Targeting Tumor Microenvironment Akt Signaling Represents a Potential Therapeutic Strategy for Aggressive Thyroid Cancer.

Effects of the tumor microenvironment (TME) stromal cells on progression in thyroid cancer are largely unexplored. Elucidating the effects and underlying mechanisms may facilitate the development of targeting therapy for aggressive cases of this disease. In this study, we investigated the impact of TME stromal cells on cancer stem-like cells (CSCs) in patient-relevant contexts where applying in vitro assays and xenograft models uncovered contributions of TME stromal cells to thyroid cancer progression. We found that TME stromal cells can enhance CSC self-renewal and invasiveness mainly via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. The disruption of Akt signaling could diminish the impact of TME stromal cells on CSC aggressiveness in vitro and reduce CSC tumorigenesis and metastasis in xenografts. Notably, disrupting Akt signaling did not cause detectable alterations in tumor histology and gene expression of major stromal components while it produced therapeutic benefits. In addition, using a clinical cohort, we discovered that papillary thyroid carcinomas with lymph node metastasis are more likely to have elevated Akt signaling compared with the ones without metastasis, suggesting the relevance of Akt-targeting. Overall, our results identify PI3K/Akt pathway-engaged contributions of TME stromal cells to thyroid tumor disease progression, illuminating TME Akt signaling as a therapeutic target in aggressive thyroid cancer.

Endonasal resection of orbital cavernous venous malformations with septal preservation.

PURPOSE: Multiple surgical approaches have been described to maximize visualization and accessibility for resection while minimizing morbidity in the patient with orbital intraconal tumors. Transnasal endoscopic approaches have become increasingly standard in select orbital cavernous venous malformations but often require a partial septectomy. The purpose of this manuscript is to communicate a septal preserving modified transseptal approach. METHODS: A 37-year old male was found to have an inferomedial intraconal orbital mass, measuring up to 2.6 cm on magnetic resonance imaging. Binarial transseptal access with septal preservation was obtained with a Killian incision on the right and a small incision in the midseptum on the left. RESULTS: Successful tumor delivery through the nasal cavity resulted in orbital relaxation. Postoperative evaluation of the septum demonstrated an intact septum with nearly no evidence of septal trauma from surgical manipulation. CONCLUSION: This technique is easily performed and affords adequate visualization and freedom of movement as traditional binarial transseptal approaches without the disadvantages of partial septal loss such as increased crusting, olfactory disturbance, and loss of nasoseptal flaps.

Thyroid Carcinoma: Phenotypic Features, Underlying Biology and Potential Relevance for Targeting Therapy.

Thyroid carcinoma consists a group of phenotypically heterogeneous cancers. Recent advances in biological technologies have been advancing the delineation of genetic, epigenetic, and non-genetic factors that contribute to the heterogeneities of these cancers. In this review article, we discuss new findings that are greatly improving the understanding of thyroid cancer biology and facilitating the identification of novel targets for therapeutic intervention. We review the phenotypic features of different subtypes of thyroid cancers and their underlying biology. We discuss recent discoveries in thyroid cancer heterogeneities and the critical mechanisms contributing to the heterogeneity with emphases on genetic and epigenetic factors, cancer stemness traits, and tumor microenvironments. We also discuss the potential relevance of the intratumor heterogeneity in understanding therapeutic resistance and how new findings in tumor biology can facilitate designing novel targeting therapies for thyroid cancer.

Subpopulations of cancer stem cells found in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer and while it has a generally good prognosis, tumor recurrence remains a major clinical challenge. Studying laboratory cell lines as well as clinical specimens indicate that PTC may follow the cancer stem cell (CSC) model. However, CSC characteristics relevant in PTC initiation and progression remain largely unknown. Here we studied a population of sphere-growing tumor cells isolated from primary cultures of clinical PTC. These sphere-growing cells consisted of aldehyde dehydrogenase positive (ALDH+) and ALDH negative (ALDH-) cell subpopulations and demonstrated a hierarchical pattern of cell division. Using combinations of selective depletion, specific inhibition and cell sorting, we found that both subpopulations of the sphere cells were able to self-renew and initiate xenograft tumors independently, and fulfilled the definition of CSC. Importantly, when the subpopulations functioned together, the cancer-initiation efficiency and the xenograft tumor progression were significantly enhanced compared to either subpopulation alone. These data revealed crucial roles of ALDH- CSC in PTC biology and suggested that CSC subpopulations function cooperatively to control PTC initiation and progression. Together, our study indicates that CSC subpopulations isolated from clinical specimens offer unprecedented opportunities for investigating PTC pathogenesis and developing effective therapies.

