Neighborhood socioeconomic status and race are associated with incidence disparities in oral cavity cancers.

OBJECTIVES: To determine the association between neighborhood socioeconomic status (nSES), race and incidence rate trends of oral cavity cancer (OCC). MATERIALS AND METHODS: We used data from the SEER (Surveillance, Epidemiology, and End Results) 18 Census Tract-level SES and Rurality Database (2006-2018) database of the National Cancer Institute to create cohorts of OCC patients between 2006 and 2018. Annual incidence rates were calculated and trends in rates were estimated using joinpoints regression. RESULTS: The incidence of OCC is the highest among low nSES White Americans (2.86 per 100 000 persons) and the lowest among high nSES Black Americans (1.17 per 100 000 persons). Incidence has significantly increased among Asian Americans (annual percent change [APC]: low nSES-2.4, high nSES-2.6) and White Americans (APC: low nSES-1.4, high nSES-1.6). Significant increases in the incidence of oral tongue cancer in these groups primarily drive this increase. Other increases were noted in alveolar ridge cancer among White Americans and hard palate cancer among Asian Americans. OCC incidence decreased significantly in Hispanic Americans of high nSES (APC: -2.5) and Black Americans of low nSES (APC: -2.7). Floor of mouth cancer incidence decreased among most groups. CONCLUSION: Despite the overall decreasing incidence of OCC, these trends are inconsistent among all OCC subsites. Differences are seen by race, nSES, and subsite, indicating intersectional barriers that extend beyond nSES and race and ethnicity alone. Further research on risk factors and developing interventions targeting vulnerable groups is needed.

TOETVA parathyroid autofluorescence detection: hANDY-i endoscopy attachment.

Background: Treatment options for thyroid pathologies have expanded to include scarless and remote access methods such as the transoral endoscopic thyroidectomy vestibular approach (TOETVA). Currently, no standardized methods exist for locating parathyroid glands (PGs) in patients undergoing TOETVA, which can lead to parathyroid injury and subsequent hypocalcemia. This early feasibility study describes and evaluates the hANDY-i endoscopic attachment for detecting PGs in transoral thyroidectomy. Methods: We used a prototype parathyroid autofluorescence imager (hANDY-i) that was mounted to a 10-mm 0-degree endoscope. The device delivers a split screen view of Red-green-blue (RGB) and near-infrared autofluorescence (NIRAF) which allows for simultaneous anatomical localization and fluorescence visualization of PGs during endoscopic thyroid dissection. Results: One cadaveric case and two patient cases were included in this study. The endoscopic hANDY-i imaging system successfully visualized PGs during all procedures. Conclusion: The ability to leverage parathyroid autofluorescence during TOETVA may lead to improved PG localization and preservation. Further human studies are needed to assess its effect on postoperative hypocalcemia and hypoparathyroidism.

Polyamine transport inhibition and cisplatin synergistically enhance tumor control through oxidative stress in murine head and neck cancer models.

Background: Surgery and/or platinum-based chemoradiation remain standard of care for patients with head and neck squamous cell carcinoma (HNSCC). While these therapies are effective in a subset of patients, a substantial proportion experience recurrence or treatment resistance. As cisplatin mediates cytotoxicity through oxidative stress while polyamines play a role in redox regulation, we posited that combining cisplatin with polyamine transport inhibitor, AMXT-1501, would increase oxidative stress and tumor cell death in HNSCC cells. Methods: Cell proliferation was measured in syngeneic mouse HNSCC cell lines treated with cisplatin ± AMXT-1501. Synergy was determined by administering cisplatin and AMXT-1501 at a ratio of 1:10 to cancer cells in vitro . Cancer cells were transferred onto mouse flanks to test the efficacy of treatments in vivo . Reactive oxygen species (ROS) were measured. Cellular apoptosis was measured with flow cytometry using Annexin V/PI staining. High-performance liquid chromatography (HPLC) was used to quantify polyamines in cell lines. Cell viability and ROS were measured in the presence of exogenous cationic amino acids. Results: The combination of cisplatin and AMXT-1501 synergize in vitro on HNSCC cell lines. In vivo combination treatment resulted in tumor growth inhibition greater than either treatment individually. The combination treatment increased ROS production and induced apoptotic cell death. HPLC revealed the synergistic mechanism was independent of intracellular polyamine levels. Supplementation of cationic amino acids partially rescued cancer cell viability and reduced ROS. Conclusion: AMXT-1501 enhances the cytotoxic effects of cisplatin in vitro and in vivo in aggressive HNSCC cell lines through a polyamine-independent mechanism.

