Miriam S. Teixeira

Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival.

To analyze the impact of resection margin status and histologic prognosticators on local recurrence (LR) and overall survival (OS) for patients with oral squamous cell carcinoma (OSCC). This study was both retrospective and prospective in design. Cohort 1 refers to the entire group of 292 patients with OSCC. The slides from the earliest resection specimens from Cohort 1 were examined in an exploratory manner for multiple parameters. Cohort 2 refers to a subset of 203 patients, who did not receive any neoadjuvant therapy and had outcome data. Cohort 3 represents a subset of Cohort 2 (n = 168) wherein the histologic resection margin status could be reconfirmed. Cohort 4 refers a subset of 85 patients with tongue/floor of mouth tumors. Margin status was designated as follows: group 1, clearance of ≥5 mm with intraoperative analysis, no need for supplemental margins (n = 46); group 2, initial margins were measured as <5 mm during intraoperative frozen section; supplemental resection margins were negative on final pathology (n = 73); group 3, the final pathology revealed resection margins <5 mm (n = 30); group 4, the final pathology revealed frankly positive resection margins (n = 19). The endpoints of LR and OS were queried with respect to T stage, tumor site, margin status, and numerous histologic variables, by Cox regression and Kaplan-Meier survival analyses. Tumor stage (T) was significantly associated with LR (P = 0.028). Kaplan-Meier analysis for stage and for intraoral site was significantly associated with LR for T4 tumors. The increased likelihood of LR was higher for T4 OSCC of the buccal mucosa (75%), sinopalate (50%), and gingiva (100%) compared with mobile tongue (27%), and oropharynx (13%) (P = 0.013). Margin status was not associated with LR or OS (Cohort 3). This was so when all tumors were grouped together and when separate analyses were performed by tumor stage and oral subsite. No significance was demonstrated when margin status was examined for patients with similar treatment (surgery alone or surgery with adjuvant RT). However, the administration of adjuvant RT did significantly increase local disease-free survival (P = 0.0027 and P = 0.001 for T1 and T2 SCC, respectively). On exploratory analyses of histologic parameters, worst pattern of invasion was significantly associated with LR (P = 0.015) and OS (P < 0.001). Perineural invasion involving large nerves (>1 mm) was associated with LR (P = 0.005) and OS (P = 0.039). Limited lymphocytic response was also significantly associated with LR (P = 0.005) and OS (P = 0.001). When used as covariates in a multivariate Cox regression model, worst pattern of invasion, perineural invasion, and lymphocytic response were significant and independent predictors of both LR and OS, even when adjusting for margin status. Thus, these factors were used to generate our risk assessment. Our risk assessment classified patients into low-, intermediate-, or high-risk groups, with respect to LR (P = 0.0004) and OS (P < 0.0001). This classification retained significance when examining patients with uniform treatment. In separate analyses for each risk group, we found that administration of adjuvant radiation therapy is associated with increased local disease-free survival for high-risk patients only (P = 0.0296) but not low-risk or intermediate-risk patients. Resection margin status alone is not an independent predictor of LR and cannot be the sole variable in the decision-making process regarding adjuvant radiation therapy. We suggest that the recommendation for adjuvant radiation therapy be based on, not only traditional factors (inadequate margin, perineural invasion, bone invasion) but also histologic risk assessment. If clinicians want to avoid the debilitation of adjuvant radiation therapy, then a 5-mm margin standard may not be effective in the presence of high-risk score.

Functional inactivation of the KLF6 tumor suppressor gene by loss of heterozygosity and increased alternative splicing in glioblastoma

