Hiroyuki Ozawa

A phase II study of temsirolimus and erlotinib in patients with recurrent and/or metastatic, platinum-refractory head and neck squamous cell carcinoma

OBJECTIVES The epidermal growth factor receptor (EGFR) is a validated target in head and neck squamous cell carcinoma (HNSCC). In recurrent and/or metastatic (R/M) HNSCC, resistance to anti-EGFR therapy inevitably occurs. Downstream activation of the PI3K/Akt/mTOR pathway is an established resistance mechanism. Concurrent mTOR blockade may improve efficacy of anti-EGFR therapy. MATERIALS AND METHODS Erlotinib 150 mg daily and temsirolimus 15 mg weekly were administered to patients with platinum-refractory R/M HNSCC and ECOG performance status 0-2. The primary endpoint was progression-free survival (PFS). Correlative studies determined PIK3CA and HRAS mutation status; p16, EGFR, pS6K, pAkt and PTEN expression; and pre- and post-treatment plasma levels of 20 immunomodulatory cytokines. RESULTS Twelve patients enrolled; six withdrew within 6 weeks due to toxicity or death, prompting early closure of the trial. Grade ≥ 3 toxicities included fatigue, diarrhea, gastrostomy tube infection, peritonitis, pneumonia, dyspnea, and HN edema. Median PFS was 1.9 months. Median overall survival was 4.0 months. Six/12 tumors were p16(+), 9/11 lacked measurable PTEN expression, and 1/12 harbored a PIK3CA mutation. On exploratory analysis, high baseline plasma VEGF and interferon-gamma levels marginally associated with tumor progression. CONCLUSIONS The combination of erlotinib and temsirolimus was poorly tolerated. Low prevalence of PTEN expression and 8% incidence of PIK3CA mutations indicate biological relevance of this pathway in R/M disease. Investigation of more tolerable combinations of EGFR and PI3K/Akt/mTOR pathway inhibitors in selected HNSCC patients is warranted.

Mucoepidermoid Carcinoma of the Head and Neck : Clinical Analysis of 43 Patients

OBJECTIVE It is well known that mucoepidermoid carcinoma (MEC) displays a variety of biological behaviors. While the high-grade type is a highly aggressive tumor, its low-grade counterpart usually demonstrates a more benign nature and several systems have, therefore, been proposed to grade this neoplasm. METHODS This report analyzes 43 patients suffering from head and neck MEC, who were treated in our department during the period from 1989 to 2005. The relationship between clinical and pathologic characteristics and survival rate was investigated. RESULTS The 5-year overall and disease-free survival rate was 62.3 and 57.2%. Multivariate analysis demonstrated that the parameters that significantly affected survival were the patients age (P = 0.040) and treatment method (P = 0.011). CONCLUSIONS The patients age and treatment method is the prognostic parameter in this study. Although complete surgical resection is the standard treatment for MEC, we should aggressively consider adjunctive radiotherapy in those cases that have a high risk of recurrence and poor prognosis.

Restoration of E-cadherin expression by selective Cox-2 inhibition and the clinical relevance of the epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma

BackgroundThe epithelial-to-mesenchymal transition (EMT) accompanied by the downregulation of E-cadherin has been thought to promote metastasis. Cyclooxygenase-2 (Cox-2) is presumed to contribute to cancer progression through its multifaceted function, and recently its inverse relationship with E-cadherin was suggested. The aim of the present study was to investigate whether selective Cox-2 inhibitors restore the expression of E-cadherin in head and neck squamous cell carcinoma (HNSCC) cells, and to examine the possible correlations of the expression levels of EMT-related molecules with clinicopathological factors in HNSCC.MethodsWe used quantitative real-time PCR to examine the effects of three selective Cox-2 inhibitors, i.e., celecoxib, NS-398, and SC-791 on the gene expressions of E-cadherin (CDH-1) and its transcriptional repressors (SIP1, Snail, Twist) in the human HNSCC cell lines HSC-2 and HSC-4. To evaluate the changes in E-cadherin expression on the cell surface, we used a flowcytometer and immunofluorescent staining in addition to Western blotting. We evaluated and statistically analyzed the clinicopathological factors and mRNA expressions of Cox-2, CDH-1 and its repressors in surgical specimens of 40 patients with tongue squamous cell carcinoma (TSCC).ResultsThe selective Cox-2 inhibitors upregulated the E-cadherin expression on the cell surface of the HNSCC cells through the downregulation of its transcriptional repressors. The extent of this effect depended on the baseline expression levels of both E-cadherin and Cox-2 in each cell line. A univariate analysis showed that higher Cox-2 mRNA expression (p = 0.037), lower CDH-1 mRNA expression (p = 0.020), and advanced T-classification (p = 0.036) were significantly correlated with lymph node metastasis in TSCC. A multivariate logistic regression revealed that lower CDH-1 mRNA expression was the independent risk factor affecting lymph node metastasis (p = 0.041).ConclusionsThese findings suggest that the appropriately selective administration of certain Cox-2 inhibitors may have an anti-metastatic effect through suppression of the EMT by restoring E-cadherin expression. In addition, the downregulation of CDH-1 resulting from the EMT may be closely involved in lymph node metastasis in TSCC.

