Decreased Levels of Soluble CD44 in a High-Risk Population following a Smoking Cessation Program.

Tobacco is a risk factor of head and neck cancer (HNC) and smoking cessation alone may reduce HNC risk by 70%. Soluble CD44 (solCD44), a cell surface receptor linked to cell proliferation and migration, and total protein (TP) levels can detect early HNC. This study aims to determine whether salivary solCD44 and TP levels in oral rinses change following a smoking cessation program. 150 smokers provided oral rinse samples at baseline and at a 12-month follow-up after participation in a smoking cessation program. Assays to measure levels of solCD44, TP, and cotinine, a metabolite used as a biomarker of tobacco exposure, were completed. A paired-samples t-test was used to determine whether there was a statistically significant (p < 0.05) mean difference in biomarker levels before and after the program. Baseline and at 12-month follow-up data were available for 88 subjects, 21 of whom quit smoking entirely. Mean levels of solCD44 significantly decreased by 0.412 ng/mL from baseline to the 12-month follow-up, p = 0.010. There was no significant difference in mean TP levels, p = 0.975. Mean cotinine levels decreased significantly by 74.7 ng/mL, p = 0.035. This is the first work demonstrating an association between smoking cessation and decreased solCD44 levels in oral rinses. Decreased expression of the tumorigenic CD44 may be one mechanism by which smoking cessation lowers cancer risk.

Current salivary biomarkers for detection of human papilloma virus-induced oropharyngeal squamous cell carcinoma.

Human papilloma virus (HPV) infection is a key risk factor and etiology for oropharyngeal squamous cell carcinoma (OPSCC). HPV-induced OPSCC is rapidly increasing in incidence, with men experiencing increased mortality. When identified at an early stage, HPV-induced OPSCC can be successfully treated. Diagnosis of HPV-related OPSCC relies on an expert physical examination and invasive biopsy. Since saliva bathes the oropharyngeal mucosa and can be collected noninvasively, saliva obtained via salivary risings is an attractive body fluid for early detection of HPV-induced OPSCC. A plethora of DNA, RNA, and protein salivary biomarkers have been explored. This review discusses these markers and their robustness for detecting oncogenic HPV in OPSCC saliva samples. Methods detecting HPV DNA were more reliable than those detecting RNA, albeit both require time-consuming analyses. Salivary HPV proteomics are a new, promising focus of HPV detection research, and while more practical, lag behind nucleic acid detection methods in their development.

Green Salad Intake Is Associated with Improved Oral Cancer Survival and Lower Soluble CD44 Levels.

Deficiencies in fruit and vegetable intake have been associated with oral cancer (oral cavity and oropharyngeal). Salivary rinses contain measurable biomarkers including soluble CD44 (solCD44) and total protein, which are known markers of oral cancer risk. This study investigates the effect of nutritional factors on solCD44 and protein levels to evaluate oral cancer risk and survival. We evaluated solCD44 and protein levels from 150 patients with oral and oropharyngeal squamous cell carcinoma and 150 frequency-matched controls. We subsequently characterized the effect of food group consumption and these biomarkers on progression-free survival (PFS) and overall survival (OS). Patients reported eating fewer servings of salad (p = 0.015), while controls reported eating fewer servings of potatoes (p < 0.001). Oral cancer patients who consumed at least one serving per week of green salad were found to have significantly lower CD44 levels than those who ate salad less frequently (mean of log2[solCD44]1.73 versus 2.25, p = 0.014). Patients who consumed at least one serving per week of "salad or other vegetables" had significantly longer PFS (median 43.5 versus 9.1 months, p = 0.003, adjusted hazard ratio (HR) = 0.39 p = 0.014) and OS (median 83.6 versus 10 months, p = 0.008, adjusted HR = 0.04 p = 0.029). These findings suggest that dietary factors, namely greater green salad and vegetable intake, may be associated with lower CD44 levels and better prognosis in oral cancer patients.

Ethical surgical triage of patients with head and neck cancer during the COVID-19 pandemic.

