Comparison of whole-body PET/CT, dedicated high-resolution head and neck PET/CT, and contrast-enhanced CT in preoperative staging of clinically M0 squamous cell carcinoma of the head and neck.

UNLABELLED: The purpose of this study was to compare optimized whole-body (WB) and dedicated high-resolution contrast-enhanced PET/CT protocols and contrast enhanced CT in the preoperative staging of primary squamous cell carcinoma of the head and neck. METHODS: A total of 44 patients with clinically M0 squamous cell carcinoma of the head and neck underwent primary tumor resection and neck dissection within 6 wk of diagnostic imaging. Imaging consisted of a standard WB PET/CT protocol without intravenous contrast enhancement, followed by a high-resolution dedicated head and neck (HN) PET/CT protocol, which included diagnostic-quality contrast-enhanced CT (CECT). Imaging results were compared with histopathology. A 5-point scale was used to designate primary tumor localization and the presence of lymph node metastasis on a per-patient and per-level basis. For cervical nodes, receiver-operating-characteristic curves were generated to determine the differences in performance between the WB and HN PET/CT protocols and CECT. Sensitivity, specificity, positive and negative predictive values, and accuracy were calculated for primary tumor and cervical nodes. RESULTS: No statistical difference was observed between WB and HN PET/CT protocols, both of which significantly outperformed CECT, in the evaluation of the primary tumor. The performance of the HN PET/CT protocol was superior to that of the WB PET/CT in the detection of cervical node metastases, achieving statistical significance on a per-level basis and approaching significance on a per-patient basis, with the greatest advantage in the detection of small positive lymph nodes (<15 mm). No significant difference was observed between the WB PET/CT protocol and CECT in nodal staging, either on a per-patient or on a per-level basis. CONCLUSION: The primary advantage of the dedicated HN PET/CT protocol over the WB protocol or CECT in the staging of head and neck cancer is in the detection of small lymph node metastases.

B-RAF V600E mutational analysis of fine needle aspirates correlates with diagnosis of thyroid nodules.

OBJECTIVE: A mutation of B-type RAF kinase (B-RAF) represents the most common genetic alteration in papillary thyroid cancer (PTC), possibly signifying a more aggressive biology. Fine needle aspiration (FNA) represents the most useful initial diagnostic tool of thyroid nodules. Molecular analysis of the mutation status of B-RAF in thyroid nodule FNAs may provide guidance for treatment planning. STUDY DESIGN: Cross-sectional study. SUBJECTS AND METHODS: A retrospective chart review was undertaken for clinically relevant data of papillary thyroid cancer (PTC), follicular variant of PTC (FV-PTC), and nonmalignant goiters. After blinded pathologic review, histologic and cytologic samples were analyzed by LightCycler PCR (LCPCR) with allele-specific fluorescent probe melting curve analysis (FMCA) for the V600E mutation of B-RAF. RESULTS: Of the 45 patient samples analyzed, B-RAF mutation was found to be significantly higher in papillary carcinomas when compared to follicular variant of papillary thyroid carcinomas (55.6% vs 14.3%, P = 0.05). Pathologic B-RAF mutational status significantly correlated with cytologic B-RAF mutational status (P < 0.0001), cytologic interpretation (P = 0.012), and histologic diagnosis (P = 0.011). CONCLUSIONS: Determination of B-RAF V600E mutation of thyroid nodule FNAs by LCPCR may be a useful tool to guide treatment planning. These data support investigating the utility of this molecular marker in a prospective manner.

The kinetics and redox state of nitric oxide determine the biological consequences in lung adenocarcinoma.

Few studies have explored the mechanistic basis for the apparent paradoxical effects of nitric oxide and its interrelated redox species (NO(X)) in cancer biology. Our aim was to determine the differential effects of the redox state and kinetics of nitrosative species on the key cancer processes of apoptosis. Therefore, a murine lung adenocarcinoma cell line was exposed to various NO(X) donor compounds differing in redox state and delivery kinetics. DNA strand breaks (DSBs) were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay) and correlated with cell viability by the MTT and soft agar colony assays, while caspase enzymatic activity was measured using an in vitro fluorogenic caspase assay. Finally, cDNA microarrays defined apoptosis-related gene expression alterations resultant from these NO(X) donors. Exogenous NO(X) differentially influences DSBs, and apoptosis-related cell death and expression based on the redox state and kinetics of NO(X) delivery. In our murine lung adenocarcinoma model we have demonstrated differential effects of NO(X) based on the mode of delivery and redox state. These data suggest that the development of NO(X)-based cancer chemotherapy must consider the redox state and kinetics of delivery into their logical design.

