Microsecretory adenocarcinoma of the skin harboring recurrent SS18 fusions: A cutaneous analog to a newly described salivary gland tumor.

BACKGROUND: Microsecretory adenocarcinoma (MSA) is a newly described salivary gland neoplasm characterized by MEF2C::SS18 fusions. MSA was previously thought to occur exclusively in salivary glands. Here, we expand the spectrum of known primary sites of this tumor by describing a series of cutaneous tumors with analogous findings. METHODS: We identified four cutaneous primary tumors with histopathologic features identical to MSA of the salivary glands. These cases were evaluated by immunohistochemistry, fluorescence in situ hybridization (FISH) for SS18 rearrangement and targeted RNA-sequencing. We also queried a pan-tumor database of advanced carcinomas for MEF2C::SS18. RESULTS: The cases occurred in men ranging from 61 to 74 years (mean, 68). They arose from the skin of the nose, chin, scalp, and external auditory canal. All included cords/microcysts of eosinophilic cells with bland oval nuclei and bluish mucin within fibromyxoid stroma. The scalp tumor also exhibited high-grade transformation (marked atypia, elevated mitotic rate, and necrosis), a feature unreported in salivary MSA. By immunohistochemistry, all cases were positive for S100. Two showed a myoepithelial component positive for p40 and smooth muscle actin or calponin. Three cases harbored MEF2C::SS18 by RNA sequencing, while one with limited tissue had SS18 rearrangement via FISH. Two patients had no evidence of recurrence or metastasis in limited follow-up (3 and 6 months). The pan-tumor database query also did not identify MEF2C::SS18 in any advanced cutaneous carcinomas. CONCLUSION: This report expands the sites that can be involved by MSA. Similar to salivary cases, MEF2C::SS18 represents a recurrent fusion in MSA of the skin. Unusual features in cutaneous cases not seen in salivary MSA include one case with high-grade transformation and two cases with a myoepithelial cell component. Identification of this fusion expands the spectrum of salivary-analog cutaneous tumors and aids in precise tumor classification.

Radiotherapy plus cetuximab or cisplatin in human papillomavirus-positive oropharyngeal cancer (NRG Oncology RTOG 1016): a randomised, multicentre, non-inferiority trial.

BACKGROUND: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma have high survival when treated with radiotherapy plus cisplatin. Whether replacement of cisplatin with cetuximab-an antibody against the epidermal growth factor receptor-can preserve high survival and reduce treatment toxicity is unknown. We investigated whether cetuximab would maintain a high proportion of patient survival and reduce acute and late toxicity. METHODS: RTOG 1016 was a randomised, multicentre, non-inferiority trial at 182 health-care centres in the USA and Canada. Eligibility criteria included histologically confirmed HPV-positive oropharyngeal carcinoma; American Joint Committee on Cancer 7th edition clinical categories T1-T2, N2a-N3 M0 or T3-T4, N0-N3 M0; Zubrod performance status 0 or 1; age at least 18 years; and adequate bone marrow, hepatic, and renal function. We randomly assigned patients (1:1) to receive either radiotherapy plus cetuximab or radiotherapy plus cisplatin. Randomisation was balanced by using randomly permuted blocks, and patients were stratified by T category (T1-T2 vs T3-T4), N category (N0-N2a vs N2b-N3), Zubrod performance status (0 vs 1), and tobacco smoking history (≤10 pack-years vs >10 pack-years). Patients were assigned to receive either intravenous cetuximab at a loading dose of 400 mg/m2 5-7 days before radiotherapy initiation, followed by cetuximab 250 mg/m2 weekly for seven doses (total 2150 mg/m2), or cisplatin 100 mg/m2 on days 1 and 22 of radiotherapy (total 200 mg/m2). All patients received accelerated intensity-modulated radiotherapy delivered at 70 Gy in 35 fractions over 6 weeks at six fractions per week (with two fractions given on one day, at least 6 h apart). The primary endpoint was overall survival, defined as time from randomisation to death from any cause, with non-inferiority margin 1·45. Primary analysis was based on the modified intention-to-treat approach, whereby all patients meeting eligibility criteria are included. This study is registered with ClinicalTrials.gov, number NCT01302834. FINDINGS: Between June 9, 2011, and July 31, 2014, 987 patients were enrolled, of whom 849 were randomly assigned to receive radiotherapy plus cetuximab (n=425) or radiotherapy plus cisplatin (n=424). 399 patients assigned to receive cetuximab and 406 patients assigned to receive cisplatin were subsequently eligible. After median follow-up duration of 4·5 years, radiotherapy plus cetuximab did not meet the non-inferiority criteria for overall survival (hazard ratio [HR] 1·45, one-sided 95% upper CI 1·94; p=0·5056 for non-inferiority; one-sided log-rank p=0·0163). Estimated 5-year overall survival was 77·9% (95% CI 73·4-82·5) in the cetuximab group versus 84·6% (80·6-88·6) in the cisplatin group. Progression-free survival was significantly lower in the cetuximab group compared with the cisplatin group (HR 1·72, 95% CI 1·29-2·29; p=0·0002; 5-year progression-free survival 67·3%, 95% CI 62·4-72·2 vs 78·4%, 73·8-83·0), and locoregional failure was significantly higher in the cetuximab group compared with the cisplatin group (HR 2·05, 95% CI 1·35-3·10; 5-year proportions 17·3%, 95% CI 13·7-21·4 vs 9·9%, 6·9-13·6). Proportions of acute moderate to severe toxicity (77·4%, 95% CI 73·0-81·5 vs 81·7%, 77·5-85·3; p=0·1586) and late moderate to severe toxicity (16·5%, 95% CI 12·9-20·7 vs 20·4%, 16·4-24·8; p=0·1904) were similar between the cetuximab and cisplatin groups. INTERPRETATION: For patients with HPV-positive oropharyngeal carcinoma, radiotherapy plus cetuximab showed inferior overall survival and progression-free survival compared with radiotherapy plus cisplatin. Radiotherapy plus cisplatin is the standard of care for eligible patients with HPV-positive oropharyngeal carcinoma. FUNDING: National Cancer Institute USA, Eli Lilly, and The Oral Cancer Foundation.