Interrogation of T Cell-Enriched Tumors Reveals Prognostic and Immunotherapeutic Implications of Polyamine Metabolism.

Metabolic features of the tumor microenvironment (TME) antagonize anti-tumor immunity. We hypothesized that T cell infiltrated tumors with a known antigen should exhibit superior clinical outcomes, though some fare worse given unfavorable metabolic features leveraging T cell-infiltrated (Thi), human papillomavirus-related (HPV+) head and neck squamous cell carcinomas (HNSC) to test this hypothesis. Expression of 2,520 metabolic genes were analyzed among Thi HPV+ HNSCs stratified by high-risk molecular subtype. RNAseq data from The Cancer Genome Atlas (TCGA; 10 cancer types), single cell RNAseq data, and an immunotherapy-treated melanoma cohort were used to test the association between metabolic gene expression and clinical outcomes and contribution of tumor versus stromal cells to metabolic gene expression. Polyamine (PA) metabolism genes were overexpressed in high-risk, Thi HPV+ HNSCs. Genes involved in PA biosynthesis and transport were associated with T cell infiltration, recurrent or persistent cancer, overall survival status, primary site, molecular subtype, and MYC genomic alterations. PA biogenesis gene sets were associated with tumor intrinsic features while myeloid cells in HPV+ HNSCs were enriched in PA catabolism, regulatory, transport, putrescine, and spermidine gene set expression. PA gene set expression also correlated with IFNγ or cytotoxic T cell ssGSEA scores across TCGA tumor types. PA transport ssGSEA scores were associated with poor survival whereas putrescine ssGSEA scores portended better survival for several tumor types. Thi melanomas enriched in PA synthesis or combined gene set expression exhibited worse anti-PD-1 responses. These data address hurdles to anti-tumor immunity warranting further investigation of divergent polyamine metabolism in the TME.

Recurrent Human Papillomavirus-Related Head and Neck Cancer Undergoes Metabolic Reprogramming and Is Driven by Oxidative Phosphorylation.

PURPOSE: Human papillomavirus (HPV) infection drives the development of some head and neck squamous cell carcinomas (HNSCC). This disease is rapidly increasing in incidence worldwide. Although these tumors are sensitive to treatment, approximately 10% of patients fail therapy. However, the mechanisms that underlie treatment failure remain unclear. EXPERIMENTAL DESIGN: We performed RNA sequencing (RNA-seq) on tissues from matched primary- (pHNSCC) and metachronous-recurrent cancers (rHNSCC) to identify transcriptional differences to gain mechanistic insight into the evolutionary adaptations of metachronous-recurrent tumors. We used HPV-related HNSCC cells lines to investigate the effect of (i) NRF2 overexpression on growth in vitro and in vivo, (ii) oxidative phosphorylation (OXPHOS) inhibition using IACS-010759 on NRF2-dependent cells, and (iii) combination of cisplatin and OXPHOS inhibition. RESULTS: The OXPHOS pathway is enriched in recurrent HPV-associated HNSCC and may contribute to treatment failure. NRF2-enriched HNSCC samples from The Cancer Genome Atlas (TCGA) with enrichment in OXPHOS, fatty-acid metabolism, Myc, Mtor, reactive oxygen species (ROS), and glycolytic signaling networks exhibited worse survival. HPV-positive HNSCC cells demonstrated sensitivity to the OXPHOS inhibitor, in a NRF2-dependent manner. Further, using murine xenograft models, we identified NRF2 as a driver of tumor growth. Mechanistically, NRF2 drives ROS and mitochondrial respiration, and NRF2 is a critical regulator of redox homeostasis that can be crippled by disruption of OXPHOS. NRF2 also mediated cisplatin sensitivity in endogenously overexpressing primary HPV-related HNSCC cells. CONCLUSIONS: These results unveil a paradigm-shifting translational target harnessing NRF2-mediated metabolic reprogramming in HPV-related HNSCC.

Neutron Therapy for High-Grade Salivary Carcinomas in the Adjuvant and Primary Treatment Setting.

