Genome-wide open reading frame profiling identifies fibroblast growth factor signaling as a driver of PD-L1 expression in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCC) are associated with significant treatment-related morbidity and poor disease-free and disease-specific survival, especially in the recurrent and metastatic (R/M HNSCC) setting. Inhibition of the programmed death-1/ligand-1 (PD-1/PD-L1) immune checkpoint is accepted as a first-line treatment strategy for R/M HNSCC and has expanded into the neoadjuvant, definitive, and adjuvant settings. To understand cellular signals modulating the PD-L1 in HNSCC, we profiled a HNSCC cell-line with a genome-wide open reading frame (ORF) library of 17,000 individual constructs (14,000 unique genes). We identified 335 ORFs enriched in PD-L1high cells and independently validated five of these ORFs (FGF6, IL17A, CD300C, KLR1C and NFKBIA) as drivers of PD-L1 upregulation. We showed that exogenous FGF ligand is sufficient to induce PD-L1 expression in multiple HNSCC cell lines and human immature dendritic cells. Accordingly, overexpression of FGFR1, FGFR3 or the FGFR3 S249C and D786N mutants common to HNSCC tumors also induced PD-L1 overexpression on tumor cells. Small molecule inhibition of FGF signaling abrogated PD-L1 upregulation in these models and also blocked "classical" IFNγ-regulated PD-L1 expression in a STAT1-independent manner. Finally, we found that FGF specifically upregulated a glycosylated form of PD-L1 in our study, and exogenous FGF led to concomitant upregulation of glycosyltransferases that may stabilize PD-L1 on the surface of HNSCC cells. Taken together, our study supports a potential role for FGF/FGFR pathway signaling as a mechanism driving immune escape and rationalizes further exploration of novel combination therapies to improve clinical responses to PD-1/PD-L1 axis inhibition in HNSCC.

Small molecule profiling to define synergistic EGFR inhibitor combinations in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a debilitating disease with poor survival. Although epidermal growth factor receptor (EGFR)-targeting antibody cetuximab improves survival in some settings, responses are limited suggesting that alternative approaches are needed. METHODS: We performed a high throughput drug screen to identify EGFR inhibitor-based synergistic combinations of clinically advanced inhibitors in models resistant to EGFR inhibitor monotherapies, and then performed downstream validation experiments on prioritized synergistic combinations. RESULTS: From our screen, we re-discovered known synergistic EGFR inhibitor combinations with FGFR or IGF-1R inhibitors that were broadly effective and also discovered novel synergistic combinations with XIAP inhibitor and DNMT inhibitors that were effective in only a subset of models. CONCLUSIONS: Conceptually, our data identify novel synergistic combinations that warrant evaluation in future studies, and suggest that some combinations, although highly synergistic, will require parallel companion diagnostic development to be effectively advanced in patients.

Genetic analysis of sinonasal undifferentiated carcinoma discovers recurrent SWI/SNF alterations and a novel PGAP3-SRPK1 fusion gene.

BACKGROUND: Sinonasal Undifferentiated Carcinoma (SNUC) is a rare and aggressive skull base tumor with poor survival and limited treatment options. To date, targeted sequencing studies have identified IDH2 and SMARCB1 as potential driver alterations, but the molecular alterations found in SMARCB1 wild type tumors are unknown. METHODS: We evaluated survival outcomes in a cohort of 46 SNUC patients treated at an NCI designated cancer center and identify clinical and disease variables associated with survival on Kaplan-Meier and Cox multivariate survival analysis. We performed exome sequencing to characterize a series of SNUC tumors (n = 5) and cell line (MDA8788-6) to identify high confidence mutations, copy number alterations, microsatellite instability, and fusions. Knockdown studies using siRNA were utilized for validation of a novel PGAP3-SRPK1 gene fusion. RESULTS: Overall survival analysis revealed no significant difference in outcomes between patients treated with surgery +/- CRT and CRT alone. Tobacco use was the only significant predictor of survival. We also confirmed previously published findings on IDH and SMARC family mutations and identified novel recurrent aberrations in the JAK/STAT and PI3K pathways. We also validated a novel PGAP3-SRPK1 gene fusion in the SNUC cell line, and show that knockdown of the fusion is negatively associated with EGFR, E2F and MYC signaling. CONCLUSION: Collectively, these data demonstrate recurrent alterations in the SWI/SNF family as well as IDH, JAK/STAT, and PI3K pathways and discover a novel fusion gene (PGAP3-SRPK1). These data aim to improve understanding of possible driver mutations and guide future therapeutic strategies for this disease.