Genomic Profiling of the Craniofacial Ossifying Fibroma by Next-Generation Sequencing.

BACKGROUND: Ossifying fibroma (OF) of the craniofacial skeleton is a fibro-osseous lesion characterized by various patterns of bone formation in a cellular fibroblastic stroma. The molecular landscape of OF remains mostly unknown. There are a few known pathogenic abnormalities in OF, including HRPT2 mutations in conventional OF and SATB2 translocations in juvenile psammomatoid OF. On the other hand, conflicting reports exist regarding MDM2 gene amplification and chromosomal copy number alterations (CNA) in OF. METHODS: Surgically removed biopsies and curettage specimens from OF patients were obtained. Clinical, radiographic, and pathologic features of tumors were reviewed. Genomic DNA was extracted from formalin-fixed, paraffin-embedded blocks of tumor tissue. Capture-based DNA next-generation sequencing targeting the coding regions 529 cancer genes and select introns was performed. RESULTS: We identified 17 OF cases from 8 male and 8 female patients with mean age of 22 years (range 1-58 years). Nine case occurred in the gnathic bones and 8 in the extragnathic craniofacial bones. These cases included 3 juvenile psammomatoid OF, 6 conventional OF and 8 juvenile trabecular OF. Large-scale CNAs were present in 6 of 17 cases. Seven cases (41%) had focal amplifications including FOSB (n = 2, 11%), FOS (n = 4, 23%), COL1A1 (n = 4, 23%) and TBX3 (n = 5, 29%). Three cases (17%) had pathogenic CDC73 mutations. No cases showed focal MDM2 amplification. CONCLUSIONS: Here, we provided a comprehensive molecular characterization of OF that reveals a heterogeneous genetic profile with occasional large-scale CNAs (n = 6, 35%). FOS, FOSB, and TBX3 genes that regulate AP-1 transcriptional complex are frequently altered in OF (n = 7, 41%), chiefly in juvenile trabecular OF. These genes encode transcription factors that act as downstream effectors of the MAP kinase signaling pathway. MDM2 amplification is an exceedingly rare event in OF, if present at all, so identification of this event should continue to raise concern for low-grade gnathic osteosarcoma. In summary, our findings suggest that OF represents a heterogeneous group of tumors at the genetic level but dysregulation of the AP-1 pathway may play a role in pathogenesis of juvenile trabecular OF.

Giant cell lesions of the jaws: does the level of vascularity and angiogenesis correlate with behavior?