Artificial intelligence-based epigenomic, transcriptomic and histologic signatures of tobacco use in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) biomarker studies rarely employ multi-omic biomarker strategies and pertinent clinicopathologic characteristics to predict mortality. In this study we determine for the first time a combined epigenetic, gene expression, and histology signature that differentiates between patients with different tobacco use history (heavy tobacco use with ≥10 pack years vs. no tobacco use). Using The Cancer Genome Atlas (TCGA) cohort (n = 257) and an internal cohort (n = 40), we identify 3 epigenetic markers (GPR15, GNG12, GDNF) and 13 expression markers (IGHA2, SCG5, RPL3L, NTRK1, CD96, BMP6, TFPI2, EFEMP2, RYR3, DMTN, GPD2, BAALC, and FMO3), which are dysregulated in OSCC patients who were never smokers vs. those who have a ≥ 10 pack year history. While mortality risk prediction based on smoking status and clinicopathologic covariates alone is inaccurate (c-statistic = 0.57), the combined epigenetic/expression and histologic signature has a c-statistic = 0.9409 in predicting 5-year mortality in OSCC patients.

Transoral Laser Resection of the Tongue Base in the Workup of Unknown Primary Head and Neck Squamous Cell Carcinoma.

Objective Failure to localize the primary tumor site in head and neck carcinoma of unknown origin after imaging and endoscopic evaluation leads to increased treatment-related morbidity. The use of transoral laser microsurgery to improve the detection of unknown primary carcinoma site identification is described in this article. Methods A retrospective cohort of 71 consecutive cases of cervical carcinoma of an unknown primary source from 2006 until 2012 from a single academic institution was analyzed. Of these, 10 patients were excluded based on our exclusion criteria. All patients underwent endoscopy with biopsies performed by fellowship-trained head and neck cancer surgeons.  Results The primary detection rate was 76% for patients who underwent laser tongue base resection versus 34% for traditional operative examination. There were no complications or prolonged recovery times in either group. Operative time was increased by the addition of the transoral base of tongue resection by 30 minutes. Conclusions Laser tongue base excision offers improved sensitivity in primary site detection without a significant increase in morbidity and only a modest increase in operative time.

Diagnostic and Management Applications of ChatGPT in Structured Otolaryngology Clinical Scenarios.

Objective: To evaluate the clinical applications and limitations of chat generative pretrained transformer (ChatGPT) in otolaryngology. Study Design: Cross-sectional survey. Setting: Tertiary academic center. Methods: ChatGPT 4.0 was queried for diagnoses and management plans for 20 physician-written clinical vignettes in otolaryngology. Attending physicians were then asked to rate the difficulty of the clinical vignettes and agreement with the differential diagnoses and management plans of ChatGPT responses on a 5-point Likert scale. Summary statistics were calculated. Univariate ordinal regression was then performed between vignette difficulty and quality of the diagnoses and management plans. Results: Eleven attending physicians completed the survey (61% response rate). Overall, vignettes were rated as very easy to neutral difficulty (range of median score: 1.00-4.00; overall median 2.00). There was a high agreement with the differential diagnosis provided by ChatGPT (range of median score: 3.00-5.00; overall median: 5.00). There was also high agreement with treatment plans (range of median score: 3.00-5.00; overall median: 5.00). There was no association between vignette difficulty and agreement with differential diagnosis or treatment. Lower diagnosis scores had greater odds of having lower treatment scores. Conclusion: Generative artificial intelligence models like ChatGPT are being rapidly adopted in medicine. Performance with curated, easy-to-moderate difficulty otolaryngology scenarios indicate high agreement with physicians for diagnosis and management. However, a decreased quality in diagnosis is associated with decreased quality in management. Further research is necessary on ChatGPT's ability to handle unstructured clinical information.