BACKGROUND: Coronavirus has serially overtaken our metropolitan hospitals. At peak, patients with acute respiratory distress syndrome may outnumber mechanical ventilators. In our Miami Hospital System, COVID-19 cases have multiplied for 4 weeks and elective surgery has been suspended. METHODS: An Otolaryngologic Triage Committee was created to appropriately allocate resources to patients. Hospital ethicists provided support. Our tumor conference screened patients for nonsurgical options. Patients were tested twice for coronavirus before performing urgent contaminated operations. N95 masks and protective equipment were conserved when possible. Patients with low-grade cancers were advised to delay surgery, and other difficult decisions were made. RESULTS: Hundreds of surgeries were canceled. Sixty-five cases screened over 3 weeks are tabulated. Physicians and patients expressed discomfort regarding perceived deviations from standards, but risk of COVID-19 exposure tempered these discussions. CONCLUSIONS: We describe the use of actively managed surgical triage to fairly balance our patient's health with public health concerns.

CD44 and associated markers in oral rinses and tissues from oral and oropharyngeal cancer patients.

OBJECTIVES: Oral and oropharyngeal squamous cell carcinoma (OOPSCC) is a debilitating disease. Salivary rinses contain soluble tumor markers including CD44 (solCD44) and total protein (TP) that may aid detection and prognosis of these aggressive tumors. Here we aim to examine the relationship between these salivary biomarkers and tissue markers p16 and CD44 and determine whether these markers can predict progression-free survival (PFS) and overall survival (OS). MATERIALS AND METHODS: Prospective study to update biomarkers using oral rinses and tissues from OOPSC patients enrolled between 2007 and 2012 at an academic tertiary referral center. 64 cases from a 300-subject case-control study with archived tissue for immunohistochemistry were included. RESULTS: 82.8% were male, 84.4% were ever smokers, 70.3% had disease stage T3-T4, and 57.8% presented with nodal disease. Nineteen patients (25%) were p16 positive. The group with strong tissue CD44 expression in membrane and cytoplasm had higher levels of solCD44 (mean 10.73 ng/ml) than other groups (5.47 ng/ml) (p = 0.033). TP levels were significantly reduced in oral rinses from subjects with p16 universal gross tumor tissue staining (mean 0.80 vs. 1.08 mg/ml; p = 0.039). On multivariate analysis, universal CD44 gross tissue staining and TP levels ≥ 1 mg/ml demonstrated poorer PFS, with the latter also affecting OS. Poorer survival was associated with soluble CD44 ≥ 5.33 ng/ml and TP ≥ 1 mg/ml. CONCLUSIONS: Direct associations were found between high solCD44 levels and strong membrane and cytoplasmic CD44 expression, and between high TP levels and peripheral/mixed p16 gross staining. Poorer PFS and OS are significantly associated with higher levels of solCD44 and protein in oral rinses.

Meta-analysis comparing outcomes of different transoral surgical modalities in management of oropharyngeal carcinoma.

BACKGROUND: Optimal transoral surgical modality for oropharyneal carcinoma is currently unclear. Transoral laser surgery (TLS), transoral robotic surgery (TORS), and conventional direct transoral (DT) oropharyngectomy are the main current transoral surgical modalities for oropharyngeal carcinoma. METHODS: MEDLINE was systematically searched through PubMed. Reference lists were reviewed. Random-effects models were used to combine studies within each group. Tests for heterogeneity were used to explore difference in effect size between groups in subgroup analysis. RESULTS: Nine studies (404 patients) in TORS arm, five studies (498 patients) in TLS arm, and three studies (335 patients) in DT arm were included. Early T classification (T1-T2) for TORS and DT were higher compared to TLS group (P < .001). There was no significant difference between groups in the rate of invaded margin, post-operative oropharyngeal bleeding, temporary tracheotomy, and gastrostomy dependence. CONCLUSION: The available data do not yet provide clear evidence of superiority of any one modality.

Effective early detection of oral cancer using a simple and inexpensive point of care device in oral rinses.

