An in vitro model and the underlying pathways of sinonasal inverted papilloma development.

Recently, the specific association between Sinonasal inverted papilloma (SIP) and EGFR exon 20 mutations has been reported. To investigate the link between specific EGFR mutations and SIP development, we established organotypic raft culture system using nasal polyp-derived immortalized NP2 (iNP2) cells expressing EGFR exon 20 mutants or an exon 19 mutant, and SIP-derived iIP4 cells harboring P772_H773insPYNP mutation. In the raft culture, iIP4 cells showed the inverted growth pattern characteristic to SIP. Interestingly, iNP2 cells expressing EGFR exon 20 duplication mutants, S768_D770dup and N771_H773dup, but not of EGFR exon 19 mutant, E746_A750del, showed the inverted growth pattern. Enhanced activation of the PI3K/AKT signaling pathway was observed in iNP2_S768_D770dup and iIP4 cells, while increased MAPK signaling was found in iNP2_N771_H773dup. Increased cell migration and invasion were found in all cells carrying EGFR mutations when compared to iNP2 cells, and this effect was inhibited by either PI3K or MEK inhibitor. Notably, iNP2 cells expressing the N771_H773dup mutant showed the highest migration and invasion abilities. These results suggest that specific mutations in EGFR exon 20 play a crucial role in SIP development, partially though hyper-activation of the PI3K/AKT and MAPK signaling pathways. This study presents the first in vitro model for SIP development, which could facilitate further investigations into SIP pathogenesis and preclinical studies for new therapeutic agents.

Establishment and genetic characterization of cell lines derived from proliferating nasal polyps and sinonasal inverted papillomas.

To better understand the pathogenesis of nasal polyps (NPs) and sinonasal inverted papillomas (SIPs), we aimed to establish cell lines from fresh tissues of NPs and SIPs and characterize them. Primary cell cultures were obtained from two NP tissues (NP2 and NP3) and one SIP tissue (IP4). All the cells were polygonal in shape, expressed cytokeratin 14, and had normal diploid chromosome status. HPV58 DNA was detected in NP3. To obtain immortal primary cells, NP2 and IP4 cells were transduced with a combination of mutant CDK4, cyclinD1 and TERT. These cells were thereafter named NP2/K4DT and IP4/K4DT, respectively. HPV58-positive NP3 cells were transduced with TERT alone, the resulting cells named NP3/T. Phenotypic and genotypic identity of original tissues and derived cells was investigated. All the cell cultures with transgenes were confirmed to be derived from their parental cells and primary tumor tissues by analysis of short tandem repeats (STR) and maintained in vitro growth, genetic profiles and gene expression characteristics of the primary cells. These virtually immortalized cells, as well as the primary cells, have potential as in vitro models for studying the pathogenesis of NPs and SIPs and for preclinical study to develop new therapeutic agents.

Neoadjuvant chemotherapy for locoregionally advanced squamous cell carcinoma of the paranasal sinuses.

BACKGROUND: Squamous cell carcinoma is the most common type of sinonasal malignancy. Despite improvements in surgical resection and adjuvant therapy, which are considered the standard of care, the outcome for patients with locoregionally advanced disease remains poor. The objective of this study was to investigate the role of induction chemotherapy in patients with locoregionally advanced sinonasal squamous cell carcinoma and to determine the oncologic outcomes in those patients. METHODS: The study included 123 consecutive patients with previously untreated, locoregionally advanced (stage III and IV) sinonasal squamous cell carcinoma who were treated with curative intent at The University of Texas MD Anderson Cancer Center between 1988 and 2017 with induction chemotherapy followed by definitive local therapy. Patient demographics, tumor staging, treatment details, and oncologic outcomes were reviewed. The outcomes of this study included response to induction chemotherapy, recurrence, organ preservation, and survival. RESULTS: The median follow-up was 32.6 months (range, 12.4-240 months). Of the 123 patients, 110 (89%) had T4 disease, and 13 (11%) had T3 disease. Lymph node metastasis at the time of presentation was observed in 36 patients (29.3%). The overall stage was stage IV in 111 patients (90.2%) and stage III in 12 patients (9.8%). The chemotherapy regimen consisted of the combination of a platinum and taxanes in most cases (109 patients; 88.6%), either as a doublet (41 patients) or in combination with a third agent, such as 5-fluorouracil (34 patients), ifosfamide (26 patients), or cetuximab (8 patients). After induction chemotherapy, 71 patients (57.8%) achieved at least a partial response, and 6 patients had a complete response. Subsequent treatment after induction chemotherapy was either: 1) definitive chemoradiation or radiation followed by surgical salvage for any residual disease, or 2) surgery followed by adjuvant radiation or chemoradiation. Overall, 54 patients (49.5%) underwent surgical resection. The 2-year overall and disease-free survival rates for the whole cohort were 61.4% and 67.9%, respectively. The rate of orbital preservation was 81.5%. The recurrence rate was 26.8% (33 patients), and distant metastases occurred in 8 patients (6.5%). Patients who had at least a partial response or stable disease had significantly better overall and disease-free survival than those who had progressive disease (P = .028 and P = .021, respectively). CONCLUSIONS: The current results indicate that a high proportion of patients with sinonasal squamous cell carcinoma achieved a favorable response to induction chemotherapy. The data suggest that response to induction chemotherapy is associated with an improved outcome and a good chance of organ preservation. The oncologic outcomes in this cohort with locally advanced (mostly T4) disease are better than those historically reported in the literature. Further study of induction chemotherapy in patients with advanced sinonasal squamous carcinoma is warranted.