Is it Anaplastic Thyroid Cancer, Primary Thyroid Lymphoma, or Rosai Dorfman Disease? An Elusive Histopathologic Diagnosis of a Thyroid Mass.

The aim of this study is to present an elusive case of primary thyroid lymphoma (PTL), initially thought to be anaplastic thyroid carcinoma, then Rosai Dorfman disease, before the final diagnosis of PTL was made. An elderly female with hypothyroidism presented with compressive airway symptoms secondary to an enlarging neck mass. Imaging was suggestive of undifferentiated thyroid cancer. The initial biopsy was unexpectedly consistent with a lymphoproliferative disorder such as Rosai-Dorfman disease. A repeat biopsy with immunohistochemical analysis yielded a diagnosis of diffuse large B-cell lymphoma of germinal center subtype. The patient was spared thyroid surgery and started on appropriate chemotherapy. PTL is within the differential diagnosis that physicians must consider in a patient with a rapidly-enlarging neck mass. A clinical index of suspicion and early accurate diagnosis may spare the patient from unnecessary surgery that is required of most other non-hematopoeitic thyroid malignancies.

Dysphagia Severity and Outcomes Following Iatrogenic High Vagal Nerve Injury.

OBJECTIVE: To examine severity of dysphagia and outcomes following iatrogenic high vagal nerve injury. METHODS: Retrospective chart review of all patients with iatrogenic high vagal nerve injury that were seen at a tertiary referral center from 2012 to 2020. RESULTS: Of 1304 patients who met criteria for initial screening, 18 met all inclusion criteria. All 18 required intervention to address postoperative dysphagia. Eleven required enteral feeding tubes with 7 eventually able to advance to exclusively per oral diets. Fourteen underwent vocal fold injection and 6 underwent laryngeal framework surgery. Sixteen pursued swallowing therapy with speech language pathology. Patients lost a mean of 8.6 kg of weight in the 6 months following the injury. Swallowing function on the Functional Outcome Swallowing Scale (FOSS) and Functional Oral Intake Scale (FOIS) was 4.4 and 2.4 respectively immediately following the injury and improved to 1.9 and 5.3 at the last follow-up. No patients had complete return of normal swallowing function at last follow up. CONCLUSION: Iatrogenic high vagal injury causes significant lasting dysphagia which improves with intervention but does not completely resolve. Interventions such as vocal fold injection, medialization laryngoplasty, cricopharyngeal myotomy, or swallowing therapy may be required to reestablish safe swallowing in these patients.

Neurotrophin Pathway Receptors NGFR and TrkA Control Perineural Invasion, Metastasis, and Pain in Oral Cancer.

Oral squamous cell carcinoma (OSCC) patients suffer from poor survival due to metastasis or locoregional recurrence, processes that are both facilitated by perineural invasion (PNI). OSCC has higher rates of PNI than other cancer subtypes, with PNI present in 80% of tumors. Despite the impact of PNI on oral cancer prognosis and pain, little is known about the genes that drive PNI, which in turn drive pain, invasion, and metastasis. In this study, clinical data, preclinical, and in vitro models are leveraged to elucidate the role of neurotrophins in OSCC metastasis, PNI, and pain. The expression data in OSCC patients with metastasis, PNI, or pain demonstrate dysregulation of neurotrophin genes. TrkA and nerve growth factor receptor (NGFR) are focused, two receptors that are activated by NGF, a neurotrophin expressed at high levels in OSCC. It is demonstrated that targeted knockdown of these two receptors inhibits proliferation and invasion in an in vitro and preclinical model of OSCC, and metastasis, PNI, and pain. It is further determined that TrkA knockdown alone inhibits thermal hyperalgesia, whereas NGFR knockdown alone inhibits mechanical allodynia. Collectively the results highlight the ability of OSCC to co-opt different components of the neurotrophin pathway in metastasis, PNI, and pain.

Combined endoscopic and transoral resection of a high-staged juvenile nasopharyngeal angiofibroma: A pictorial essay.