PURPOSE: To compare vascularity and angiogenic activity in aggressive and nonaggressive giant cell lesions (GCLs) of the jaws. MATERIALS AND METHODS: This is a retrospective study of 14 GCLs treated at the University of California, San Francisco. Immunohistochemistry was used to determine of the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), CD34, and CD31. VEGF and bFGF expression in giant cells (GCs) and surrounding mononuclear stroma was classified into 1) high immunoreactivity (>50% staining) and 2) low immunoreactivity (<50% staining). CD31- and CD34-stained vessels were counted at 200× magnification. Clinical and radiographic records were reviewed to classify lesions as aggressive or nonaggressive. RESULTS: Of the lesions, 8 were aggressive and 6 were nonaggressive. High VEGF expression was found within the GCs in 4 of 8 aggressive lesions compared with 1 of 6 nonaggressive lesions. The stroma in both groups had low staining. High staining of the GCs for bFGF was found in 6 of 8 aggressive lesions compared with 3 of 6 nonaggressive lesions. The stroma of all aggressive cases showed high expression of bFGF compared with 3 of 6 nonaggressive cases. The aggressive group had a mean of 20.1 ± 5.4 vessels/high-powered field (hpf) stained for CD31 compared with 11.5 ± 5.6 vessels/hpf in the nonaggressive group. The aggressive group had 24.6 ± 7.0 vessels/hpf stained with CD34 compared with 18.5 ± 4.0 vessels/hpf in the nonaggressive group. CONCLUSIONS: The vascularity and level of angiogenesis within aggressive GCLs are higher than those in nonaggressive lesions.

MDM2 Gene Amplification and Expression of MDM2 and CDK4 are Rare in Ossifying Fibroma of Craniofacial Bones.

Ossifying fibroma of the craniofacial bones is a fibro-osseous lesion characterized by varied patterns of bone formation in a fibroblastic stroma. Ossifying fibroma is a putatively benign lesion with no reports of malignant transformation or metastasis. Differentiation from other fibro-osseous lesions can be challenging necessitating synthesis of clinical, radiological and pathological findings. The molecular pathogenesis of ossifying fibroma is poorly understood but recent studies have reported MDM2 gene amplification and chromosomal copy number changes in a subset of ossifying fibromas. MDM2 amplification in ossifying fibroma, if true, presents a diagnostic problem because this genetic event, at least among craniofacial fibro-osseous lesions, was previously considered specific for low-grade osteosarcoma. In the present study, we investigated the utility of MDM2 and CDK4 immunohistochemistry, and fluorescence in situ hybridization for MDM2 gene amplification, in the diagnosis of 44 craniofacial bone ossifying fibromas. Focal MDM2 and CDK4 nuclear immunoreactivity was found in 11 and 1 ossifying fibromas, respectively, but none demonstrated MDM2 amplification by fluorescence in situ hybridization. A single tumor displayed MDM2 amplification without nuclear immunoreactivity to either MDM2 or CDK4. Our data suggest that while focal MDM2 and CDK4 nuclear expression may be detected in a minority of ossifying fibromas, this expression does not correlate with MDM2 amplification. In addition, MDM2 amplification is extremely rare in ossifying fibroma so the detection of this genetic abnormality should continue to raise concern for osteosarcoma.

Soft Tissue Special Issue: Gnathic Fibro-Osseous Lesions and Osteosarcoma.

Gnathic fibro-osseous lesions are a diverse group of disease processes which share overlapping microscopic features characterized by fibroblastic stroma with variable cellularity and a range of bone forming pathological processes leading to woven, sclerotic and cementum-like structures. Some of the lesions are unique to craniofacial location and a combination of clinical, radiological and pathological correlation is often necessary for diagnostic accuracy. Gnathic osteosarcomas are rare tumors with differences in age distribution and behavior as compared to osteosarcoma of long bones. This review will discuss the clinicopathological and radiological features of gnathic fibro-osseous lesions and osteosarcoma with updates on current genetics and molecular pathogenesis.

Use of nonsteroidal anti-inflammatory drugs predicts improved patient survival for PIK3CA-altered head and neck cancer.

