Erika Reategui

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. (Cancer Epidemiol Biomarkers Prev 2007;16(7):1348–55)

Salivary Soluble CD44: A Potential Molecular Marker for Head and Neck Cancer

Objective: Head and neck squamous cell carcinoma (HNSCC) is a debilitating disease which is cured only 50% of the time. If diagnosed early, survival rates could reach 80%, but there is currently no practical method for early detection. CD44 comprises a family of isoforms that, in certain tumors, are alternatively spliced and overexpressed in tissues and circulation. Here we examine salivary soluble CD44 (solCD44) expression in HNSCC patients and normal controls to determine its potential as a screening tool. Method: We did a solCD44 ELISA on saliva from 26 HNSCC patients, 10 normal volunteers, conditioned media (CM) of 4 HNSCC cell lines, and 1 CD44-negative cell line (COS-7). Western blot was done on CM from 2 HNSCC cell lines (UMSS11B and FaDu), COS-7, 3 HNSCC, and 2 normal saliva specimens to verify ELISA antibody specificity. SolCD44 levels were significantly elevated in HNSCC patients compared with normal controls (7.85 ng/mL for HNSCC patients and 1.09 ng/mL for normal controls, P < 0.001). Results: The test detected 79% of mucosally invasive HNSCC using preliminary cutoff points. SolCD44 levels did not vary significantly with tumor size, stage, recurrence, history of radiation treatment, or tobacco and alcohol risk factors. A 65 to 75 kDa band, corresponding to solCD44, was detected in all of the HNSCC cell line CM and saliva whereas normal samples showed a fainter band or were undetectable. Conclusion: In this preliminary analysis, the salivary solCD44 ELISA seems to effectively detect HNSCC at all stages. Further study is indicated because early detection is clearly important in this disease.

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.

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 RTPCR 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 reversetranscription, 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

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

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.

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.

Abstract LB-272: Effect of smoking cessation on soluble CD44 levels in head and neck cancer

In the past year, an estimated 55,070 individuals were diagnosed with head and neck cancer and approximately 12,000 related deaths occurred. This disease is devastating to the patients and families it affects due to its significant impact on swallowing, speech and appearance. Tobacco, the single largest risk factor for head and neck cancer, accounts for an estimated 85% of head and neck cancers. Carcinogens found in tobacco smoke induce damage to DNA leading to mutations and DNA strand breaks. When not correctly repaired, overexpression of gene products including CD44, a cancer stem cell marker, occurs. CD44 can be cleaved into a soluble form (solCD44) that is found in body fluids. CD44 levels in oral rinses distinguish head and neck cancer patients from controls with around 62% sensitivity and 88% specificity. CD44 becomes abnormally expressed in dysplasia and elevated levels of solCD44 have been found prior to clinical evidence of disease, in some cases. In this study we investigate changes in solCD44 levels with smoking cessation. 150 smokers from a South Florida community known to be at high-risk for oral cancer were recruited and enrolled in smoking cessation programs. At baseline, questions on smoking status, frequency and history were administered to participants. Oral rinses and saliva samples were also collected to determine levels of solCD44 and cotinine, which was used to confirm quit status, using ELISA assays. One year after enrollment (± 3 weeks), samples and questionnaires were taken again from participants to identify quitters and measure their CD44 levels. Results were then computed and analyzed by paired t-test using SAS University Edition. So far, thirty-seven participants (24.6%) reported quitting smoking but only half of them (n = 13) were confirmed by cotinine ( Based on this data, a larger number of quitters and/or an additional time points post quitting are needed as it might reveal more profound results. Such results would be critical, offering smoking cessation as a means for subjects with high levels of CD44 to decrease their risk of progression. Citation Format: Faisal F. Alotaibi, Turki M. Almuhaimid, Erika Reategui, Elizabeth Franzmann, Jarrard Goodwin. Effect of smoking cessation on soluble CD44 levels in head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-272. doi:10.1158/1538-7445.AM2015-LB-272