Planned neck dissection after definitive radiotherapy or chemoradiation for base of tongue cancers.

OBJECTIVES: The study goal was to analyze the role of planned neck dissection for squamous cell carcinoma of the base of the tongue treated with definitive radiotherapy or chemoradiation. STUDY DESIGN, SETTING: We conducted a retrospective study of patients with squamous cell carcinoma of the base of the tongue undergoing planned neck dissection after definitive radiotherapy or chemoradiation. RESULTS: Twenty-two of 41 (53.7%) patients had one to six positive residual lymph nodes after receiving definitive radiotherapy or chemoradiation. Neck control rates were 92.3% and 88.3% at two and five years, respectively. Three of 22 (13.6%) patients with pathological residual nodal disease had regional or locoregional failures, compared with 1 of 19 (5.3%) patients with a pathologically complete response (P = 0.39). CONCLUSIONS: We observed a high incidence of pathologically residual lymph nodes after definitive radiotherapy or chemoradiation. SIGNIFICANCE: Planned neck dissection following definitive radiotherapy or chemoradiation is highly effective in achieving regional control of squamous cell carcinoma of the base of the tongue.

Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules.

BACKGROUND: Fine needle aspiration (FNA) is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC). In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC. METHODS: Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE) surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 mum FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR). Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing. RESULTS: A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8%) FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4%) FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells. CONCLUSION: When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.

The magnanimous professional life and tragic demise of J. H. Douglas, MD.

OBJECTIVES/HYPOTHESIS: Many of the hallmarks of a very successful medical career, such as recognition as a leader in a field of medicine, making important contributions to medical knowledge of the day, and a steadfast dedication to patient care, had already been achieved by Dr. John Hancock Douglas. Therefore, the mystery surrounding his dismissal from membership of the newly formed American Laryngology Society (now the ALA) and his tragic demise stand in stark contrast. We discuss the model professional life and mysterious but tragic final days of this very important laryngologist of the 19th century. STUDY DESIGN/METHODS: A historical vignette. RESULTS: Dr. Douglas's professional qualities of leadership of the American Sanitary Commission, his various contributions to the advancement of medical knowledge during that era, and his steadfast dedication to the care of his patients represent highlights of a very honorable professional career. His final demise, bankrupt, in an ill state of health, and stripped of his professional appointment to the ALA, seems an unjust end to the life of this notable and magnanimous laryngologist. CONCLUSIONS: We offer this historical review of the life and demise of Dr. John H. Douglas as a tribute to this important figure in our profession's history.

Nitrosative stress induces DNA strand breaks but not caspase mediated apoptosis in a lung cancer cell line.

BACKGROUND: Key steps crucial to the process of tumor progression are genomic instability and escape from apoptosis. Nitric oxide and its interrelated reactive intermediates (collectively denoted as NOX) have been implicated in DNA damage and mutational events leading to cancer development, while also being implicated in the inhibition of apoptosis through S-nitrosation of key apoptotic enzymes. The purpose of this study was to explore the interrelationship between NOX-mediated DNA strand breaks (DSBs) and apoptosis in cultured tumor cell lines. METHODS: Two well-characterized cell lines were exposed to increasing concentrations of exogenous NOX via donor compounds. Production of NOX was quantified by the Greiss reaction and spectrophotometery, and confirmed by nitrotyrosine immunostaining. DSBs were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay), and correlated with cell viability by the MTT assay. Apoptosis was analyzed both by TUNEL staining and Annexin V/propidium iodine FACS. Finally, caspase enzymatic activity was measured using an in-vitro fluorogenic caspase assay. RESULTS: Increases in DNA strand breaks in our tumor cells, but not in control fibroblasts, correlated with the concentration as well as rate of release of exogenously administered NOX. This increase in DSBs did not correlate with an increase in cell death or apoptosis in our tumor cell line. Finally, this lack of apoptosis was found to correlate with inhibition of caspase activity upon exposure to thiol- but not NONOate-based NOX donor compounds. CONCLUSIONS: Genotoxicity appears to be highly interrelated with both the concentration and kinetic delivery of NOX. Moreover, alterations in cell apoptosis can be seen as a consequence of the explicit mechanisms of NOX delivery. These findings lend credence to the hypothesis that NOX may play an important role in tumor progression, and underscores potential pitfalls which should be considered when developing NOX-based chemotherapeutic agents.