Juvenile nasopharyngeal angiofibromas (JNAs) are highly vascular and benign tumors that can expand into the skull base. Delay of treatment can result in intracranial invasion, requiring extensive open approaches such as a facial translocation, maxillary swing, or an orbitozygomatic craniotomy. We describe a single-stage, combined endoscopic and transoral approach on a 14-year-old male with extensive high-stage dumbbell-shaped JNA involving the infratemporal fossa, orbit, buccal space, and intracranial extension into Meckel's cave. Successful resection of the tumor and good postoperative outcome was achieved. A transoral approach allowed for greater access to the infratemporal fossa, where endonasal resection was not possible, allowing for improved visualization, greater traction, and dissection. In select highly staged JNAs with significant lateral extension and intracranial involvement, successful and complete resection may be accomplished with this combined approach. Utilization of this approach avoids the morbidity of more invasive open approaches.

Brush swab as a noninvasive surrogate for tissue biopsies in epigenomic profiling of oral cancer.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has poor survival rates. There is a pressing need to develop more precise risk assessment methods to tailor clinical treatment. Epigenome-wide association studies in OSCC have not produced a viable biomarker. These studies have relied on methylation array platforms, which are limited in their ability to profile the methylome. In this study, we use MethylCap-Seq (MC-Seq), a comprehensive methylation quantification technique, and brush swab samples, to develop a noninvasive, readily translatable approach to profile the methylome in OSCC patients. METHODS: Three OSCC patients underwent collection of cancer and contralateral normal tissue and brush swab biopsies, totaling 4 samples for each patient. Epigenome-wide DNA methylation quantification was performed using the SureSelectXT Methyl-Seq platform. DNA quality and methylation site resolution were compared between brush swab and tissue samples. Correlation and methylation value difference were determined for brush swabs vs. tissues for each respective patient and site (i.e., cancer or normal). Correlations were calculated between cancer and normal tissues and brush swab samples for each patient to determine the robustness of DNA methylation marks using brush swabs in clinical biomarker studies. RESULTS: There were no significant differences in DNA yield between tissue and brush swab samples. Mapping efficiency exceeded 90% across all samples, with no differences between tissue and brush swabs. The average number of CpG sites with at least 10x depth of coverage was 2,716,674 for brush swabs and 2,903,261 for tissues. Matched tissue and brush swabs had excellent correlation (r = 0.913 for cancer samples and r = 0.951 for normal samples). The methylation profile of the top 1000 CpGs was significantly different between cancer and normal samples (mean p-value = 0.00021) but not different between tissues and brush swabs (mean p-value = 0.11). CONCLUSIONS: Our results demonstrate that MC-Seq is an efficient platform for epigenome profiling in cancer biomarker studies, with broader methylome coverage than array-based platforms. Brush swab biopsy provides adequate DNA yield for MC-Seq, and taken together, our findings set the stage for development of a non-invasive methylome quantification technique for oral cancer with high translational potential.

The REASON score: an epigenetic and clinicopathologic score to predict risk of poor survival in patients with early stage oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a capricious cancer with poor survival rates, even for early-stage patients. There is a pressing need to develop more precise risk assessment methods to appropriately tailor clinical treatment. Genome-wide association studies have not produced a viable biomarker. However, these studies are limited by using heterogeneous cohorts, not focusing on methylation although OSCC is a heavily epigenetically-regulated cancer, and not combining molecular data with clinicopathologic data for risk prediction. In this study we focused on early-stage (I/II) OSCC and created a risk score called the REASON score, which combines clinicopathologic characteristics with a 12-gene methylation signature, to predict the risk of 5-year mortality. METHODS: We combined data from an internal cohort (n = 515) and The Cancer Genome Atlas (TCGA) cohort (n = 58). We collected clinicopathologic data from both cohorts to derive the non-molecular portion of the REASON score. We then analyzed the TCGA cohort DNA methylation data to derive the molecular portion of the risk score. RESULTS: 5-year disease specific survival was 63% for the internal cohort and 86% for the TCGA cohort. The clinicopathologic features with the highest predictive ability among the two the cohorts were age, race, sex, tobacco use, alcohol use, histologic grade, stage, perineural invasion (PNI), lymphovascular invasion (LVI), and margin status. This panel of 10 non-molecular features predicted 5-year mortality risk with a concordance (c)-index = 0.67. Our molecular panel consisted of a 12-gene methylation signature (i.e., HORMAD2, MYLK, GPR133, SOX8, TRPA1, ABCA2, HGFAC, MCPH1, WDR86, CACNA1H, RNF216, CCNJL), which had the most significant differential methylation between patients who survived vs. died by 5 years. All 12 genes have already been linked to survival in other cancers. Of the genes, only SOX8 was previously associated with OSCC; our study was the first to link the remaining 11 genes to OSCC survival. The combined molecular and non-molecular panel formed the REASON score, which predicted risk of death with a c-index = 0.915. CONCLUSIONS: The REASON score is a promising biomarker to predict risk of mortality in early-stage OSCC patients. Validation of the REASON score in a larger independent cohort is warranted.

