neck. Further studies are required for better understanding these disease entities and their response to treatment modalities.

The clinical importance of cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC) is well recognized. However, a reliable method for the detection of functioning CSC has not yet been established. We hypothesized that YAP1, a transcriptional coactivator, and SOX2, a master transcription factor of SCC, may cooperatively induce stemness through transcriptional reprogramming. We immunohistochemically examined the expression of SOX2 and YAP1 in the CD44 variant 9 (CD44v9)-positive invasion front. A CSC-inducible module was identified through a combination of siRNAs and sphere formation assays. YAP1 and SOX2 interactions were analyzed in vitro. The triple overexpression of SOX2, YAP1, and CD44v9 was significantly associated with poor prognosis. TCGA data revealed that the CSC-inducible module, which was related to EMT and angiogenesis, was significantly correlated with poor prognosis. The KLF7 expression, representatively chosen from the module, also correlated with poor prognosis and was essential for sphere formation and CSC propagation. Sphere stress-activated YAP1 enhanced SOX2 activity. The stress-triggered activation of YAP1/SOX2 transcriptionally reprograms HNSCC for the acquisition of stemness. Triple SOX2, YAP1, and CD44v9 immunostaining assays may be useful for the selection of high-risk patients with functioning CSCs, and YAP1 targeting may lead to the development of a CSC-targeting therapy.

Supplemental material, Table_1s20190420 for Small Cell Carcinoma in the Head and Neck by Takahiro Wakasaki, Ryuji Yasumatsu, Muneyuki Masuda, Mioko Matsuo, Akihiro Tamae, Kazuhiko Kubo, Ryunosuke Kogo, Ryutaro Uchi, Masahiko Taura and Takashi Nakagawa in Annals of Otology, Rhinology & Laryngology

Objectives The aim of the present study was to investigate the molecular basis for the use of immune checkpoint inhibitors to treat salivary gland carcinomas (SGC). Materials and methods We examined the clinical and prognostic significance of programed death ligands 1 and 2 (PD-L1 and -L2) expression using immunohistochemistry and in situ hybridization, as well as microsatellite instability (MSI) status using polymerase chain reaction, along with tumor-infiltrating lymphocytes (TILs) in 30 cases of SGC. Results The SGC cases studied included adenoid cystic carcinoma (AdCC, 36.7%), salivary duct carcinoma (SDC, 26.7%), mucoepidermoid carcinoma (MEC, 23.3%), and carcinoma ex pleomorphic adenoma (CxPA, 13.3%). Either PD-L1 or PD-L2 overexpression was observed in 36.7% patients. PD-L2 expression was associated with reduced disease-specific survival (DSS) and disease-free survival (DFS) (P = 0.0266 and P = 0.0209, respectively). Simultaneous PD-L1 and PD-L2 overexpression was detected in 13.3% of cases, and was correlated with reduced DSS (P = 0.0113). Among non-AdCCs, all cases that developed distant metastasis were positive for PD-L2 (P = 0.001). Cases showing low-TILs that were positive for either PD-L1 or L2 were associated with poor DFS. No MSI was detected in the SGC cases studied. Conclusion To our knowledge, this is the first comprehensive study examining PD-L1 and PD-L2 status, MSI status, and TILs in SGC. Our results indicate that the PD-1/PD-L1 or PD-L2 pathway, which is associated with poor clinical outcomes, may provide promising therapeutic targets against SGC in selected patients. Further experimental and clinical studies are encouraged.