PIK3CA is the most commonly altered oncogene in head and neck squamous cell carcinoma (HNSCC). We evaluated the impact of nonsteroidal anti-inflammatory drugs (NSAIDs) on survival in a PIK3CA-characterized cohort of 266 HNSCC patients and explored the mechanism in relevant preclinical models including patient-derived xenografts. Among subjects with PIK3CA mutations or amplification, regular NSAID use (≥6 mo) conferred markedly prolonged disease-specific survival (DSS; hazard ratio 0.23, P = 0.0032, 95% CI 0.09-0.62) and overall survival (OS; hazard ratio 0.31, P = 0.0043, 95% CI 0.14-0.69) compared with nonregular NSAID users. For PIK3CA-altered HNSCC, predicted 5-yr DSS was 72% for NSAID users and 25% for nonusers; predicted 5-yr OS was 78% for regular NSAID users and 45% for nonregular users. PIK3CA mutation predicted sensitivity to NSAIDs in preclinical models in association with increased systemic PGE2 production. These findings uncover a biologically plausible rationale to implement NSAID therapy in PIK3CA-altered HNSCC.

Peripheral cannabinoids attenuate carcinoma-induced nociception in mice.

We investigated the cannabinoid receptor (CBr) agonists Win55,212-2 (non-selective) and AM1241 (CBr2 selective) and the peripheral receptor (CBr1) in carcinoma-induced pain using a mouse model. Tumors were induced in the hind paw of female mice by local injection of a human oral squamous cell carcinoma (SCC). Significant pain, as indicated by reduction in withdrawal thresholds in response to mechanical stimulation, began at 4 days after SCC inoculation and lasted to 18 days. Local administration of Win55,212-2 (10 mg/kg) and AM1241 (10 mg/kg) significantly elevated withdrawal thresholds, indicating an antinociceptive effect. Ipsilateral expression of CBr1 protein in L5 DRG was significantly upregulated compared to ipsilateral L4 DRG and in normal tissue. These findings support the suggestion that cannabinoids are capable of producing antinociception in carcinoma-induced pain.

Peripheral endothelin A receptor antagonism attenuates carcinoma-induced pain.

In this study we investigated the role of endothelin-1 (ET-1) and its peripheral receptor (ET-A) in carcinoma-induced pain in a mouse cancer pain model. Tumors were induced in the hind paw of female mice by local injection of cells derived from a human oral squamous cell carcinoma (SCC). Significant pain, as indicated by reduction in withdrawal thresholds in response to mechanical stimulation, began at four days after SCC inoculation and lasted to 28 days, the last day of measurement. Intra-tumor expression of both ET-1 mRNA and ET-1 protein were significantly upregulated compared to normal tissue, and local administration of the ET-A receptor selective antagonist, BQ-123 (100 microM) significantly elevated withdrawal thresholds, indicating the induction of an antinociceptive effect. These findings support the suggestion that ET-1 and ET-A receptors contribute to the severity of carcinoma-induced soft tissue cancer pain.

Elevated salivary endothelin levels in oral cancer patients--a pilot study.

The analysis of saliva has been proposed as a potentially rapid, non-invasive method to monitor and diagnose patients with oral disease. In this study we measured salivary endothelin-1 (ET-1) levels in patients diagnosed with oral squamous cell carcinoma (SCC) prior to treatment. We demonstrate significantly elevated salivary ET-1 levels in the oral SCC group (4.37+/-1.35pg/ml), relative to the control group (1.16+/-0.29pg/ml). ET-1 and ET-1 mRNA were also measured in oral SCC tissue specimens and compared to normal oral epithelial controls. The concentration of ET-1 in the oral SCC specimens was 17.87+/-4.0pg/ml and in the normal epithelial controls the concentration of ET-1 was 5.43+/-2.5pg/ml. ET-1 mRNA was significantly overexpressed in 80% (8/10) of the oral SCC specimens. Our results demonstrate the potential utility of salivary analysis for ET-1 levels to monitor patients at risk for oral SCC.

Racial disparity in stage at diagnosis and survival among adults with oral cancer in the US.

OBJECTIVES: To explore distribution of stage at diagnosis and relative survival rates among US adults with oral cavity cancer in relation to race, and over time. METHODS: We obtained 1973-2002 oral cancer incidence data from the Surveillance, Epidemiology, and End Results (SEER) Program, and computed proportions for each oral cavity site by stage at diagnosis, tumor size, and 5-year relative survival rates among Whites and Blacks. RESULTS: A total of 46 855 cases of oral cavity cancer were reported to the SEER registry among adults > or =20 years between 1973 and 2002. African-Americans had a significantly higher proportion of cancer, mainly in the tongue, that had spread to a regional node or to a distant site at diagnosis than Whites: 67% versus 49% of tongue cancers reported from 1973 to 1987 (P < 0.001), and 70% versus 53% of those reported from 1988 to 2002 (P < 0.001). They had a significantly higher proportion of tongue cancer that were >4 cm in diameter at time of diagnosis (59% versus 44%; P < 0.001), and black men in particular experienced lower 5-year relative survival rates than white men, in particular, for tongue cancer (25% versus 43% from 1973 to 1987, and 31% versus 53% from 1988 to 2002). CONCLUSION: There are significant racial disparities with respect to stage at diagnosis and survival among adults with oral cancer reported to the SEER registry from 1973 to 2002. One possible explanation for the lower survival among Blacks may be a difference in access to, and utilization of, healthcare services.