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor and possesses a high incidence of 10p loss. The KLF6 (Kruppel‐like transcription factor) tumor suppressor gene on 10p15 is inactivated by loss of heterozygosity (LOH) and/or somatic mutation in a number of human cancers and forced expression of KLF6 in GBM lines inhibits their growth and transformation. In addition, increased expression of its alternatively spliced, cytoplasmic isoform KLF6‐SV1 has now been shown to play a role in cancer pathogenesis. On the basis of these findings we examined the role of KLF6 and KLF6‐SV1 in the development and progression of GBM. LOH analysis of 17 primary GBM patient samples using KLF6‐specific microsatellite markers revealed that 88.2% (15/17) had LOH of the KLF6 locus. Interestingly, no KLF6 somatic mutations were identified. RNA analysis revealed concomitant decreases in all primary GBM tumors (n = 11) by ∼80% in KLF6 expression (p < 0.001) coupled with increased KLF6‐SV1 expression (p < 0.001) when compared to normal astrocytes. To determine the biological relevance of these findings, we examined the effect of KLF6 expression and KLF6‐SV1 knockdown in A235 and CRL2020 cell lines. Reconstitution of KLF6 decreased cell proliferation by almost 50%, whereas targeted KLF6 reduction increased cell proliferation 2.5–4.5 fold. Conversely, targeted KLF6‐SV1 reduction decreased cell proliferation by 50%. Taken together, our findings demonstrate that KLF6 allelic imbalance and decreased KLF6 and increased KLF6‐SV1 expression are common findings in primary GBM tumors, and these changes have antagonistic effects on the regulation of cellular proliferation in GBM cell lines.

KLF6 allelic loss is associated with tumor recurrence and markedly decreased survival in head and neck squamous cell carcinoma

The Krüppel‐like transcription factor (KLF6) gene is a tumor suppressor gene (TSG) reported to be dysregulated and inactivated through loss of heterozygosity (LOH) and/or somatic mutation in a number of major human cancers. The aim of the present study was to examine KLF6 gene status and expression in head and neck squamous cell carcinomas (HNSCC). A collection of 81 well‐characterized oral and oropharyngeal HNSCC samples were analyzed for evidence of KLF6 LOH and mutation and differences in expression patterns between normal and cancerous tissues and these findings were correlated with clinicopathological variables. We also tested the effect of KLF6 inhibition in HNSCC cell lines on proliferation and p21 expression. LOH was found in approximately 30% of cases and was strongly correlated with cancer progression, tumor recurrence and decreased patient survival. Overall, median survival of patients with LOH was less than half (19 vs. 41 months, p = 0.036, stratified on stage) than those without loss. Risk of death for patients with LOH was 8 times greater independent of tumor size, nodal status, tobacco smoking or treatment modality (HR 7.89, 95% CI: 1.9–32.4). Subsequent analyses revealed KLF6 mutations in only 2 of 20 samples, but altered subcellular protein localization in 64% of tumors. Targeted stable reduction of KLF6 in HNSCC cell lines increased cellular proliferation while decreasing p21 expression. Taken together, these findings suggest that KLF6 LOH represents a clinically‐relevant biomarker predicting patient survival and tumor recurrence and that dysregulation of KLF6 function plays an important role in HNSCC progression.

Eustachian tube function in young children without a history of otitis media evaluated using a pressure chamber protocol.

Abstract Conclusions: Protocol limitations were identified and accounted for in the analysis. Percent gradient equilibrated (PGE) was affected by driving gradient direction in a similar manner to other efficiency measures. A finer resolution of possible age-related changes in eustachian tube opening efficiency is expected with the application of more sophisticated statistical models to the complete dataset at study end. Objective: To report the results of an interim analysis for an ongoing study designed to characterize the age-related changes in eustachian tube opening efficiency measured using a pressure chamber protocol in children without a history of middle ear disease. Methods: To date, 41 children aged 3 years without a history of otitis media have been enrolled in a longitudinal study of the age-related changes in eustachian tube function and evaluated at yearly intervals between 3 and 7 years of age. Eustachian tube opening efficiency, the percent of the applied pressure gradient equilibrated by swallowing, was measured by repeat tympanometry during a pressure chamber protocol. Data (120 tests) were analyzed using an ANOVA with variance partitioned by age (3 through 6 years), gradient direction (positive/negative), and ear (left/right). Results: PGE was higher for left ears and positive driving gradients, but was not different among age groups.