Uncommon presentations of parathyroid adenoma.

BACKGROUND: Patients with parathyroid adenomas (PAs) are commonly encountered in otolaryngology and may present with asymptomatic hypercalcemia. Retropharyngeal hematoma and recurrent laryngeal nerve (RLN) paralysis are less commonly encountered presentations and may be harbingers of a malignant process. METHODS AND RESULTS: We present 2 patients with rare presentations of parathyroid adenoma. The first is a 57-year-old man with retropharyngeal hemorrhage and progressive airway compromise. The second is a 51-year-old woman presenting with dysphonia who was found to have RLN paralysis. Both of these patients were found to have benign disease. CONCLUSIONS: Although most patients with PA present with asymptomatic hypercalcemia, this disease entity must be considered in patients with other unusual presentations including hemorrhagic neck masses and dysphonia. The importance of a broad differential diagnosis and thorough workup is emphasized.

IMRT with simultaneous integrated boost and concurrent chemotherapy for locoregionally advanced squamous cell carcinoma of the head and neck.

PURPOSE: To evaluate the efficacy and toxicity of accelerated radiotherapy with concurrent chemotherapy in advanced head-and-neck squamous cell carcinoma. METHODS AND MATERIALS: Between April 2003 and May 2008, 43 consecutive patients with advanced head-and-neck squamous cell carcinoma received accelerated chemoradiation with concurrent cisplatin or cetuximab. The doses for intensity-modulated radiotherapy with simultaneous integrated boost were 67.5, 60.0, and 54 Gy in 30 daily fractions of 2.25, 2.0, and 1.8 Gy to the planning target volumes for gross disease, high-risk nodes, and low-risk nodes, respectively. RESULTS: Of the patients, 90.7% completed chemoradiotherapy as prescribed. The median treatment duration was 43 days (range, 38-55 days). The complete response rate was 74.4%. With median follow-up of 36.7 months (range, 16.8-78.1 months) in living patients, the estimated 1-, 2-, and 5-year locoregional control, overall survival, and disease-free survival rates were 82%, 82%, and 82%; 73%, 65%, and 61%; and 73%, 73%, and 70%, respectively. One treatment-related death occurred from renal failure. Grade 3 mucositis and dermatitis occurred in 13 patients (30.2%) and 3 patients (6.9%), respectively. Grade 2 xerostomia occurred in 12 patients (27.9%). In patients with adequate follow-up, 82% were feeding tube free by 6 months after therapy; 13% remained feeding tube dependent at 1 year. Grade 3 soft-tissue fibrosis, esophageal stricture, osteoradionecrosis, and trismus occurred in 3 patients (6.9%), 5 patients (11.6%), 1 patient (2.3%), and 3 patients (6.9%), respectively. CONCLUSIONS: Our results show that intensity-modulated radiotherapy with simultaneous integrated boost with concurrent chemotherapy improved local and regional control. Acute and late toxicities were tolerable and acceptable. A prospective trial of this fractionation regimen is necessary for further assessment of its efficacy and toxicity compared with other approaches.

Fiberoptic resonance Raman spectroscopy to measure carotenoid oxidative breakdown in live tissues.