Clinical characteristics, HIV status, and molecular biomarkers in squamous cell carcinoma of the conjunctiva in Ghana.

BACKGROUND AND AIMS: Conjunctival squamous cell carcinoma (CSCC) varies in incidence geographically from 0 to 1 case per 100 000 per year globally. Additionally, the incidence of CSCC is known to increase 49% for every 10° decrease in latitude. Since the onset of the AIDS epidemic, there has been a trend of increasing incidence of CSCC in Africa, and despite relatively stable levels of ultraviolet (UV) exposure, there is an observed 12 times greater risk of developing CSCC when individuals are infected with HIV. In this study, we aim to analyze the clinical characteristics and biomarkers of CSCC in Ghana. METHODS: In this study, a registry review of patients from January 2011 to May 2016 with CSCC at Komfo-Anokye Teaching Hospital in Kumasi, Ghana, was performed (n = 64). Tumor blocks of the CSCC were analyzed for the expression of various biomarkers. RESULTS: In this study, the median age of onset of CSCC is 46.5 years old (range of 20-90 y old). Fifty one and a half percent (n = 33) of the cohort is female. There is a low rate of smoking and alcohol use in our CSCC cohort. Thirty-nine percent (n = 12) of Ghanaian men with CSCC are HIV-, while only 12% (n = 4) of women are HIV-. Fifteen patients had metastasis to lymph nodes or other tissues, and we observed a statistically significant relationship between HIV infection and metastasis (P = 0.027, chi-squared test). We observed no statistically significant relationship between known prognostic CSCC biomarkers and HIV status, age, or tumor stage. CONCLUSION: Better characterization of CSCC could have a profound impact on the prevention, early identification, and treatment of CSCC in Africa. A retrospective chart analysis and collection of tumor samples can be challenging in this region due to methods of record keeping and stigma attached to clinical data such as HIV testing and smoking and alcohol use. As a result, in this study, data were often incomplete leading to inconclusive results and analysis that should be interpreted with caution. Future studies should consider a prospective study design that gathers clinical data in a standardized format and ensures fresh tissue from CSCC tumors.

Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma.

BACKGROUND: We sought to describe targeted DNA sequencing data of persistent/recurrent laryngeal squamous cell carcinoma (LSCC) and to compare gene-specific alteration frequencies with that of primary, untreated LSCC specimens from The Cancer Genome Atlas (TCGA). METHODS: The tumors of 21 patients with persistent/recurrent LSCC were subjected to targeted DNA sequencing using the Ion AmpliSeq Comprehensive Cancer Panel. Gene-specific alteration frequencies were compared (Chi-Square test) to primary, untreated LSCC sequencing data from TCGA using the cBioPortal platform. RESULTS: Persistent/recurrent LSCC was characterized by a high rate of inactivating alterations in TP53 (38.1%) and CDKN2A (33%), amplification events of CCND1 (19.1%), and ERBB2 (14.3%), and NOTCH1 (19.1%) mutations. Comparison of primary vs persistent/recurrent LSCC revealed significant differences in alteration frequencies of eight critical genes: BAP1, CDKN2A, DCUN1D1, MSH2, MTOR, PIK3CA, TET2, and TP53. CONCLUSIONS: Our results provide preliminary support for a distinct mutational profile of persistent/recurrent LSCC that requires validation in larger cohorts.

The genomic landscape of UM-SCC oral cavity squamous cell carcinoma cell lines.

OBJECTIVES: We sought to describe the genetic complexity of 14 UM-SCC oral cavity cancer cell lines that have remained uncharacterized despite being used as model systems for decades. MATERIALS AND METHODS: We performed exome sequencing on 14 oral cavity UM-SCC cell lines and denote the mutational profile of each line. We used a SNP array to profile the multiple copy number variations of each cell line and use immunoblotting to compare alterations to protein expression of commonly amplified genes (EGFR, PIK3CA, etc.). RNA sequencing was performed to characterize the expression of genes with copy number alterations. RESULTS: The cell lines displayed a highly complex network of genetic aberrations that was consistent with alterations identified in the HNSCC TCGA project including PIK3CA amplification, CDKN2A deletion, as well as TP53 and CASP8 mutations, enabling genetic stratification of each cell line in the panel. Copy number FISH and spectral karyotyping analysis demonstrate that cell lines retain chromosomal heterogeneity. CONCLUSIONS: Collectively, we developed an important resource for future oral cavity HNSCC cell line studies and highlight the complexity of genomic aberrations in cell lines.