NF-κB and STAT3 Transcription Factor Signatures Differentiate HPV-positive and HPV-negative Head and Neck Squamous Cell Carcinoma

Using high‐throughput analyses and the TRANSFAC database, we characterized TF signatures of head and neck squamous cell carcinoma (HNSCC) subgroups by inferential analysis of target gene expression, correcting for the effects of DNA methylation and copy number. Using this discovery pipeline, we determined that human papillomavirus‐related (HPV+) and HPV− HNSCC differed significantly based on the activity levels of key TFs including AP1, STATs, NF‐κB and p53. Immunohistochemical analysis confirmed that HPV− HNSCC is characterized by co‐activated STAT3 and NF‐κB pathways and functional studies demonstrate that this phenotype can be effectively targeted with combined anti‐NF‐κB and anti‐STAT therapies. These discoveries correlate strongly with previous findings connecting STATs, NF‐κB and AP1 in HNSCC. We identified five top‐scoring pair biomarkers from STATs, NF‐κB and AP1 pathways that distinguish HPV+ from HPV− HNSCC based on TF activity and validated these biomarkers on TCGA and on independent validation cohorts. We conclude that a novel approach to TF pathway analysis can provide insight into therapeutic targeting of patient subgroup for heterogeneous disease such as HNSCC.

Diagnosis and management of cervical sympathetic chain schwannoma: A review of 9 cases

Conclusions. Cervical sympathetic chain schwannoma (CSCS) sometimes mimics carotid body tumor (CBT). Differential diagnosis between these tumors is sometimes difficult using MRI alone. MRA, color Doppler ultrasonography, and fine needle aspiration (FNA) after imaging may be helpful to rule out CBT. Surgical resection of CSCS is relatively effortless, and Horners syndrome is an expected but acceptable postoperative complication. Intratumoral hemorrhage and vasodilation may be the main reasons for significant enhancement on MRI. Objectives. CSCSs are rare and known to mimic carotid body tumors. We report 9 cases of CSCS with an emphasis on imaging, surgical management, and pathological findings. Moreover, we describe the differential diagnosis of CSCS and CBT, and speculate the reasons behind significant enhancement on MRI. Patients and methods. Nine cases of CSCS treated at a tertiary referral center between 1996 and 2008 were reviewed. Results. MRI revealed 3 of 9 cases (33%) splayed the carotid bifurcation and displayed marked contrast enhancement with gadolinium. All patients underwent surgical excision of the mass with minimal blood loss. Postoperative Horners syndrome was encountered in all patients, which required no treatment. Marked gadolinium enhancement tended to be associated with histological findings such as intratumoral hemorrhage and vasodilation.

Decreased SMAD4 expression is associated with induction of epithelial-to-mesenchymal transition and cetuximab resistance in head and neck squamous cell carcinoma

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting this receptor, is widely used to treat these patients. In the following investigation, we examined the role of SMAD4 down-regulation in mediating epithelial-to-mesenchymal transition (EMT) and cetuximab resistance in HNSCC. We determined that SMAD4 downregulation was significantly associated with increased cell motility, increased expression of vimentin, and cetuximab resistance in HNSCC cell lines. In the HNSCC genomic dataset obtained from The Cancer Genome Atlas, SMAD4 was altered in 20/279 (7%) of HNSCC via homozygous deletion, and nonsense, missense, and silent mutations. When SMAD4 expression was compared with respect to human papillomavirus (HPV) status, HPV-positive tumors had higher expression compared to HPV-negative tumors. Furthermore, higher SMAD4 expression also correlated with higher CDKN2A (p16) expression. Our data suggest that SMAD4 down-regulation plays an important role in the induction of EMT and cetuximab resistance. Patients with higher SMAD4 expression may benefit from cetuximab use in the clinic.