Background: In this era of precision medicine, a clinically effective molecular target of HNSCC has not been identified so far. This is mainly because the predominant genetic alterations in HNSCC are loss-of-function (LOF) mutations in tumor-suppressor genes (TSGs), which are generally undruggable targets. We hypothesized that certain effector molecules liberated from the TSGs restriction may play an essential role in HNSCC oncogenesis and evolution. In this context, we analyzed the oncogenic function of transcription coactivator YAP1, which is the essential downstream effector protein of the Hippo pathway. Experiments and Results: We first examined the effect of mouse tongue epithelium specific conditional double knockout (cDOK) of Mob1a/b, the core component of the Hippo pathway that inhibits YAP1, employing CreERT; Mob1aflox/flox; Mob1b−/− mouse. Consequent YAP1 activation (but not TAZ) developed extremely rapid and highly reproducible (100%) tongue tumors: carcinoma in situ (2 weeks) and invasive carcinoma (4 weeks). The tongue epithelium cells isolated from the cDOK mouse demonstrated YAP1-dependent aggressive phenotypes. In human oral HNSCC samples, expanding accumulation of nuclear YAP1 protein was observed from the basal stem/progenitor cells to invasive cancer cells, and this spreading overexpression was significantly correlated with poor prognosis of patients. The YAP1 target module expression, established thorough the microarray assays with cDKO mouse cells, also correlated with the unfavorable prognosis of patients in the TCGA data. To elucidate the epigenetic mechanism that underlies this extremely rapid carcinogenesis, we then conducted chip-seq for cDKO mouse cells with or without YAP1 activation. Our preliminary data indicated that YAP1-targeted genes predominantly reside in the euchromatin islands and, surprisingly, their enhancers are marked with active H3K27ac, even in the YAP1-off condition, thus ready to respond to YAP1 nuclear translocation and thereby to start transcription, contradicting our expectation that genome-wide chromatin remodeling must be required in this rapid carcinogenesis model. Given that the presumed physiologic roles of YAP1 are a monitor of microenvironment and an emitter of regenerative cue under emergent cellular stressors (e.g., tissue injury) in the normal stem/progenitor cells, it is plausible that YAP1-target genes are kept in the ready-to-go status and constitutive activation of YAP1 and subsequent expression of targeted genes lead to the rapid carcinogenesis, causing the condition of “wounds that never heal.” Finally, our data and a recent study by Prof. Gutkind’s laboratory indicate that the major LOF of TSGs (e.g., p53 and FAT1) in HNSCC may convergently lead to the liberation of YAP1 activity. Conclusions: Collectively our data suggest that both the onset and the evolution of HNSCC might be highly dependent on (i.e., addicted to) the activation of YAP1-induced transcription. Citation Format: Muneyuki Masuda, Hirofumi Omori, Kuniaki Sato, Kenichi Taguchi, Masashige Bando, Satoshi Toh, Katsuhiko Shirahige, Koshi Mimori, Akira Suzuki. Transcriptional addiction to YAP1—a major driving force of oral cancer carcinogenesis and evolution? [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A31.