Correction: Expression of immunohistochemical markers in non-oropharyngeal head and neck squamous cell carcinoma in Ghana.

[This corrects the article DOI: 10.1371/journal.pone.0202790.].

Expression of immunohistochemical markers in non-oropharyngeal head and neck squamous cell carcinoma in Ghana.

BACKGROUND: Head and neck cancers include carcinomas of the oral cavity, larynx, sinonasal tract and nasopharynx. Studies on molecular expression of prognostic tumour markers in Ghana are scarce. The purpose of this study was to determine the expression of p53, p16, EGFR, Cyclin-D1 and HER2 among patients with non-oropharyngeal head and neck squamous cell carcinoma (HNSCC). METHODOLOGY: Tissue microarrays from 154 histologically confirmed non-oropharyngeal HNSCC at the Komfo Anokye Teaching Hospital from 2006-2014 were constructed using duplicate cores of representative and viable areas from tumours. Expression of EGFR, p53, p16, Cyclin-D1 and HER2 was evaluated using immunohistochemistry. RESULTS: For non-oropharyngeal HNSCC, majority of the cases (66.2%; 102/154) had stage IV disease. EGFR was the most expressed molecular marker (29.4%; 25/85) followed by p53 (24.0%; 29/121), p16 (18.3%; 23/126) and Cyclin-D1 (10.0%; 12/120). HER2 was not expressed in any of the cases. There was a significantly (p = 0.022) higher expression of Cyclin-D1 in tumours of the oral cavity (19.6%; 9/46) than in those of the larynx (4.7%; 2/43) and nose (3.2%; 1/31). Tumours in stages I-III were more frequently positive for p16 (28.6%; 12/42) than tumours in stage IV (13.1%; 11/84). CONCLUSION: Expression of p53, EGFR, p16 and Cyclin-D1 in non-oropharyngeal HNSCC in Ghana is largely similar to what has been reported in published studies from other countries.

Correlation of Crtc1/3-Maml2 fusion status, grade and survival in mucoepidermoid carcinoma.

OBJECTIVE: Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the salivary glands. Tumor stage and grade have historically been important predictors of survival. An oncogenic CRTC1- or CRTC3-MAML2 gene fusion has been identified in a number of MECs. Historically, these gene fusions have been associated with lower grade tumors and better survival. However, reported gene fusion rates and prognosis varies widely across studies, and have not controlled for tumor grade. We sought to identify gene fusion rates and outcomes in our cohort of MEC patients. MATERIALS AND METHODS: An IRB-approved retrospective cohort of patients with MEC was identified at the University of Michigan. Clinical, histologic, and outcome data was collected from medical records. RNA was isolated from formalin fixed paraffin-embedded tumor sections, and qRT-PCR was performed to identify CRTC1/3-MAML2 gene fusions. Sanger sequencing of qRT-PCR products was used to confirm gene fusions. RESULTS: Overall, 90 patient MEC tumors were collected (58 low-grade, 25 intermediate-grade, and 7 high-grade). Gene fusions were identified in 59% (53/90) of tumors. On univariate and bivariate analysis, fusion status did not significantly associate with grade or survival. CONCLUSION: We have identified a high rate of CRTC1/3-MAML2 gene fusions in a large cohort of MEC. We do not identify any correlation between fusion status with tumor grade or survival. These findings suggest further characterization of MECs is needed before considering the CRTC1/3-MAML2 gene fusion as a prognostic biomarker. Additional genetic drivers may account for survival and grade in MECs.

Identification of Targetable ERBB2 Aberrations in Head and Neck Squamous Cell Carcinoma.