Biomarker immunoprofile in salivary duct carcinomas: clinicopathological and prognostic implications with evaluation of the revised classification

Salivary duct carcinoma (SDC) is an uncommon, aggressive malignant neoplasm histologically resembling high-grade mammary ductal carcinoma. SDC can arise de novo or ex pleomorphic adenoma. To clarify the correlation of biomarker immunoprofile with clinicopathological findings and clinical outcome of SDC, we conducted immunohistochemistry for EGFR, HER2, HER3, AR, CK5/6, p53, and Ki-67, along with HER2 fluorescence in situ hybridization in 151 SDCs. SDCs ex pleomorphic adenoma more commonly overexpressed EGFR, HER2, HER3, and Ki-67 than de novo SDCs (P = 0.015, < 0.001, 0.045, and 0.02, respectively). In multivariate analysis, AR− and CK5/6+ were associated with shorter progression-free survival (P = 0.027 and 0.004, respectively). Moreover, patients with p53-extreme negative/positive demonstrated poorer overall survival (P = 0.007). On assessing the revised classification by the combination of biomarker expression, the percentages of each subtype were as follows: ‘apocrine A’ (AR+/HER2−/Ki-67-low) (24%), ‘apocrine B’ (AR+/HER2−/Ki-67-high) (18%), ‘apocrine HER2’ (AR+/HER2+) (35%), ‘HER2-enriched’ (AR−/HER2+) (12%), and ‘double negative’ (AR−/HER2−) (11%). ‘Double negative’ was further subclassified into ‘basal-like’ (EGFR and/or CK5/6+) (7%) and ‘unclassified’ (3%). Consequently, patients with ‘apocrine A’ showed a better progression-free survival than those with any other subtypes. Our revised immunoprofiling classification was valuable for predicting the survival and might be useful in personalized therapy for patients with SDC.

SMAD4 Loss Is Associated with Cetuximab Resistance and Induction of MAPK/JNK Activation in Head and Neck Cancer Cells

Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC. Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivo. Results: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4-low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo. We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss. Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. Clin Cancer Res; 23(17); 5162–75. ©2017 AACR.

Impact of hematological inflammatory markers on clinical outcome in patients with salivary duct carcinoma: a multi-institutional study in Japan

The prognostic role of modified Glasgow Prognostic Score (mGPS), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in patients with salivary duct carcinoma (SDC) remains unclear. We conducted a multi-institutional retrospective cohort study of 140 SDC patients. The survival impact of these hematological markers was evaluated using multivariate proportional hazard models.High mGPS (≥1) was significantly associated with worse survival (3-year overall survival (OS): 16.7% vs 66.1%, p-value=0.003; 3-year progression-free survival (PFS): 0.0% vs 27.9%, p-value<0.001). Additionally, high C-reactive protein (CRP) (≥0.39 mg/dl) was significantly associated with worse survival (3-year OS: 32.1% vs 68.2%, p-value=0.001; 3-year PFS: 7.1% vs 31.1%, p-value<0.001). These associations were consistent with multivariate analysis adjusted for established prognostic factors. Although we also found significant association of high NLR (≥2.5) with OS (HR 1.80; 95% confidence interval, 1.05-3.08) in multivariate analysis, this association were inconsistent with the results of PFS. In addition, we found no significant associations of PLR with survival. In conclusion, we found that mGPS, CRP and NLR were identified as prognostic factors associated with survival in SDC patients.

CoGAPS matrix factorization algorithm identifies transcriptional changes in AP-2alpha target genes in feedback from therapeutic inhibition of the EGFR network

Patients with oncogene driven tumors are treated with targeted therapeutics including EGFR inhibitors. Genomic data from The Cancer Genome Atlas (TCGA) demonstrates molecular alterations to EGFR, MAPK, and PI3K pathways in previously untreated tumors. Therefore, this study uses bioinformatics algorithms to delineate interactions resulting from EGFR inhibitor use in cancer cells with these genetic alterations. We modify the HaCaT keratinocyte cell line model to simulate cancer cells with constitutive activation of EGFR, HRAS, and PI3K in a controlled genetic background. We then measure gene expression after treating modified HaCaT cells with gefitinib, afatinib, and cetuximab. The CoGAPS algorithm distinguishes a gene expression signature associated with the anticipated silencing of the EGFR network. It also infers a feedback signature with EGFR gene expression itself increasing in cells that are responsive to EGFR inhibitors. This feedback signature has increased expression of several growth factor receptors regulated by the AP-2 family of transcription factors. The gene expression signatures for AP-2alpha are further correlated with sensitivity to cetuximab treatment in HNSCC cell lines and changes in EGFR expression in HNSCC tumors with low CDKN2A gene expression. In addition, the AP-2alpha gene expression signatures are also associated with inhibition of MEK, PI3K, and mTOR pathways in the Library of Integrated Network-Based Cellular Signatures (LINCS) data. These results suggest that AP-2 transcription factors are activated as feedback from EGFR network inhibition and may mediate EGFR inhibitor resistance.