OBJECTIVES/HYPOTHESIS: Our primary objective was to compare differences in survival of patients with high-grade salivary gland carcinomas (SGCs) receiving adjuvant neutron versus photon radiotherapy using a hospital-based national cohort and restricted mean survival time (RMST) analysis. Our secondary objective was to compare survival of similar patients treated with primary neutron versus photon radiation. STUDY DESIGN: Multicenter, retrospective population-based study of patients within the National Cancer Database from 2004 to 2014. METHODS: One thousand eight hundred forty-four patients were selected on diagnosis of high-grade parotid and submandibular malignancies. One thousand seven hundred seventy-seven patients receiving photon and 67 patients receiving neutron therapy were identified who met inclusion criteria. Patients were then categorized as having primary surgery with adjuvant radiation or primary radiation without prior surgery. Bivariate analysis was performed to assess for differences between groups, and RMST analysis was performed at 1-, 2-, and 5-year timepoints with controlling for available covariate data. RESULTS: There was no significant difference in RMST for patients receiving neutrons over photons at 1, 2, and 5 years in the adjuvant setting. Among patients undergoing primary radiotherapy, there was a difference in RMST of 2.29 months at 1 year and 5.05 months at 2 years for neutrons over photons, though this benefit was not observed at 5 years post-therapy. CONCLUSIONS: For patients with high grade SGCs undergoing adjuvant photon versus neutron radiotherapy, there was no difference in RMST. There was observed to be a significant difference in RMST at 1 and 2 years among patients undergoing primary neutron therapy of up to 5 months. Given the benefit observed with primary neutron therapy, it should be considered in both the primary and adjuvant treatment setting. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:541-547, 2021.

The role of elective neck dissection in high-grade parotid malignancy: A hospital-based cohort study.

OBJECTIVES/HYPOTHESIS: The role of elective neck dissection (END) in patients with clinically N0 (cN0), high-grade parotid carcinoma is unclear. The objective of this study was to assess the association between END and survival in patients with cN0, high-grade parotid carcinoma. STUDY DESIGN: Retrospective, multicenter cohort study. METHODS: A review of hospital-based cases from the National Cancer Data Base was performed. Participants included patients diagnosed with cN0, high-grade parotid cancer between January 1, 2004 and December 31, 2013. The primary exposure was receipt of neck dissection. Secondary exposures included receipt of adjuvant radiation and/or chemotherapy. Univariate and multivariate survival analyses were performed. Unadjusted and adjusted survival estimates were determined. RESULTS: Overall, 1,547 patients were included, with a median follow-up time of 48 months. END did not have a statistically significant effect on 3-year survival (3-year: 69.9%, 95% confidence interval [CI]: 67.2 to 72.6). Survival at 3-years among those not receiving END was 66.1% (95% CI: 62.7 to 69.5). Parotidectomy and adjuvant radiotherapy had the strongest effect on mortality. There was no difference in 3-year survival among patients who underwent parotidectomy and adjuvant radiation stratified by receipt of END nor did END have a statistically significant effect on survival in mucoepidermoid carcinoma, adenocarcinoma, high-risk histology, high T stage, or academic center treatment subgroups. CONCLUSIONS: END did not have a statistically significant effect on survival among cN0 patients with high-grade parotid cancer when taking into account receipt of adjuvant therapy and confounding. The role of END on survival and locoregional control remains to be further elucidated in prospective studies. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:1487-1495, 2020.

The mutational landscape of recurrent versus nonrecurrent human papillomavirus-related oropharyngeal cancer.

BACKGROUND: Human papillomavirus-related (HPV-related) oropharyngeal squamous cell carcinomas (OPSCCs) have an excellent response rate to platinum-based chemoradiotherapy. Genomic differences between primary HPV-related OPSCCs that do or do not recur are unknown. Furthermore, it is unclear if HPV-related OPSCCs that recur share a genomic landscape with HPV-negative head and neck cancers (HNCs). METHODS: We utilized whole exome sequencing to analyze somatic nucleotide (SNVs) and copy number variants (CNVs) among a unique set of 51 primary HPV-related OPSCCs, including 35 that did not recur and 16 that recurred. We evaluated 12 metachronous recurrent OPSCCs (7 with paired primary OPSCCs) and 33 primary HPV-unrelated oral cavity and OPSCCs. RESULTS: KMT2D was the most frequently mutated gene among primary HPV-related OPSCCs (n = 51; 14%) and among metachronous recurrent OPSCCs (n = 12; 42%). Primary HPV-related OPSCCs that recurred shared a genomic landscape with primary HPV-related OPSCCs that did not recur. However, TSC2, BRIP1, NBN, and NFE2L2 mutations occurred in primary OPSCCs that recurred but not in those that did not recur. Moreover, primary HPV-related OPSCCs that recur harbor features of HPV-unrelated HNCs, notably including MAPK, JAK/STAT, and differentiation signaling pathway aberrations. Metachronous recurrent OPSCCs shared a genomic landscape with HPV-unrelated HNCs, including a high frequency of TP53, CASP8, FAT1, HLA-A, AJUBA, and NSD1 genomic alterations. CONCLUSION: Overall, primary HPV-related OPSCCs that recur share a genomic landscape with nonrecurrent OPSCCs. Metachronous recurrent OPSCCs share genomic features with HPV-negative HNCs. These data aim to guide future deescalation endeavors and functional experiments. FUNDING: This study is supported by the American Cancer Society (RSG TBG-123653), funding support for RAH (T32DC00018, Research Training in Otolaryngology, University of Washington), funds to EM from Seattle Translational Tumor Research (Fred Hutchinson Cancer Research Center), and center funds from the Fred Hutchinson Cancer Research Center to EM. UD is supported by the Department of Veterans Affairs, Biomedical Laboratory Research and Development (BLR&D), grant IO1-oo23456, and funds from the Pittsburgh Foundation and PNC Foundation.