Mixed olfactory neuroblastoma and neuroendocrine carcinoma: An unusual case report and literature review.

BACKGROUND: The aim of the study was to present a case of mixed olfactory neuroblastoma (ONB) and carcinoma, an extremely rare tumor with only a few cases in the published literature. CASE DESCRIPTION: An otherwise healthy 27-year-old male presented with sinus complaints, headache, and unilateral eye discharge. Imaging and endoscopy revealed a mass presumed to represent a juvenile nasopharyngeal angiofibroma. Unexpectedly, the final pathology report revealed high grade mixed ONB and carcinoma. This tumor is the sixth and youngest documented patient with mixed ONB and carcinoma. CONCLUSION: Physicians should remain vigilant for the possibility of malignancy in their approach to nasal cavity masses, even in young otherwise healthy patients. Careful review of the immunohistopathology should also be taken, as mixed olfactory tumors such as these are aggressive, rare entities that require multidisciplinary oncologic care.

Cost effectiveness of intraoperative pathology in the management of indeterminate thyroid nodules.

Objective This study aims to determine the cost effectiveness of rapid frozen section (RFS) for indeterminate thyroid nodules. Materials and methods A retrospective chart review was conducted between January 2009 and June 2013 at a tertiary care institution. Main outcomes were number needed to treat, RFS efficacy, and cost-savings of avoiding second completion thyroidectomy. Cost-effectiveness was estimated using 2015 Medicare reimbursement rate. Results Out of 1,114 patients undergoing thyroid surgery, 314 had preoperative AUS/FLUS cytopathology and subsequent thyroid lobectomy with RFS. RFS identified 13 of the 32 patients with malignancy resulting in a total thyroidectomy. 19 of the 29 malignancies not detected by RFS were papillary microcarcinomas. Conclusions Completion thyroidectomy was avoided in 1 out of every 24 patients resulting in cost-savings of $ 80.04 per patient. In the era of outpatient thyroid surgery, intraoperative RFS for indeterminate thyroid nodules is cost-effective.

Role of polymorphic Fc gamma receptor IIIa and EGFR expression level in cetuximab mediated, NK cell dependent in vitro cytotoxicity of head and neck squamous cell carcinoma cells.

Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy, although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore, in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells, cetuximab concentration, and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets, respectively, in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore, supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level, cetuximab concentration, and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P < 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a, and secreted significantly (P < 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab mediated clinical responses. Cellular and secreted immune profiles and FcgammaR genotypes from patients' lymphocytes may provide clinically useful biomarkers of immune activation in cetuximab treated patients.

Immunotherapy of head and neck cancer using tumor antigen-specific monoclonal antibodies.

Monoclonal antibodies (mAbs) are now commonly used therapeutic agents in cancer patients. Since US Food and Drug Administration approval of cetuximab for head and neck squamous cell carcinoma, it has been used increasingly in this disease. Several other mAbs also are in development or in clinical -trials. Recently, evidence has accumulated that mAbs induce activation of cellular immunity, including natural killer and T cells and that this may contribute to clinical response. mAbs have been shown to mediate antibody-dependent cellular cytotoxicity, complement-dependent lysis, and activation of tumor antigen-specific T cells. Various patient and tumor factors, such as polymorphisms in Fcgamma receptors expressed by immune cells, activity of T-regulatory cells, and tumor escape through downregulation of antigen-processing machinery in tumor cells, are likely to modulate the immune activation mediated by therapeutic mAbs. Understanding the interplay of these factors is likely to improve the selection of the most appropriate candidates for mAb-based immunotherapy, prediction of clinical response, and our understanding of mechanisms of tumor escape from therapeutic mAbs.