Based on compelling epidemiologic and corroboratory in vitro studies, carotenoids are thought to have great potential as dietary prevention against cancer. Yet, carotenoid-based chemopreventive trials have found very contradictory results. Definitive conclusions from these trials are hampered by an inability to accurately and safely measure carotenoids in specific tissues at risk of cancer development. Raman spectroscopy has been proposed as an optical technology with which to analyze various molecules in live tissues. One major obstacle that impedes the clinical use of this powerful technology is the lack of a fiberoptic Raman probe suitable for endoscopic tissue evaluation. A single-fiber resonance Raman Spectroscope capable of noninvasive "optical biopsies" to measure carotenoid concentrations in live tissues has been developed. The accuracy of this Raman instrument was confirmed by comparison with more standard methods of spectrophotometry and high-pressure liquid chromatography using solubilized beta-carotene (BC) and BC-loaded cells before use in a small patient cohort. This Raman instrument detected intact BC as well as BC oxidative breakdown as a decrement of its Raman signal in cells. Use of the Raman instrument in our small cohort study showed its feasibility for measuring human tissues and raised some potentially intriguing possibilities about BC tissue pharmacokinetics and oxidative biology. Based on these results, our newly developed single fiberoptic resonance Raman instrument may provide a very useful method of measuring carotenoids and their oxidative breakdown within live tissue during future carotenoid chemopreventive trials. This proof-of-concept study provides the foundation to justify future validation of our Raman prototype.

Relative contributions of radiation and cisplatin-based chemotherapy to sensorineural hearing loss in head-and-neck cancer patients.

PURPOSE: To investigate the risk of sensorineural hearing loss (SNHL) in patients with head-and-neck cancer and treated with radiation therapy (RT) or concomitant cisplatin-based chemoradiation, the relationship among SNHL and radiation dose to the cochlea, the use of two common cisplatin dose regimens. METHODS AND MATERIALS: A total of 62 head-and-neck cancer patients treated with curative intent were included in this prospective study. Of the patients, 21 received RT alone, 27 received 40 mg/m(2) weekly cisplatin, 13 received 100 mg/m(2) every 3 weeks during RT, and 1 received RT with weekly epidermal growth factor receptor inhibitor antibody. The effect of chemotherapy and RT dose on hearing was determined using a model that accounted for the age and variability between each ear for each patient. RESULTS: We constructed a model to predict dose-dependent hearing loss for RT or cisplatin-based chemotherapy either alone or in combination. For patients only receiving RT, no significant hearing loss was found at doses to the cochlea of less than 40 Gy. Patients receiving 100 mg/m(2) or 40 mg/m(2) of cisplatin chemotherapy had an estimated +21.5 dB and +9.5 dB hearing loss at 8,000 Hz with low radiation doses (10 Gy), which rose to +38.4 dB and +18.9 dB for high radiation doses (40 Gy). CONCLUSIONS: Use of RT alone with doses of less than 40 Gy did not result in clinically significant hearing loss. High-frequency SNHL was profoundly damaged in patients who received concomitant cisplatin when doses of 100 mg/m(2) were used. The threshold cochlear dose for hearing loss with cisplatin-based chemotherapy and RT was predicted to be 10 Gy. The inner ear radiation dose constraints and cisplatin dose intensity should be considered in the treatment of advanced head-and-neck cancer.

SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma.

Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given the well-established role of mitochondrial defects in human disease, functional characterization of these proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex, a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide cofactor). Germline loss-of-function mutations in the human SDH5 gene, located on chromosome 11q13.1, segregate with disease in a family with hereditary paraganglioma, a neuroendocrine tumor previously linked to mutations in genes encoding SDH subunits. Thus, a mitochondrial proteomics analysis in yeast has led to the discovery of a human tumor susceptibility gene.

Parosteal osteosarcoma: case report and review of the literature.

BACKGROUND: The majority of osteosarcoma cases of the head and neck are high-grade lesions. We present a case and discuss the diagnostic and therapeutic implications of a rare low-grade parosteal osteosarcoma of the maxilla. METHODS: A 32-year-old man presenting to the Head and Neck Surgical Oncology clinic with a 1-year history of a firm palatal mass. Evaluation clinically and radiographically raised the suspicion of an osteosarcoma. RESULTS: A partial maxillectomy revealed a parosteal osteosarcoma with negative margins. No adjuvant therapy was recommended, and the patient remains without evidence of local recurrence after 3 years. CONCLUSIONS: Parosteal osteosarcomas of the head and neck region are rare, low-grade variants of osteosarcoma, but have the potential to recur with simple local excision. Clinical and radiographic features are diagnostically helpful. Definitive diagnosis comes from histopathology, and wide local resection should be employed as the optimal treatment.

Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis.

BACKGROUND: A single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence of 29-83%. AIMS: To use a PCR assay to molecularly characterise the BRAF activating point mutation in a series of PTC and benign thyroid cases and correlate the mutation results with histological findings. METHODS: Formalin-fixed paraffin-embedded (FFPE) sections were evaluated for the BRAF V600E mutation using LightCycler PCR with allele-specific fluorescent probe melting curve analysis (LCPCR). RESULTS: 42 (37 PTC; 5 benign) surgical tissue samples were analysed for the BRAF V600E activating point mutation. Using LCPCR and direct DNA sequencing, the BRAF mutation was identified in 23/37 (62.2%) PTC FFPE samples. DNA sequencing results demonstrated confirmation of the mutation. CONCLUSIONS: Detection of BRAF-activating mutations in PTC suggests new approaches to management and treatment of this disease that may prove worthwhile. Identification of the BRAF V600E activating mutation in routine FFPE pathology samples by a rapid laboratory method such as LCPCR could have significant value.

Treatment of oropharyngeal squamous cell carcinoma with external beam radiation combined with interstitial brachytherapy.

BACKGROUND: We reviewed the outcomes of oropharyngeal squamous cell carcinoma treated with external beam radiation and interstitial brachytherapy. METHODS: Ninety patients with squamous cell carcinoma of the oropharynx were treated with interstitial brachytherapy at the University of Utah between 1984 and 2001. Seventy-two patients received external beam radiotherapy (EBRT) followed by brachytherapy boost, 11 had surgery followed by EBRT and brachytherapy, 4 had surgery and brachytherapy, and 3 were treated with brachytherapy alone. Median doses for EBRT and brachytherapy were 50 and 24 Gy, respectively. RESULTS: Median follow-up after brachytherapy was 48.3 months for all patients. Five-year local control, disease-free survival, and overall survival were 76%, 61%, and 55%. For T1, T2, T3, and T4, 5-year local control rates were 83%, 79%, 79%, and 64%, respectively. Severe complications occurred in 13 patients, including 2 treatment-related deaths. CONCLUSIONS: EBRT combined with interstitial brachytherapy provide good local control rates for locally advanced oropharyngeal squamous cell carcinoma.

Black thyroid resulting from short-term doxycycline use: case report, review of the literature, and discussion of implications.

BACKGROUND: Black thyroid pigmentation has been considered pathognomonic for chronic minocycline ingestion for more than 30 years. Although never conclusively linked to overt disease, evidence clearly exists that minocycline is a competitive inhibitor of thyroid peroxidase in metabolically active thyroid tissue. This offers a potential mechanism of pigment accumulation, which can account for the occasional finding of hypopigmentation in thyroid carcinomas. To our knowledge, an association with tetracycline derivatives other than minocycline has not been documented. METHODS: Herein is a case report of a patient with gross black thyroid tissue containing a hypopigmented papillary thyroid carcinoma. Twelve days before surgery, the patient was placed on doxycycline, a tetracycline derivative, to optimize an in vitro fertilization regimen. RESULTS: The gross specimen was diffusely black in color with a 1.5-cm hypopigmented focus of papillary thyroid cancer. Hematoxylin-eosin staining, as well as electron micrographs, was consistent with findings associated with minocycline ingestion. CONCLUSIONS: Black thyroid is rare and only previously associated with chronic minocycline ingestion. This report documents a black thyroid in a patient after short-term exposure to doxycycline. Pigment accumulation in normal tissue is thought to occur by inhibition of thyroid peroxidase. Minocycline is a competitive inhibitor of this enzyme in its native configuration. Thyroid carcinomas are known to have abnormal thyroid peroxidase, which could account for reports of hypopigmented tumors within grossly darkened thyroid tissue. Hypopigmented foci within such "black thyroid" deserve through pathologic examination.