Abstract 3548: CD44, protein, demographics and risk factor data: A combined approach to detect head and neck squamous cell carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease. The main risk factors are tobacco and alcohol use and human papillomavirus (HPV) infection. Early detection tests are needed because the majority of patients present in late stage when cure rates reach only 40%. Our group has developed a simple, inexpensive, noninvasive diagnostic test based on soluble CD44 (solCD44) and total protein levels. We used a case-control design to evaluate soluble markers for HNSCC in 150 oropharyngeal (OP) and lip/oral cavity (Lip/OC) patients and 150 controls frequency matched for age, gender, race, ethnicity, tobacco use and socioeconomic status. We compared patient groups with respect to important covariates using the chi-square, Fishers exact test, or t-test. Markers’ mean levels were compared either by t-test or ANOVA, followed by pairwise multiple comparisons. Logistic regression analysis was used to evaluate predictivity of the salivary markers univariately and multivariately with adjustment for demographics and risk factors. We report odds ratio (OR) estimates with corresponding 95% confidence interval (95% CI) and area under the curve (AUC) of the operating characteristic curve (ROC) for fitted models. The case-control groups did not differ in regards to age, gender, race, ethnicity, history of ever vs. never smoking, current alcohol use, number of teeth removed, or county vs. private hospital system. The mean log2CD44 and protein levels were elevated in cases (log2 CD44= 1.94 ng/ml, protein=0.93 mg/ml) compared to controls (log2 CD44= 1.28 ng/ml, protein= 0.76 mg/ml), (p<.0001; p=.003, respectively). Log2CD44 levels were significantly elevated in older vs. younger cases (p<0.05). There were no significant mean log2 CD44 level differences between Lip/OC and OP cases, TNM or HPV status in cancer patients. In the cases for which HPV status was available (as measured by the surrogate marker p16), log 2 CD44 levels varied by smoking status (lower in never smokers), by drinking status in HPV + cases (lower in non-drinkers) and N-stage (higher levels in N0, Nx vs. N1-N3 in HPV-positive and the opposite effect in HPV-negative tumors). For protein, there were no differences in either the case or the control group based on demographic or risk variables. When we stratified by HPV status, race/ethnicity and drinking status did have an effect (higher levels in blacks vs. WNH and higher in current vs. former and never/mild drinkers in HPV positive tumors only). A specific model was developed for each of the 3 race/ethnicity groups with the highest AUC for blacks (AUC= .835) and WNH (AUC= .831) followed by HW (AUC= .777). Models included log2CD44, protein, smoking, gender and age; strongly suggesting that these factors play an important role for HNSCC early detection studies. Citation Format: Lutecia H. Mateus Pereira, Isildinha Reis, Robert Duncan, Judy Wen, Erika Reategui, Stephanie Bayers, Laurian Walters, Aymee Perez, Jennifer Hu, W Jarrard Goodwin, Elizabeth J. Franzmann. CD44, protein, demographics and risk factor data: A combined approach to detect head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3548. doi:10.1158/1538-7445.AM2013-3548

Abstract B83: Acceptability of a rinse technique for diagnosing head and neck squamous cell carcinoma among African Americans.

Background/Objective: Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease, particularly among African Americans. Cure rates increase from 30% to 80% if the disease is detected early. This study evaluated the acceptability of a non-invasive, easy to administer, very affordable, and innovative rinse technique that assays the soluble form of CD44 and total protein as HNSCC early detection markers in a sample of African American men and women. The aim of the study was to determine whether the rinse was acceptable and whether the outcome of the rinse was likely to promote screening behavior. Methods: Participants (N=127) were from underserved, low-income (83% of sample reported annual income of less than

10:10 AM: Salivary Soluble CD44 Levels in Head and Neck Cancer

15,000), African American backgrounds (mean age = 51.24). Men (n=73) and women (n=53) were administered a questionnaire to capture satisfaction, intention to repeat the test, and whether the results of the test would affect screening behavior after using the rinse. Nearly 100% of the sample was currently smoking or using nicotine products. Descriptive statistics, t-tests and Analysis of Variance (ANOVA) were employed using SPSS 19 (IBM). Results: Findings showed that using the rinse was highly acceptable to African American men and women (i.e., philosophy behind use, ease of rinse, willingness to repeat, perceived willingness of others in their social group to use rinse, all mean scores = strongly agree). With regard to the rinses potential effects of behavior, mean scores indicate that participants strongly agreed that: (a) they would act on a positive cancer finding based on the rinse by scheduling an appointment for treatment, (b) they would perform the rinse as prescribed, (c) they would engage in preventative behaviors if the rinse indicated a high probability of getting HNC, (d) they would stop smoking if the rinse indicated a high probability of getting HNC, and (e) people who are at high risk of HNC should stop smoking. Acceptability of the rinse and the rinses potential effects on behavior did not differ between males and females (all p-values of t-tests > .05) or based on educational attainment categories (all p-values of ANOVAs > .05). Compared to currently unemployed participants, those who were employed reported that: the rinse was harder to do (t(1,124)=-2.37, p=.019); were more likely to schedule a treatment appointment based on the rinses outcome (t(1,124)=3.54, p=.001); they were more likely to rinse as prescribed (t(1,124)=2.84, p=.003); and they were more likely to take action to prevent HNSCC based on the determination of the rinse (t(1,124)=3.04, p=.003). Interestingly, individuals with health care coverage reported that the test was harder to do (F(2, 123)=4.92, p=.009), but did not differ from uninsured participants on any other variables (all p-values of ANOVAs > .05). Discussion: Findings indicate that a simple, easy to use rinse technique is acceptable to patients and is highly likely to promote screening behavior among African Americans at risk for HNSCC. Differences were noted between insured versus uninsured participants, employed versus unemployed participants but no differences were observed between men and women and between groups based on educational attainment. Citation Format: Suzanne C. Lechner, Lutecia H. Mateus Pereira, Erika Reategui, Claudia Gordon, Jamie Studts, Margaret Byrne, Monica Webb Hooper, David J. Lee, Elizabeth J. Franzmann. Acceptability of a rinse technique for diagnosing head and neck squamous cell carcinoma among African Americans. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B83.