INTRODUCTION: Head and neck cancer remains a challenging disease that is increasing in incidence with the majority of patients diagnosed at an advanced stage where 5-year survival is approximately 50%. Current approaches including oral-brush biopsies, fluorescence-based technologies,  and salivary molecular profiling have demonstrated some success; however, cost, ease of use, and accuracy remain limiting factors. Areas covered: This is a profile of a novel, easy to use oral rinse point-of-care (POC) test to aid in the diagnosis of oral and oropharyngeal cancer. Background science related to the challenge of oral and oropharyngeal cancer and natural history of diagnostic aids for this disease are provided. Results of studies performed for validation of a POC and laboratory test are also discussed. Expert commentary: The POC test has been validated through a case : control clinical study and a prospective European trial, using version 1.0 (v1.0), which have demonstrated consistent performance including a > 90% negative predictive value, with a sensitivity of 80%. The assay was designed to identify malignant lesions in the oral cavity and oropharynx by improving upon standard clinical assessment.

Immunohistochemistry Analysis of CD44, EGFR, and p16 in Oral Cavity and Oropharyngeal Squamous Cell Carcinoma.

Objectives We analyze the relationship between CD44, epidermal growth factor receptor (EGFR), and p16 expression in oral cavity and oropharyngeal cancers in a diverse population. We also describe whether particular patterns of staining are associated with progression-free survival and overall survival. Study Design Prospective study, single-blind to pathologist and laboratory technologist. Setting Hospital based. Subjects and Methods Immunohistochemistry, comprising gross staining and cellular expression, was performed and interpreted in a blinded fashion on 24 lip/oral cavity and 40 oropharyngeal cancer specimens collected between 2007 and 2012 from participants of a larger study. Information on overall survival and progression-free survival was obtained from medical records. Results Nineteen cases were clinically p16 positive, 16 of which were oropharyngeal. Oral cavity lesions were more likely to exhibit strong CD44 membrane staining ( P = .0002). Strong CD44 membrane and strong EGFR membrane and/or cytoplasmic staining were more common in p16-negative cancers ( P = .006). Peripheral/mixed gross p16 staining pattern was associated with worse survival than the universal staining on univariate and multivariate analyses ( P = .006, P = .030). This held true when combining gross and cellular localization for p16. For CD44, universal gross staining demonstrated poorer overall survival compared with the peripheral/mixed group ( P = .039). CD44 peripheral/mixed group alone and when combined with universal p16 demonstrated the best survival on multivariate analysis ( P = .010). Conclusion In a diverse population, systematic analysis applying p16, CD44, and EGFR gross staining and cellular localization on immunohistochemistry demonstrates distinct patterns that may have prognostic potential exceeding current methods. Larger studies are warranted to investigate these findings further.

Effect of actionable somatic mutations on racial/ethnic disparities in head and neck cancer prognosis.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and minorities have the worst survival. However, the molecular mechanisms underlying survival disparities have not been elucidated. METHODS: In a retrospective study, we assessed association between HNSCC early death (<2 years) and 208 somatic mutations of 10 cancer-related genes in 214 patients: 98 non-Hispanic whites (46%), 72 Hispanic whites (34%), and 44 African Americans (20%). RESULTS: Hispanic whites and African Americans had significantly higher mutation rates for EGFR, HRAS, KRAS, and TP53. HNSCC early death was significantly associated with 3+ mutations (odds ratio [OR] = 2.78, 95% confidence interval [CI] = 1.16, 6.69), NOTCH1 mutations in non-Hispanic whites (OR = 5.51; 95% CI = 1.22-24.83) and TP53 mutations in Hispanic whites (OR = 3.84; 95% CI = 1.08-13.68) in multivariable analysis adjusted for age, sex, tumor site, and tumor stage. CONCLUSION: We have provided the proof-of-principal data to link racial/ethnic-specific somatic mutations and HNSCC prognosis and pave the way for precision medicine to overcome HNSCC survival disparities. © 2016 Wiley Periodicals, Inc. Head Neck 38:1234-1241, 2016.

Risk Stratification System for Oral Cancer Screening.