Background: In this era of precision medicine, the clinically effective molecular target of HNSCC has not been identified so far. This is mainly because the predominant genetic alterations in HNSCC are loss-of-function (LOF) mutations in tumor suppressor genes (TSGs), which are generally undruggable targets. We hypothesized that certain effector molecules liberated from the TSGs restriction may play an essential role in HNSCC oncogenesis and evolution. In this context, we analyzed the oncogenic function of transcription coactivator YAP1, the essential downstream effector protein of the Hippo pathway. Experiments and Results: We first examined the effect of mouse tongue epithelium specific conditional double knockout (cDOK) of Mob1a/b, the core component of the Hippo pathway that inhibits YAP1, employing CreERT; Mob1aflox/flox; Mob1b−/− mouse. Consequent YAP1 activation (but not TAZ) developed extremely rapid and highly reproducible (100%) tongue tumors: carcinoma in situ (2 weeks) and invasive carcinoma (4 weeks). The tongue epithelium cells isolated from the cDOK mouse demonstrated YAP1-dependent aggressive phenotypes. In human oral HNSCC samples, expanding accumulation of nuclear YAP1 protein was observed from the basal stem/progenitor cells to invasive cancer cells and this spreading overexpression was significantly correlated with poor prognosis of patients. The YAP1 target module expression, established thorough the microarray assays with cDKO mouse cells, also correlated with the unfavorable prognosis of patients in the TCGA data. To elucidate epigenetic mechanism that underlies this extremely rapid carcinogenesis, we then conducted chip-seq for cDKO mouse cells with or without YAP1 activation. Our preliminary data indicated that YAP1-targeted genes predominantly reside in the euchromatin islands and, surprisingly, their enhancers are marked with active H3K27ac, even in the YAP1-off condition, thus ready to respond to YAP1 nuclear translocation and thereby to start transcription, contradicting our expectation that that genome-wide chromatin remodeling must be required in this rapid carcinogenesis model. Given that the presumed physiologic roles of YAP1 are a monitor of microenvironment and an emitter of regenerative cue under emergent cellular stressors (e.g., tissue injury) in the normal stem/progenitor cells, it is plausible that YAP1-target genes are kept in the ready-to-go status and constitutive activation of YAP1 and subsequent expression of targeted genes lead to the rapid carcinogenesis, causing the condition of “wounds that never heal.” Finally, our data and a recent study by Prof. Gutkind’s laboratory indicate that the major LOF of TSGs (e.g., p53 and FAT1) in HNSCC may convergently lead to the liberation of YAP1 activity. Conclusions: Collectively, our data suggest that for both the onset and the evolution, HNSCC might be highly dependent on (i.e., addicted to) the activation of YAP1-induced transcription. Citation Format: Muneyuki Masuda, Hirofumi Omori, Kuniaki Sato, Kenichi Taguchi, Masashige Bando, Satoshi Toh, Katsuhiko Shirahige, Koshi Mimori, Akira Suzuki. Transcriptional addiction to YAP1—the major driving force of oral cancer carcinogenesis and evolution [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B16.

Immune-related adverse events (irAEs) have been shown to be associated with higher antitumor responses and a clinical benefit in non-small cell lung carcinoma, renal cell carcinoma, and melanoma patients. However, little is known regarding the association between irAEs and the clinical effect of nivolumab for recurrent/metastatic head and neck squamous cell carcinoma (R/MHNSCC).We evaluated 108 patients treated with nivolumab for R/MHNSCC at 2 participating institutions. IrAEs were identified and profiled. We analyzed the association of each immune-related adverse effect with the clinical outcome of the patients.Among 108 patients, the objective response rate (ORR) was 29.6% (32/108 patients), and the disease control rate (DCR) was 50.0% (54/108 patients). IrAEs were observed in 41 patients (38.0%). Patients with irAEs had a significantly higher ORR and DCR than those without irAEs (46.3% vs. 19.4%, P = 0.004 and 75.6% vs. 34.3%, P 0.001, respectively). The median progression-free and overall survival rates in patients with irAEs were significantly longer than in those without irAEs.There was a significant relationship between irAEs and efficacy in R/MHNSCC patients treated with nivolumab. Our results indicate that the development of irAEs may aid in the earlier prediction of anticancer effects in patients with recurrent or metastatic HNSCC during nivolumab monotherapy.

Objective In some cases, the exposure and safeguarding of the internal carotid artery (ICA) are not easy by the maxillary swing approach that is used as a mainstay for the removal of nasopharyngeal tumors. To address this issue, we have developed a new combined transcervical and orbitozygomatic approach. Methods A nasopharyngeal adenocarcinoma arose in a 52-year-old patient and occupied the right middle skull base extending to the ICA. We first identified and dissected the ICA from the posterolateral part of the tumor using a transcervical approach. Then, the tumor was approached and removed by an orbitozygomatic technique with hemifacial dismasking. The surgical defect was filled using a temporal muscle flap, which was divided into two parts according to the blood supply from either the anterior or the posterior deep temporal artery. Results The postoperative course was uneventful and favorable cosmetic results were obtained. The patient has been free of carcinoma for more than 40 months after the surgery. Conclusion Our new combined approach might be a good option for selected patients with nasopharyngeal tumors.