Increasing prevalence of HPV in oropharyngeal carcinoma suggests adaptation of p16 screening in Southeast Asia.

BACKGROUND: Human papillomavirus (HPV) is etiologically linked to increasing oropharyngeal squamous cell carcinoma (OPSCC) rates in the Western world. However, the role of HPV in Southeast Asia, a high incidence region, hasn't been assessed. METHODS: 96 formalin-fixed, paraffin-embedded (FFPE) tissue blocks and corresponding patient data were obtained from Srinagarind Hospital, Thailand from 2012-2017. DNA from areas of 70 %+ cellularity were genotyped using polymerase chain reaction (PCR) and stained for p16, a surrogate marker for HPV. Inverse probability weights based on data from the hospital-based cancer registry were used in statistical analyses. Adjusted linear regression was used to assess changes in OPSCC HPV prevalence and conduct projections. Kaplan-Meier and Cox proportional hazard models were used to determine HPV-specific survival differences. RESULTS: 14 patients exhibited monoinfection with HPV16, two with HPV18 and one was HPV16/18 coinfected. PCR results were in agreement with p16 staining. On average, HPV + patients were more likely to have tonsil cancer (p-value:0.002). HPV prevalence increased by 2% annually (pvalue: 0.01), from 16 % in 2012 to 26 % in 2017. At the current rate, OPSCC HPV positivity will exceed 50 % by 2030. HPV positivity was shown to be protective in Kaplan-Meier (log-rank p = 0.02) and sex, age and stage adjusted Cox models (HR:0.34 [95 %CI:0.22, 0.52]). CONCLUSION: Given the increased prevalence and similarities in presentation of HPV + OPSCC to those observed in Western countries, the data suggest the adaptation of p16 staining and subsequent restaging of OPSCC tumors as suggested by the American Joint Committee on Cancer in Southeast Asia.

Prevalence and association of Epstein-Barr virus infection with sinonasal inverted papilloma and sinonasal squamous cell carcinoma in the northeastern Thai population.

AIMS: Sinonasal inverted papillomas (SIP) and sinonasal squamous cell carcinomas (SNSCC) are sinonasal tumors with unclear etiology and pathogenesis. Epstein-Barr virus (EBV) has been detected in these tumors but information concerning their association is still limited. This study aimed to investigate the prevalence in, and association of EBV infection with SIP and SNSCC in northeastern Thailand. METHODS: DNA was extracted from 226 formalin-fixed, paraffin-embedded tissues including 80 nasal polyps (NP; the control group), 64 SIP and 82 SNSCC samples. Presence of EBV in these tissues was investigated using real-time PCR and their localization within tissues was confirmed using in situ hybridization (ISH). Characteristics of patients and the association of EBV prevalence with sinonasal tumors were analyzed. RESULTS: SIP and SNSCC were frequently found in people aged > 50 years and more often in males than in females (3:1 ratio). EBV infection was detected in 33.75, 64.06 and 37.80% of NP, SIP and SNSCC tissues, respectively, by real-time PCR. There was a statistically significant association between EBV infection and SIP (odds ratio [OR] = 3.52). This was not the case for SNSCC when compared to the NP group (OR = 1.83). Interestingly, EBV infection tended to be associated with inflammation and dysplasia in SIP. In SNSCC, EBV was mostly found in samples with undifferentiated or poorly differentiated cell types as well as in recurrent cases and lymph-node metastasis. Using ISH, EBV was detected only in infiltrating lymphocytes within the tumor stroma, not in the tumor epithelial cells. CONCLUSIONS: Infiltrating lymphocytes containing EBV in the tumor microenvironment might enhance tumorigenesis of SIP and SNSCC. The mechanism by which EBV promotes development of SIP and SNSCC needs to be elucidated in the future.

Peroxiredoxin-2 and zinc-alpha-2-glycoprotein as potentially combined novel salivary biomarkers for early detection of oral squamous cell carcinoma using proteomic approaches.

No effective screening method is available for oral squamous cell carcinoma (OSCC) that is recognized to influence by environmental factors as well as human papillomavirus (HPV) and Epstein-Barr virus (EBV). Therefore, we sought to identify salivary biomarkers for screening of OSCC with or without HPV and/or EBV infection. Saliva, lesion and oral exfoliated cells were collected from OSCC patients and cancer-free controls (CFCs) and grouped depending on their HPV- and EBV-infection status. Salivary protein was precipitated and subjected to 2-dimensional gel electrophoresis. Differential expression of proteins was identified by mass spectrometry and validated by Western blotting. Distinctive expression patterns of salivary proteins were detected in OSCC as compared with CFCs. Levels of peroxiredoxin-2 (PRDX-2) and zinc-alpha-2-glycoprotein (ZAG) were significantly up-regulated in OSCC cases (p<0.001) relative to CFCs. Similarly, these proteins were also up-regulated in lesion cells compared with oral exfoliated cells (p<0.001). However, the expression patterns of these proteins were not significantly influenced by patient histories (risk factors). In combination, these proteins yielded the highest discriminatory power (AUC=0.999), sensitivity (100%), and specificity (98.77%) in distinguishing the early stages of OSCC. The detection of PRDX-2 combining with ZAG protein could potentially be used as salivary biomarkers for early screening of OSCC. SIGNIFICANCE: Our findings demonstrate a useful of combined detection of PRDX-2 and ZAG as a salivary biomarker for the early detection of OSCC.