Oral cavity and oropharyngeal cancer (oral cancer) is a deadly disease that is increasing in incidence. Worldwide 5-year survival is only 50% due to delayed intervention with more than half of the diagnoses at stage III and IV, whereas earlier detection (stage I and II) yields survival rates up to 80% to 90%. Salivary soluble CD44 (CD44), a tumor-initiating marker, and total protein levels may facilitate oral cancer risk assessment and early intervention. This study used a hospital-based design with 150 cases and 150 frequency-matched controls to determine whether CD44 and total protein levels in oral rinses were associated with oral cancer independent of age, gender, race, ethnicity, tobacco and alcohol use, and socioeconomic status (SES). High-risk subjects receiving oral cancer prevention interventions as part of a community-based program (n = 150) were followed over 1 year to determine marker specificity and variation. CD44 ≥5.33 ng/mL was highly associated with case status [adjusted OR 14.489; 95% confidence interval (CI), 5.973-35.145; P < .0001, vs. reference group CD44 <2.22 ng/mL and protein <1.23 mg/mL]. Total protein aided prediction above CD44 alone. Sensitivity and specificity in the frequency-matched study was 80% and 48.7%, respectively. However, controls were not representative of the target screening population due, in part, to a high rate of prior cancer. In contrast, specificity in the high-risk community was 74% and reached 95% after annual retesting. Simple and inexpensive salivary CD44 and total protein measurements may help identify individuals at heightened risk for oral cancer from the millions who partake in risky behaviors. Cancer Prev Res; 9(6); 445-55. ©2016 AACR.

CD44 interacts with EGFR and promotes head and neck squamous cell carcinoma initiation and progression.

OBJECTIVES: CD44 is a promising target for therapy in head and neck squamous cell carcinoma (HNSCC) and has two defined roles in tumorigenesis: it is a cancer stem cell (CSC) marker and it promotes migration and proliferation through interaction with many signaling molecules. The purpose of this study was to investigate the role of CD44 in HNSCC carcinogenesis. MATERIALS AND METHODS: The effects of CD44 in cell proliferation, migration, apoptosis and cisplatin resistance were studied by its overexpression in HNSCC cells. We also evaluated the effect of CD44 on tumor progression by siRNA methodology, immunohistochemistry (IHC) and western blot analysis. CD44 and EGFR colocalization were examined in CAL 27 cells by laser scanning confocal microscopy. The interaction between CD44 and EGFR was analyzed by immunoprecipation. RESULTS: Overexpression of CD44 enhances cell proliferation and migration and correlates with increased cisplatin resistance and apoptosis inhibition in SCC25 cells. Downregulation of CD44 in CAL27 cells inhibited constitutive EGFR phosphorylation and significantly reduced tumor growth in nude mice. CD44 and EGFR colocalized in CAL 27 cells. CD44 coimmunoprecipated with EGFR in CAL 27 cells, indicating that these proteins interact with each other. CONCLUSION: CD44 therapy in HNSCC may target the CSC population and alter EGFR signaling.

Salivary protein and solCD44 levels as a potential screening tool for early detection of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a devastating disease usually diagnosed at a late stage when cure rates are 40%. We examined a simple and inexpensive molecular tool that may aid HNSCC detection. METHODS: Building on prior findings that total protein levels are elevated in 102 HNSCC cases versus 84 control subjects, we further analyzed these levels with respect to important risk and demographic variables and compared the results to soluble CD44 (solCD44). Using multivariate adaptive regression splines (MARSs)-logit modeling and logistic regression, we determined whether total protein, solCD44, or the combination best identifies HNSCC. RESULTS: Combined higher levels of solCD44 and protein were significantly associated with HNSCC (odds ratio [OR] = 24.90; 95% confidence interval [CI], 9.04-68.57; area under the curve [AUC] = 0.786). A model including protein plus solCD44 resulted in a better area (AUC 0.796) than either marker alone. CONCLUSION: Oral rinse levels of solCD44 and protein seem to hold promise for detection of HNSCC.