IMPORTANCE: ERBB2 (formerly HER2) is an important drug target in breast cancer, where anti-ERBB2 therapy has been shown to lead to improvements in disease recurrence and overall survival. ERBB2 status in head and neck squamous cell carcinoma (HNSCC) has not been well studied. Identification of ERBB2-positive tumors and characterization of response to ERBB2 therapy could lead to targeted treatment options in HNSCC. OBJECTIVE: To identify ERBB2 aberrations in HNSCCs and investigate the potential for ERBB2-targeted therapy in HNSCCs. DESIGN, SETTING, AND PARTICIPANTS: A retrospective case series of patients with laryngeal (42 tumor specimens) and oral cavity (94 tumor specimens) SCC enrolled in the University of Michigan Head and Neck Specialized Program of Research Excellence was conducted. Publicly available sequencing data (The Cancer Genome Atlas), as well as data from other studies, were reviewed to identify additional mutations and overexpression in ERBB2 in HNSCC. Established HNSCC cell lines were used for follow-up in vitro analysis. The study was conducted from October 1, 2014, to August 30, 2015. INTERVENTIONS: With the use of targeted, amplicon-based sequencing with the Oncomine Cancer Panel, the copy number and mutation status of commonly altered genes in HNSCCs were assessed. Immunohistochemical staining was performed on tissue microarrays of HNSCCs to assess the expression of ERBB2. Western blotting for HNSCC cell line ERBB2 expression and cell survival assays after treatment with ERBB2 inhibitors were performed. MAIN OUTCOMES AND MEASURES: The prevalence of ERBB2 genetic aberrations and ERBB2 overexpression in laryngeal and oral cavity SCCs, prevalence of ERBB2 aberrations in HNSCC in The Cancer Genome Atlas, ERBB2 protein expression in HNSCC cell lines, and response of HNSCC cell lines to targeted ERBB2 inhibitors. RESULTS: Of the 42 laryngeal SCC samples screened by targeted sequencing, 4 (10%) were positive for ERBB2 amplification. Two of these samples showed ERBB2 overexpression on immunohistochemistry. Two of the 94 oral cavity SCC samples (2%) were positive for ERBB2 on immunohistochemistry. Analysis of 288 patients from publicly available HNSCC sequencing data revealed 9 amplifications (3%) in ERBB2. Protein expression was variable across HNSCC cell lines, and a subset of these cell lines showed responsiveness to anti-ERBB2 therapy. CONCLUSIONS AND RELEVANCE: ERBB2 aberrations were identified in a subset of HNSCCs. These tumors may be responsive to targeted therapy against ERBB2. Screening for ERBB2 aberrations and applying targeted therapy in ERBB2-positive patients may be useful in personalized therapy trials, particularly in patients who are refractory to current treatment paradigms.

Leptin acts independently of food intake to modulate gut microbial composition in male mice.

Shifts in the composition of gut bacterial populations can alter host metabolism and may contribute to the pathogenesis of metabolic disorders, including obesity. Mice deficient in leptin action are obese with altered microbiota and increased susceptibility to certain intestinal pathogens. Because antimicrobial peptides (AMPs) secreted by Paneth cells represent a major mechanism by which the host influences the gut microbiome, we examined the mRNA expression of gut AMPs, several of which were decreased in leptin receptor (LepR)-deficient db/db mice, suggesting a potential role for AMP modulation of microbiota composition. To address the extent to which the alterations in gut microbiota and AMP mRNA expression in db/db mice result from increased food intake vs other defects in leptin action, we examined the effects of pair feeding and gut epithelial LepRb ablation on AMP mRNA expression and microbiota composition. We found that the phylum-level changes in fecal microbial content and AMP gene expression persist in pair-fed db/db mice, suggesting that these differences do not stem from hyperphagia alone. In addition, despite recent evidence to support a role for intestinal epithelial LepRb signaling in pathogen susceptibility, ablation of LepRb from the intestinal epithelium fails to alter body weight, composition of the microbiota, or AMP expression, suggesting a role for LepRb elsewhere for this regulation. Indeed, gut LepRb cells are not epithelial but rather constitute a previously uncharacterized population of perivascular cells within the intestinal submucosa. Overall, our data reveal a role for LepRb signaling extrinsic to the intestinal epithelium and independent of food intake in the control of the gut microbiome.

Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 2. Discovery of 7-azaspiro[3.5]nonane urea PF-04862853, an orally efficacious inhibitor of fatty acid amide hydrolase (FAAH) for pain.

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degradation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spirocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using an iterative design and optimization strategy, lead compounds were identified with a remarkable reduction in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltetrazole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remarkable potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was advanced as a clinical candidate.

Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 1: identification of 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane as lead scaffolds.

Herein we report the identification of two new fatty acid amide hydrolase (FAAH) inhibitor lead series with FAAH k(inact)/K(i) potency values greater than 1500M(-1)s(-1). The two novel spirocyclic cores, 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane, clearly distinguished themselves from the other spirocyclic cores on the basis of their superior potency for FAAH. Lead compounds from these two series have suitable FAAH potency and selectivity for additional medicinal chemistry optimization.