A Phase I Clinical Trial of AZD1775 in Combination with Neoadjuvant Weekly Docetaxel and Cisplatin before Definitive Therapy in Head and Neck Squamous Cell Carcinoma.

Purpose: The WEE1 tyrosine kinase regulates G2-M transition and maintains genomic stability, particularly in p53-deficient tumors which require DNA repair after genotoxic therapy. Thus, a need arises to exploit the role of WEE1 inhibition in head and neck squamous cell carcinoma (HNSCC) mostly driven by tumor-suppressor loss. This completed phase I clinical trial represents the first published clinical experience using the WEE1 inhibitor, AZD1775, with cisplatin and docetaxel.Patients and Methods: We implemented an open-label phase I clinical trial using a 3+3 dose-escalation design for patients with stage III/IVB HNSCC with borderline-resectable or -unresectable disease, but who were candidates for definitive chemoradiation. Escalating AZD1775 was administered orally twice a day over 2.5 days on the first week, then in combination with fixed cisplatin (25 mg/m2) and docetaxel (35 mg/m2) for 3 additional weeks. The primary outcome measure was adverse events to establish MTD. Secondary measures included response rates, pharmacokinetics (PK), pharmacodynamics, and genomic data.Results: The MTD for AZD1775 was established at 150 mg orally twice per day for 2.5 days. RECISTv1.1 responses were seen in 5 of 10 patients; histologic adjustment revealed three additional responders. The only drug-limiting toxicity was grade 3 diarrhea. The PK C8hr target of 240 nmol/L was achieved on day 4 at all three doses tested. Pharmacodynamic analysis revealed a reduction in pY15-Cdk, and increases in γH2AX, CC3, and RPA32/RPA2 were noted in responders versus nonresponders.Conclusions: The triplet combination of AZD1775, cisplatin, and docetaxel is safe and tolerable. Preliminary results show promising antitumor efficacy in advanced HNSCC, meriting further investigation at the recommended phase II dose. Clin Cancer Res; 24(12); 2740-8. ©2018 AACR.

Skills transfer to sinus surgery via a low-cost simulation-based curriculum.

BACKGROUND: Surgical skill development outside the operating room aims to improve technique and subsequent patient safety. The purpose of this study was to evaluate the correlation between technical and cognitive skills with cadaveric endoscopic sinus surgery (ESS) performance and change in ESS performance before and after implementation of a dedicated ESS simulation-based and knowledge-based curriculum. METHODS: A before-after study design was implemented among 10 medical students and 10 junior otolaryngology residents. Participants completed a knowledge-based, multiple-choice ESS pretest and watched an ESS prosection video. Participants performed 9 tasks on a previously validated low-cost, low-technology, nonbiologic sinus surgery task trainer followed by cadaveric maxillary antrostomy and anterior ethmoidectomy. Participants then completed a simulation-based and knowledge-based ESS curriculum followed by a repeat cadaveric maxillary antrostomy and anterior ethmoidectomy. Performance was graded with a 5-point global rating scale (GRS) and a 5-point ESS-specific checklist. RESULTS: We observed a stronger correlation between the multiple-choice, knowledge-based, ESS pretest scores and cadaveric ESS GRS score (r = 0.73) than between task trainer performance and cadaveric ESS GRS score (r = 0.43). We also noted a significant increase in precurriculum vs postcurriculum mean ± standard deviation (SD) cadaveric ESS checklist scores for both medical students (1.18 ± 0.25 vs 2.58 ± 0.57; p = 0.0002) and residents (2.09 ± 0.78 vs 2.88 ± 0.54; p = 0.023). The greatest improvements for residents were in performance of uncinectomy, enlargement of maxillary os, and identification of the bulla. CONCLUSION: These findings provide evidence supporting the use of ESS training curricula outside the operating room.