Salivary markers and risk factor data: a multivariate modeling approach for head and neck squamous cell carcinoma detection.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease largely due to late stage diagnosis. Prior work indicates that soluble CD44 (solCD44) and total protein may be useful diagnostic markers for HNSCC. In this study we combine the markers solCD44, IL-8, HA, and total protein with demographic and risk factor data to derive a multivariate logistic model that improves HNSCC detection as compared to our previous data using biomarkers alone. METHODS: We performed the solCD44, IL-8, HA, and total protein assays on oral rinses from 40 HNSCC patients and 39 controls using ELISA assays. Controls had benign diseases of the upper aerodigestive tract and a history of tobacco or alcohol use. All subjects completed a questionnaire including demographic and risk factor data. RESULTS: Depending on cancer subsite, differences between cases and controls were found for all markers. A multivariate logistic model including solCD44, total protein and variables related to smoking, oral health and education offered a significant improvement over the univariate models with an AUC of 0.853. Sensitivity ranged from 75-82.5% and specificity from 69.2-82.1% depending on predictive probability cut points. CONCLUSION: A multivariate model, including simple and inexpensive molecular tests in combination with risk factors, results in a promising tool for distinguishing HNSCC patients from controls. IMPACT: In this case-control study, the resulting observations led to an unprecedented multivariate model that distinguished HNSCC cases from controls with better accuracy than the current gold standard which includes oral examination followed by tissue biopsy. Since the components are simple, noninvasive, and inexpensive to obtain, this model combining biomarkers, risk factor and demographic data serves as a promising prototype for future cancer detection tests.

Molecular markers of micrometastasis in oral cavity carcinomas.

OBJECTIVE: To examine the expression of candidate markers for micrometastasis. STUDY DESIGN: Cross-sectional analysis of subjects with oral cavity carcinomas who underwent sentinel lymph node biopsy (SLNB) and subsequent immunohistochemical (IHC) analysis. SUBJECTS AND METHODS: Two groups were identified based on SLNB status: negative SLNB (19/30) and positive SLNB (11/30). Specimens underwent IHC using conjugated monoclonal antibodies for membrane type-1 matrix metalloproteinase (MT1-MMP), CD44, focal adhesion kinase-1, and E-cadherin. Staining results were evaluated to determine if a particular marker was associated with SLNB status or other histopathologic prognosticators. RESULTS: For MT1-MMP, 21 percent (3/14) of evaluable specimens stained positively in the SLNB(-) group and 67 percent (4/6) stained positively in the SLNB(+) group (P=0.12). No statistically significant association was seen between any marker's staining pattern and SLNB status alone. When MT1-MMP staining was evaluated in tumors with SLNB(+) or perineural invasion (PNI) present on histopathology, six of nine specimens (67%) stained positively for MT1-MMP, vs one of 11 (9%) in specimens lacking either negative prognosticator (P=0.016, RR=7.33). CONCLUSION: Preliminary results suggest that MT1-MMP positivity in primary tumor specimens may identify aggressive tumor types, evidenced by the presence of micrometastasis or PNI.

Soluble CD44 is a potential marker for the early detection of head and neck cancer.

INTRODUCTION: Head and neck squamous cell carcinoma (HNSCC) is a devastating and deadly disease, largely because it is diagnosed in late stage. Cure rates, currently at 50%, could increase to >80% with early detection. In this study, we evaluate soluble CD44 (solCD44) as an early detection tool for HNSCC by determining whether it reliably distinguishes HNSCC from benign disease of the upper aerodigestive tract. METHODS: We carried out the solCD44 ELISA on oral rinses from 102 patients with HNSCC and 69 control patients with benign diseases of upper aerodigestive tract to determine the sensitivity and specificity of the test for differentiating HNSCC from benign disease. Furthermore, we did a pilot study using methylation-specific PCR primers on oral rinses from 11 HNSCC patients with low solCD44 levels and 10 benign disease controls. RESULTS: Mean salivary solCD44 levels were 24.4 +/- 32.0 ng/mL for HNSCC patients (range, 0.99-201 ng/mL) and 9.9 +/- 16.1 ng/mL (range, 0.73-124 ng/mL) for the patients with benign disease (P < 0.0001). Depending on cutoff point and HNSCC site, sensitivity ranged from 62% to 70% and specificity ranged from 75% to 88%. Nine of 11 HNSCC and 0 of 10 controls with low solCD44 levels showed hypermethylation of the CD44 promoter. CONCLUSIONS: SolCD44 is elevated in the majority of HNSCC and distinguishes cancer from benign disease with high specificity. Whereas the solCD44 test lacks sensitivity by itself, methylation status of the CD44 gene seems to complement the solCD44 test. Our pilot data indicate that, together, these markers will detect HNSCC with very high sensitivity and specificity.