Head and neck soft tissue sarcomas: a multivariate analysis of outcomes.

BACKGROUND: Soft tissue sarcomas of the head and neck region represent a rare group of tumors of which a limited number of published individual- and institution-based experiences exist. METHODS: We performed an analysis of head and neck sarcoma patients identified from our institution between 1973 and 1999. Exclusion criteria included pediatric rhabdomyosarcomas, sarcomas of the neuromeningeal axis or non-head and neck primary disease sites, and bone sarcomas. All cases underwent pathologic re-review before statistical analysis. RESULTS: After pathologic review, 111 head and neck sarcoma patients remained (mean age, 47 +/- 20 years). The median duration of follow-up was 51 months; the actuarial 5-year relapse-free, disease-specific, and overall survivals were 55%, 52%, and 44%, respectively. Forty-six percent remained free of recurrence at the most recent follow-up, and the most common site of recurrence was local followed by distant sites. By multivariate analysis, size and grade significantly influenced relapse-free, disease-specific, and overall survivals, whereas margin status additionally influenced relapse-free survival. Subset analysis of the fibrosarcoma/malignant fibrous histiocytoma and desmoid/dermatofibrosarcoma protuberans histologies was undertaken. CONCLUSIONS: Size >5 cm and high-grade histology are considered poor prognostic indicators. Patients with either of these characteristics should be considered for adjuvant trials.

Anterior skull base surgery for malignant tumors: a multivariate analysis of 27 years of experience.

INTRODUCTION: Few studies have examined prognostic factors that have an impact on outcomes in anterior skull base surgery by multivariate analysis. METHODS: We retrospectively examined our institution's skull base experience from 1973-2000. RESULTS: During this time, 166 patients underwent an anterior skull base resection for malignancy (median age, 53 years; range, 6-92 years). The 5-year relapse-free and disease-specific survival was 41% and 57% (median follow-up, 53 months). Multivariate analysis found that dural invasion, primary histologic diagnosis, and margin status had a significant impact on relapse-free and disease-specific survival. CONCLUSIONS: These data indicate that patients with anterior skull base malignancies are treated successfully with skull base surgery. Patients demonstrating adverse prognostic variables such as dural invasion, adverse histologic findings, and/or positive margins should be considered for the addition of adjuvant therapy or innovative therapies as they become available in the future.

Nitric oxide and apoptosis during human head and neck squamous cell carcinoma development.

PURPOSE: Apoptosis index (AI), Bcl-2, and Bax have shown prognostic significance in head and neck squamous cell carcinoma (HNSCCa). Other areas of research have implicated nitric oxide (NO) or its various intermediate species in both proapoptotic and antiapoptotic processes. We have previously shown that NO-generating enzymes are significantly increased during the stepwise progression to HNSCCa. The aim of this study was to explore the interrelationship of NO and a known consequence of NO-related oxidative stress, apoptosis, during this step-wise process. MATERIALS AND METHODS: Formalin fixed-paraffin embedded tissue samples of 10 normal oral mucosa, 15 reactive/dysplastic lesions, and 17 HNSCCa lesions studied previously were subjected to the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP labeling (TUNEL) assay as well as immunohistochemical staining against Bcl-2, Bax, and p53. Patient charts were reviewed and clinical data were compared. The study pathologist (G.K.H) reviewed these slides blinded to patient identifiers or clinical data. The number of immunopositive cell nuclei or staining intensity was graded, noting the pattern of immunostaining. These staining characteristics were compared with the results of immunostaining previously obtained for endothelial constitutive NO synthase (ecNOS) and nitrotyrosine. RESULTS: Compared with normal oral mucosa, the AI, Bcl-2, Bax, Bcl-2/Bax intensity and frequency ratios, and mutant p53 intensity significantly changed in reactive/dysplastic and HNSCCa lesions (P <.001 for all). Correlations between the staining characteristics of the antigens studied are presented. Furthermore, perilesional inflammatory cells showed staining in the TUNEL assay. CONCLUSIONS: In a set of tissue samples previously well characterized, these new findings implicate a link between NO and the induction of apoptotic cell death in HNSCCa development.