Anatomical Region Segmentation for Objective Surgical Skill Assessment with Operating Room Motion Data.

Background Most existing objective surgical motion analysis schemes are limited to structured surgical tasks or recognition of motion patterns for certain categories of surgeries. Analyzing instrument motion data with respect to anatomical structures can break the limit, and an anatomical region segmentation algorithm is required for the analysis. Methods An atlas was generated by manually segmenting the skull base into nine regions, including left/right anterior/posterior ethmoid sinuses, frontal sinus, left and right maxillary sinuses, nasal airway, and sphenoid sinus. These regions were selected based on anatomical and surgical significance in skull base and sinus surgery. Six features, including left and right eye center, nasofrontal beak, anterior tip of nasal spine, posterior edge of hard palate at midline, and clival body at foramen magnum, were used for alignment. The B-spline deformable registration was adapted to fine tune the registration, and bony boundaries were automatically extracted for final precision improvement. The resultant deformation field was applied to the atlas, and the motion data were clustered according to the deformed atlas. Results Eight maxillofacial computed tomography scans were used in experiments. One was manually segmented as the atlas. The others were segmented by the proposed method. Motion data were clustered into nine groups for every dataset and outliers were filtered. Conclusions The proposed algorithm improved the efficiency of motion data clustering and requires limited human interaction in the process. The anatomical region segmentations effectively filtered out the portion of motion data that are out of surgery sites and grouped them according to anatomical similarities.

An Automated Methodology for Assessing Anatomy-Specific Instrument Motion during Endoscopic Endonasal Skull Base Surgery.

Objectives Describe instrument motion during live endoscopic skull base surgery (ESBS) and evaluate kinematics within anatomic regions. Design Case series. Setting Tertiary academic center. Participants A single skull base surgeon performed six anterior skull base approaches to the pituitary. Main Outcomes and Measures Time-stamped instrument coordinates were recorded using an optical tracking system. Kinematics (i.e., mean cumulative instrument travel, velocity, acceleration, and angular velocity) was calculated by anatomic region including nasal vestibule, anterior and posterior ethmoid, sphenoid, and lateral opticocarotid recess (lOCR) regions. Results We observed mean (standard deviation, SD) velocities of 6.14 cm/s (1.55) in the nasal vestibule versus 1.65 cm/s (0.34) near the lOCR. Mean (SD) acceleration was 7,480 cm/s 2 (5790) in the vestibule versus 928 cm/s 2 (662) near the lOCR. Mean (SD) angular velocity was 17.2 degrees/s (8.31) in the vestibule and 5.37 degrees/s (1.09) near the lOCR. We observed a decreasing trend in the geometric mean velocity, acceleration, and angular velocity when approaching the pituitary ( p < 0.001). Conclusion Using a novel method for analyzing instrument motion during live ESBS, we observed a decreasing trend in kinematics with proximity to the pituitary. Additional characterization of surgical instrument motion is paramount for optimizing patient safety and training.

Region-Specific Objective Signatures of Endoscopic Surgical Instrument Motion: A Cadaveric Exploratory Analysis.

Objectives The objective of this study was to evaluate region-specific surgical instrument kinematics among novice and experienced surgeons performing endoscopic endonasal skull base surgery. Design Cadaveric experimental study. Setting Tertiary academic center. Participants Two novice and two experienced surgeons performed eight endoscopic total ethmoidectomies and sphenoidotomies using an optically tracked microdebrider. Main Outcome Measures Time-stamped Euclidian coordinates were recorded. Cumulative instrument travel, mean linear velocity and acceleration, and mean angular velocities were calculated in the anterior ethmoid, posterior ethmoid, and sphenoid sinus regions. Results Mean cumulative instrument travel (standard deviation) was highest in the posterior ethmoid region for both novice and experienced surgeons (9,795 mm [1,664] vs. 3,833 mm [1,080]). There was a trend in mean linear and angular velocities, and acceleration with increasing magnitudes for experienced surgeons compared with novices. Among experienced surgeons, we observed a trend of decreasing yaw velocity during the approach to the surgical target. Conclusions We present a novel method of evaluating surgical instrument motion with respect to anatomical regions of the skull base during endoscopic endonasal skull base surgery. These data may be used in the development of surgical monitoring and training systems to optimize patient safety.