Characterization of CD44v3-containing isoforms in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease that is only cured 50% of the time. A better understanding of the molecular mechanisms involved in HNSCC progression may lead to earlier detection and improved cure rates. CD44 is a ubiquitous transmembrane glycoprotein comprising a family of alternatively spliced isoforms involved in cell migration and cell proliferation. CD44 isoforms containing the variant 3 (v3) exon include a growth factor binding site and may be involved in tumor progression. To characterize CD44v3-containing isoforms expression in HNSCC we purified RNA from four HNSCC cell lines and performed RT-PCR using junction primer strategies followed by gel elecrophoresis. Cloning and sequencing of HNSCC cell line PCR products revealed two isoforms. One of these, CD44v3-10, has been previously described. The other isoform, CD44v3, has not been characterized in HNSCC tissues. To further study this isoform, we purified RNA from 19 HNSCC tissues, 7 normal margin tissues and 5 true normal tissues. Following reverse-transcription, we performed quantitative PCR using junction primers specific for CD44v3. Results show that HNSCC tumor tissues expressed mean CD44v3 levels that were elevated 4.5 times more than true normal tissues (p < 0.01). Mean CD44v3 values for HNSCC tumors were 0.43 +/- 0.44 while mean levels for true normal tissues were 0.10 +/- 0.11. Levels in tumor tissue did not vary significantly with tumor characteristics such as site, stage, prior treatment, or nodal status. In addition, to characterize the role of this molecule plays in tumor progression, we overexpressed CD44v3 in a HNSCC cell line. Our results indicate that although higher levels of CD44v3 did not affect the rate of proliferation, a significant increase in migration was observed. CD44v3 may provide a target for future diagnostic and therapeutic interventions for HNSCC.

Complete hypopharyngeal obstruction by mucosal adhesions: a complication of intensive chemoradiation for advanced head and neck cancer.

BACKGROUND: Severe swallowing dysfunction is the dominant long-term complication observed in patients treated for head and neck squamous cell carcinoma (HNSCC) with treatment protocols using intensive concurrent chemotherapy with radiation therapy (chemo/XRT). We identified a subset of these patients, who were seen with complete obstruction of the hypopharynx distal to the site of the primary cancer, and in whom we postulate that the obstruction was caused by separable mucosal adhesions rather than obliteration by a mature fibrous stricture. METHODS: Seven patients were referred to the senior author with a diagnosis of complete hypopharyngeal obstruction between 1992 and 2001. The diagnosis was confirmed by barium swallow imaging and/or endoscopy before referral in all patients. Patients underwent recanalization by passing a Jesberg esophagoscope under general anesthesia, followed by serial dilations and intensive swallowing therapy. Patient charts were reviewed retrospectively after institutional review board approval. RESULTS: All seven patients were successfully recanalized. No patient had a perforation or other significant complication related to the recanalization procedure or subsequent dilations. Five of the seven patients showed improvement in swallowing at some point after the initial procedure, but just two patients recovered sufficiently to have their gastrostomy tube removed permanently. CONCLUSIONS: We conclude that complete hypopharyngeal obstruction secondary to mucosal adhesions is one cause of gastrostomy tube dependence in patients who have been treated with chemo/XRT for HNSCC. It is a difficult problem to treat, but most patients can recover useful swallowing function without undergoing laryngectomy or major surgical reconstruction. The postulated pathophysiology has implications for prevention as well as treatment.