Quantitative Description of Eustachian Tube Movements During Swallowing as Visualized by Transnasal Videoendoscopy

IMPORTANCE Eustachian tube (ET) dysfunction predisposes ears to otitis media, tympanic membrane retraction, retraction pocket and perforation, or cholesteatoma. OBJECTIVE To develop a method to quantitatively measure the eustachian tube (ET) component movements and their interactions captured by transnasal videoendoscopy of the ET during swallowing. DESIGN, SETTING, AND PARTICIPANTS A blinded analysis of ET mechanics in 33 adults, aged 18 to 54 years, with no middle ear disease at present but without (group 1 [n = 16]) or with (group 2 [n = 17]) history of disease, conducted at a clinical research laboratory. INTERVENTIONS Videoendoscopy of the ET orifice at the nasopharynx. MAIN OUTCOMES AND MEASURES Eustachian tube component translations and structural interactions during a swallow and the between-group differences in those variables. After topical anesthesia of the nose, a 45° telescope was introduced unilaterally and focused on the ipsilateral ET orifice. A video recording of ET component movements was made during 3 swallows. Swallow and ET opening durations and times to selected events were calculated. Images at 3 time points were analyzed by measuring the apex angle, the medial-lateral luminal width, and the medial angles between a frame-normal horizontal line through the apex and fixed points on the torus and medial and lateral luminal walls. Linear and angular variables during a swallow were expressed as change from baseline. RESULTS Luminal opening was driven by soft palate elevation-related medial rotation of the torus and medial wall, coupled with lateral wall fixedness. The magnitude of the change from baseline for most variables was statistically greater than 0. Swallow time, palatal elevation time, time interval between maximum palatal elevation, and maximum eustachian tube opening time were not different between groups 1 and 2. Opening time was longer (mean [SD], 0.49 [0.28] vs 0.67 [0.51] seconds; P = .03) in group 2. Higher magnitude of torus rotation (mean [SD], 36.05° [12.96°] vs 27.72° [9.45°]; P = .002) with maximum soft palate elevation in group 1 resulted in greater degree of eustachian tube orifice widening (mean [SD], 0.34% [0.47%] vs -0.02% [0.49%]; P = .001) compared with the resting position in that group. CONCLUSIONS AND RELEVANCE This methodology has application in developing quantitative descriptions of ET mechanics in groups of persons without and with history or suspected ET dysfunction. A lesser degree of soft palate elevation during swallow that derives the ET medial lamina rotation and widening of the ET orifice may be associated with poor ET function and higher risk for otitis media. Videoendoscopic evaluation of the ET orifice may assist in diagnosing presence and mechanism of ET dysfunction.

Eustachian Tube Function in 6-Year-Old Children with and without a History of Middle Ear Disease

Objective To test the hypothesis that eustachian tube opening efficiency, measured as the fractional gradient equilibrated (FGE), is lower in 6-year-old children with no middle ear disease but a well-documented history of recurrent acute otitis media, as compared with children with a negative disease history (control). Study Design Cross-sectional study. Setting Tertiary care pediatric hospital. Subjects and Methods Bilateral eustachian tube function was evaluated in 44 healthy 6-year-old children (19 boys, 29 white). None had middle ear disease at the time of testing, but 23 had a history of recurrent acute otitis media. Twenty-one had no significant past otitis media. Eustachian tube function was measured with a pressure chamber protocol that established negative middle ear gauge pressures (referenced to the chamber pressure) and recorded that pressure before and after a swallow. FGE was calculated as the change in middle ear gauge pressure with swallowing divided by the preswallow pressure. Between-group comparisons of the preswallow pressures and FGEs were made with a 2-tailed Student’s t test. Results FGE was independent of the preswallow middle ear gauge pressure. For the 39 and 44 evaluable ears in the control and recurrent acute otitis media groups, the mean preswallow pressures were −194 daPa (95% confidence interval [95% CI] = −211 to −177) versus −203 (95% CI = −216 to −190; P > .40), and FGEs were 0.32 (95% CI = 0.21-0.43) vs 0.16 (95% CI = 0.08-0.24; P = .016), respectively. Conclusion In children with past recurrent acute otitis media, residual eustachian tube opening inefficiency is maintained after they have “outgrown” their middle ear disease.

Oral pseudoephedrine decreases the rate of transmucosal nitrous oxide exchange for the middle ear

Determine if oral treatment with a vasoconstrictor decreases the blood to middle ear exchange rate of the perfusion‐limited gas, nitrous oxide (N2O).

Oxymetazoline Applied Topically to the Nasal Mucosa Decreases Trans-Mucosal Nitrous Oxide Exchange for the Middle Ear.