Cancer stem cell self-renewal as a therapeutic target in human oral cancer.

Tumor recurrence following treatment remains a major clinical challenge in oral cavity cancer. Cancer stem cells (CSCs) have been isolated from human oral cancers and been considered as the driving force of tumor recurrence and metastasis. However, it still remains unclear whether targeting CSCs in oral cancer is a clinically relevant strategy to combat cancer recurrence and metastasis. Here, using clinical cancer specimens and patient-derived xenografts, we show that the self-renewal regulator BMI1 is highly expressed in CSCs of oral cavity squamous cell carcinoma. Inhibition of BMI1 decreases oral CSCs' self-renewal and tumor-initiating potential. Treatment of pre-established human oral cancer xenografts with a BMI1 inhibitor resulted in abrogation of tumor progression and reduced the frequency of CSCs in the xenografts. Remarkably, the BMI1 inhibitor has therapeutic effects in cisplatin-resistant tumors and can reduce metastases initiated by circulating CSCs. Mechanistically, BMI1-inhibition leads to oral CSC necroptotic cell death, which underlies the self-renewal impairment after inhibiting BMI1. Our data provide a pre-clinical proof-of-concept that targeting BMI1-related CSC self-renewal is a clinically relevant anti-cancer therapy in human oral cavity squamous cell carcinoma.

Plain films in the evaluation of batteries as esophageal foreign bodies.

OBJECTIVE: To determine the sensitivity and specificity of plain films in differentiating coin batteries from coins. SETTING: Study was conducted at a tertiary referral university medical center. METHODS: Eleven radiographs were taken of various objects and independently reviewed by 14 radiologists and otolaryngologists. Reviewers were asked to identify the object filmed as either a battery or not a battery. In addition, otolaryngologists were asked if they would immediately proceed to the operative suite for removal based on the film. Results were tabulated and analyzed using a spreadsheet. RESULTS: Overall, plain films had a sensitivity and specificity of 80.4% and 79.1%, respectively with an overall accuracy of 79.8%. When used as a test to determine urgency of removal, sensitivity increases to 94.4% while specificity decreases to 67.1% with an overall accuracy of 83.1%. CONCLUSIONS: Plain films are an effective method of evaluating for the possibility of batteries as esophageal foreign bodies.

Death pathways in noise-damaged outer hair cells.

Using morphological criteria, death pathways in outer hair cells (OHCs) were determined in chinchilla organs of Corti that had been exposed to a high- or moderate-level octave band of noise (OBN) centered at either 0.5 or 4-kHz. The specimens were part of our large collection of plastic-embedded flat preparations of chinchilla cochleae. Three death pathways were identified: (1) oncotic - swollen, pale-staining cell with a swollen nucleus, (2) apoptotic - shrunken, dark-staining cell with a pyknotic nucleus and (3) a newly defined third pathway - no basolateral plasma membrane but cellular debris arranged in the shape of an intact OHC with a nucleus deficient in nucleoplasm. To minimize the secondary loss of OHCs from the entrance of endolymph into the organ of Corti, the specimens used for quantitative analysis of death pathways had the following characteristics: (1) the level to which they were exposed was less than or equal to 95dB SPL, (2) the exposure duration was 6-216h, (3) fixation for microscopic examination took place in vivo 1-2h post-exposure and (4) there were no focal OHC lesions in the organs of Corti. Fifty-eight noise-exposed cochleae met these criteria. In these specimens, degenerating and missing OHCs were classified as to which death pathway the cells had followed or were following. Nine non-noise-exposed cochleae were also evaluated for OHC death pathways. The number of OHCs following the third death pathway was significantly greater in the noise-exposed cochleae than the non-noise-exposed cochleae for total exposure energies greater than those produced by 75dB SPL for 216h to a 0.5-kHz OBN and 57dB SPL for 48h to a 4-kHz OBN. In cochleae exposed to either octave band, OHCs dying by oncosis or apoptosis were uncommon.