DNA promoter hypermethylation in saliva for the early diagnosis of oral cancer.

Oral health care professionals could drastically improve the quality of life for patients with potentially malignant oral lesions by using a noninvasive test that could be used to detect cancer using saliva. Promoter DNA hypermethylation is a critical step in oral carcinogenesis and has a number of significant advantages over genetic and protein diagnostic markers. Methylight is a recently developed assay that rapidly quantifies promoter hypermethylation and could potentially be applied into a clinical setting.

Human Papillomavirus Regulates HER3 Expression in Head and Neck Cancer: Implications for Targeted HER3 Therapy in HPV+ Patients.

Purpose: Human papillomavirus (HPV) 16 plays an etiologic role in a growing subset of head and neck squamous cell carcinomas (HNSCC), where viral expression of the E6 and E7 oncoproteins is necessary for tumor growth and maintenance. Although patients with HPV+ tumors have a more favorable prognosis, there are currently no HPV-selective therapies. Recent studies identified differential receptor tyrosine kinase (RTK) profiles in HPV+ versus HPV- tumors. One such RTK, HER3, is overexpressed and interacts with phosphoinositide-3-kinase (PI3K) in HPV+ tumors. Therefore, we investigated the role of HPV oncoproteins in regulating HER3-mediated signaling and determined whether HER3 could be a molecular target in HPV+ HNSCC.Experimental Design: HER3 was investigated as a molecular target in HPV+ HNSCC using established cell lines, patient-derived xenografts (PDX), and human tumor specimens. A mechanistic link between HPV and HER3 was examined by augmenting E6 and E7 expression levels in HNSCC cell lines. The dependency of HPV+ and HPV- HNSCC models on HER3 was evaluated with anti-HER3 siRNAs and the clinical stage anti-HER3 monoclonal antibody KTN3379.Results: HER3 was overexpressed in HPV+ HNSCC, where it was associated with worse overall survival in patients with pharyngeal cancer. Further investigation indicated that E6 and E7 regulated HER3 protein expression and downstream PI3K pathway signaling. Targeting HER3 with siRNAs or KTN3379 significantly inhibited the growth of HPV+ cell lines and PDXs.Conclusions: This study uncovers a direct relationship between HPV infection and HER3 in HNSCC and provides a rationale for the clinical evaluation of targeted HER3 therapy for the treatment of HPV+ patients. Clin Cancer Res; 23(12); 3072-83. ©2016 AACR.

Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma.

Genomes of solid tumors are characterized by gains and losses of regions, which may contribute to tumorigenesis by altering gene expression. Often the aberrations are extensive, encompassing whole chromosome arms, which makes identification of candidate genes in these regions difficult. Here, we focused on narrow regions of gene amplification to facilitate identification of genetic pathways important in oral squamous cell carcinoma (SCC) development. We used array comparative genomic hybridization (array CGH) to define minimum common amplified regions and then used expression analysis to identify candidate driver genes in amplicons that spanned <3 Mb. We found genes involved in integrin signaling (TLN1), survival (YAP1, BIRC2), and adhesion and migration (TLN1, LAMA3, MMP7), as well as members of the hedgehog (GLI2) and notch (JAG1, RBPSUH, FJX1) pathways to be amplified and overexpressed. Deregulation of these and other members of the hedgehog and notch pathways (HHIP, SMO, DLL1, NOTCH4) implicates deregulation of developmental and differentiation pathways, cell fate misspecification, in oral SCC development.

Frequent allelic imbalances at 8p and 11q22 in oral and oropharyngeal epithelial dysplastic lesions.