Navigation Guidance During Free Flap Mandibular Reconstruction: A Cadaveric Trial.

Importance: Segmental mandibulectomy for tumors that distort the buccal surface of the mandible present a reconstructive challenge. Objective: To determine whether mandible alignment after navigation-guided mandible reconstruction is better than alignment after non-template-assisted freehand reconstruction and as good as template-assisted reconstruction in a cadaveric trial. Design, Setting, and Participants: A cadaveric trial using 10 specimens was conducted at a tertiary academic center. Fiducials were created on the ramus to compare alignment with each intervention. Segmental mandibulectomy was performed on each cadaver. Each cadaver underwent navigation-guided reconstruction, template-assisted reconstruction using a manually shaped plate, and non-template-assisted freehand reconstruction with plate contouring performed after mandibulectomy. The study was conducted from October 1, 2015, to January 1, 2016; data analysis was performed from February 1, 2016, to March 1, 2016. Interventions: Segmental mandibulectomy, navigation-guided reconstruction, template-assisted reconstruction using a manually shaped plate, and non-template-assisted freehand reconstruction. Main Outcomes and Measures: Ramus fiducial coordinates were recorded at baseline and after each intervention. Mandible dimensions were measured using cephalometric landmarks. Postintervention and baseline differences in ramus and mandible position were calculated. Results: Ramus alignment was not significantly different between navigation-guided and template-assisted reconstruction, differing by 0.54 mm (98.3% CI, -0.38 to 1.47 mm). Non-template-assisted freehand reconstruction was associated with a 3.14-mm difference in alignment compared with template-assisted reconstruction (98.3% CI, 1.09 to 5.19 mm). Navigation-guided alignment resulted in a 3.69-mm improvement in alignment compared with non-template-assisted freehand reconstruction (98.3% CI, 1.79 to 5.58 mm). There was some improvement in the gonion-gonion and lingula mandibulae-lingula mandibulae (Lm-Lm) alignment for navigation-assisted compared with non-template-assisted freehand reconstruction by 1.97 mm (98.3% CI, -0.65 to 4.58 mm) and 1.39 mm (98.3% CI, -0.17 to 2.95 mm), respectively. There was marginal evidence of better Lm-Lm alignment for navigation-guided than template-assisted reconstruction (0.44 mm; 98.3% CI, -0.06 to 0.95 mm). Conclusions and Relevance: Mandible alignment following navigation-guided reconstruction is similar to template-assisted reconstruction. Navigation-guided alignment is likely better than non-template-assisted freehand reconstruction, and navigation guidance offers a reliable technique for real-time adjustment when reconstructing complex surgical defects, such as tumors effacing the buccal cortex of the mandible.

Green tea epigallocatechin 3-gallate accumulates in mitochondria and displays a selective antiapoptotic effect against inducers of mitochondrial oxidative stress in neurons.

Epigallocatechin-3-gallate (EGCG) is a major flavonoid component of green tea that displays antiapoptotic effects in numerous models of neurotoxicity. Although the intrinsic free radical scavenging activity of EGCG likely contributes to its antiapoptotic effect, other modes of action have also been suggested. We systematically analyzed the antiapoptotic action of EGCG in primary cultures of rat cerebellar granule neurons (CGNs). The dose-dependent protective effects of EGCG were determined after coincubation with eight different stimuli that each induced neuronal apoptosis by distinct mechanisms. Under these conditions, EGCG provided significant neuroprotection only from insults that induce apoptosis by causing mitochondrial oxidative stress. Despite this selective antiapoptotic effect, EGCG did not significantly alter the endogenous activities or expression of Mn(2+)- superoxide dismutase, glutathione peroxidase, Nrf2, or Bcl-2. Subfractionation of CGNs after incubation with (3)H-EGCG revealed that a striking 90-95% of the polyphenol accumulated in the mitochondrial fraction. These data demonstrate that EGCG selectively protects neurons from apoptosis induced by mitochondrial oxidative stress. This effect is likely due to accumulation of EGCG in the mitochondria, where it acts locally as a free radical scavenger. These properties of EGCG make it an interesting therapeutic candidate for neurodegenerative diseases involving neuronal apoptosis triggered by mitochondrial oxidative stress.