Objective: Determine if the middle ear (ME) trans-mucosal nitrous oxide (N2O) gas exchange rate can be pharmacologically modulated by the nasal application of a vasoconstrictor. Methods: In a randomized, double-blind, crossover study, 20 adults received a nasal spray challenge containing either oxymetazoline or saline (placebo). At each session, subjects were fitted with a non-rebreathing mask and breathed room air for 20 minutes, 50% N2O:50% O2 for 20 minutes, and 100% O2 for 10 minutes. Throughout, heart rate, blood pressure (BP), and blood O2 saturation were monitored, and bilateral ME pressure was recorded by tympanometry every minute. The primary outcome measure was the slope of the ME pressure-time function for the experimental period, a direct measure of the transMEM N2O exchange constant. The effects of treatment, session, and period on the measured vital signs and of treatment, session, disease history, and ear on the ME pressure-time slopes were evaluated for statistical significance using repeated measures ANOVAs. Results: The analysis documented a significant effect of period on O2 saturation (N2O > room air, P = .03) and of treatment on blood pressure (oxymetazoline > placebo, P < .02) and the ME pressure-time slope (placebo > oxymetazoline, P = .05). Conclusion: The exchange rate across the ME mucosa of inert gases can be decreased by topical treatment of the nasal mucosa with oxymetazoline.

A Novel Functional Imaging Method of the Eustachian Tube

Objectives: (1) Image the Eustachian tube (ET) lumen by computed tomography (CT) scanning during ET function (ETF) testing, (2) characterize the differences in image quality for different scanning protocols, and (3) establish a novel research methodology for studying ET anatomy and physiology. Methods: In a cadaver head without craniofacial or otologic abnormalities, the tympanic membrane was perforated and ETF test was done using the forced response test (FRT) in a CT scanner. Opening (OP), steady (PS), and closing (CP) pressures were measured during forced air flow from the middle ear (ME) to the nasopharynx across the open ET. Temporal bone CT scans with continuous 0.625 mm thickness were done at a low and standard radiation doses before and during the steady flow (SF) phase of the FRT, after instilling iodinated contrast into the ME and ET, and after the FRT cleared the contrast from the ET. Image analysis was done using the CT scanner console software to view the ET and peritubal structures in standard views and after image reconstructions. Results: The average OP, PS, and CP values were 488 ± 249, 376 ± 101, and 211 ± 62 daPa. While a distinct ET lumen could not be demonstrated during the FRT done with air, CT with intra-luminal contrast clearly demonstrated the entire ET lumen. Post-contrast FRT demonstrated residual contrast outlining the lumen. Conclusions: Standard temporal bone CT doses provided a slightly better signal-to-noise than the low-dose CT for the air but not for the contrast. Combining ETF testing and CT imaging has potential research applications.

Accuracy of the ETDQ-7 for Identifying Persons with Eustachian Tube Dysfunction:

Objective To compare the accuracy of the Eustachian Tube Dysfunction Questionnaire (ETDQ-7) in identifying people with eustachian tube (ET) dysfunction based on symptoms and an objective ET function test. Study Design Cross-sectional study. ObjSettingective Tertiary referral center. Subjects and Methods Fifty-five subjects with and without symptoms suggestive of ET dysfunction completed the ETDQ-7 and had their ET function evaluated by the percentage of middle ear pressure equilibrated after 5 swallows (PEq5) either during a pressure chamber test (intact tympanic membranes) or by the inflation-deflation test (nonintact tympanic membranes). ETDQ-7 score ≥14.5 and PEq5 <60% were used to define ET dysfunction, and sensitivity, specificity, and receiver operating characteristic curves were used to assess the level of association between ETDQ-7 scores and PEq5. Results Twenty-five asymptomatic subjects (group 1 = 15 females, 15 white; mean ± SD age, 32 ± 12.8 years) and 30 subjects with ET dysfunction symptoms (group 2 = 17 females, 25 white; age, 27 ± 16.3 years) were included in the analysis. ETDQ-7 sensitivity and specificity regarding correct group assignment were 70% and 100%, respectively, and with respect to predicting PEq5<60%, 54% and 78%. An area under the curve (AUC) of 0.68 (95% CI, 0.53-0.83) at the participant level and 0.64 (95% CI, 0.50-0.77) at the ear level indicated a moderate level of association that was lower, though not statistically significant, for nonintact tympanic membranes (AUC = 0.63 at the participant level and AUC = 0.49 at the ear level). Conclusion The ETDQ-7 score had a higher correlation with the ET dysfunction symptoms than with an objective measure of ET function.