Allelic imbalance is characteristic of oral squamous cell carcinoma (SCC) and contributes to the tumorigenesis of this disease. Our previous studies suggest that chromosome regions 8p and 11q22.2 approximately q22.3 are frequent sites of loss of heterozygosity (LOH) in head and neck SCC. Here, we explored the allelic imbalance pattern of these regions in 27 cases of oral epithelial dysplastic lesions. A previously reported frequent LOH (9p21) in head and neck dysplasia was also examined. Laser capture microdissection (LCM) technology was utilized to harvest homogenous cell populations from archived clinical tissues and thus greatly enhancing the sensitivity, accuracy and reliability of genetic assessment. The allelic imbalance (LOH and microsatellite instability) on 8p, 11q22.2 approximately q22.3, and 9p21 were observed at one or more loci in 66.7%, 63.0%, and 63.0% of cases, respectively. Our results demonstrate that 8p, 11q22.2 approximately q22.3, and 9p21 are frequent allelic imbalance regions in oral premalignant dysplasia and suggest the presence of tumor suppressor genes in these regions.

Beta-6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms.

Beta-6 Integrin, tenascin-C, and MMP-1 (matrix metalloproteinase-1) are invasion-related proteins that are frequently overexpressed in many human malignancies. The objective of this study was to determine whether there is overexpression of these molecules in three types of salivary neoplasms showing markedly different behavior. A total of 55 formalin-fixed, paraffin-embedded archived specimens comprising 19 adenoid cystic carcinomas (ACC), 18 polymorphous low-grade adenocarcinomas (PLGA) and 18 pleomorphic adenomas (PA) were utilized in this study. A standard immunohistochemical technique was used to determine the expression levels of beta-6 integrin, tenascin-C, and matrix metalloproteinase-1 (MMP-1) proteins. Sections were assessed semiquantitatively, and tumors were divided into two groups, low-expressors (0-1+) and high-expressors (2-3+) for statistical analysis. Staining was graded as 0 (<1% positive tumor cells), 1+ (<25% positive tumor cells), 2+ (25-50% positive tumor cells), and 3+ (>50% positive cells). The results showed that the malignant tumors were higher expressors of beta-6 than the benign tumors. ACCs showed significantly higher expression of beta-6 than PAs (p=0.04). No significant difference was observed between ACCs and PLGAs. beta-6 expression was rarely seen in normal salivary gland epithelium and was occasionally present in mucosa overlying the tumors. PAs were high-expressors of tenascin-C with a significant difference relative to ACCs (p=0.03). A majority of tumors in all three tumor types showed high expression of MMP1 with expression significantly greater in the PAs compared to ACCs (p=0.008). We conclude that ACCs and PLGAs express beta-6, tenascin-C, and MMP-1, but that their expression patterns are not significantly different. beta-6 appears to be more closely associated with the malignant tumors, and MMP-1 more closely associated with the benign tumors. We believe that beta-6, tenascin-C, and MMP-1 proteins are part of the molecular repertoire used by salivary tumors for malignant invasion and benign tumor expansion.

Increased nitric oxide levels and iNOS over-expression in oral squamous cell carcinoma.

Inducible nitric oxide synthase (iNOS) is responsible for generating high levels of nitric oxide (NO) in tissues. Increased iNOS expression has been demonstrated in a number of carcinomas including head and neck squamous cell carcinoma (SCC). However, iNOS levels have not been evaluated specifically in oral cavity SCC, or in the precancerous lesions that progress to oral SCC. Also, NO levels have not been measured in oral precancerous or cancerous tissues. We therefore measured iNOS mRNA, iNOS protein and NO in oral SCC, oral dysplasias and normal oral epithelium. We used RT-PCR to quantify and compare iNOS mRNA levels in these oral tissue specimens. We found that iNOS mRNA was overexpressed in 41% of oral SCC but in only 8% of dysplasia specimens (P = 0.003). Immunohistochemistry was used to evaluate iNOS protein levels in oral SCC, oral dysplasias and normal oral epithelium. A significantly higher percentage of oral SCC specimens showed the highest level of iNOS staining relative to the oral dysplasias and normal oral epithelial samples (95% of oral SCC, 50% of dysplasias, and only 0% of normal epithelial controls, P < 0.0001). The positive staining for iNOS was limited to the SCC cells. Production of NO from iNOS was quantified using HPLC and found to be significantly higher in oral SCC (1.45 +/- 0.56 microg/ml) than normal epithelial controls (0.43 +/- 0.26 microg/ml) (P = 0.0013). We conclude that iNOS mRNA levels and NO production are significantly increased, in oral SCC compared to oral dysplasias and normal epithelial controls. These findings suggest that increased iNOS expression and the generation of high NO levels might have a role in oral SCC development.