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.

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.

Superimposing polymorphism: the case of a point mutation within a polymorphic Alu insertion.

The COL3A1 Alu insertion is a member of the AluY subfamily. It has been found to be absent in non-human primates and polymorphic in worldwide human populations. The integration of the element into the human genome seems to have preceded the initial migration(s) of anatomically modern humans out of the African continent. Although the insertion has been detected in populations from all the continents, its highest frequency values are located within sub-Saharan Africa. The sequence alignment of the COL3A1 insertion from several African individuals revealed a bi-allelic single nucleotide polymorphism (SNP) at the downstream terminus of the element's poly-A tract. Once discovered, a selective PCR procedure was designed to determine the frequency of both alleles in 19 worldwide populations. The A-allele in this binary SNP experiences a clinal increase in the eastward direction from Africa to Southeast Asia and Mongolia, reaching fixation in the two latter regions. The T variant, on the other hand, exhibits a westward clinal increase outside of Africa, with its lowest frequency in Asia and achieving fixation in northern Europe. The presence of this internal SNP extends the usefulness provided by the polymorphic Alu insertion (PAI). It is possible that superimposing polymorphisms like this one found in the COL3A1 locus may accentuate signals from genetic drift events allowing for visualization of recent dispersal patterns.

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.

Single-nucleotide variant in multiple copies of a deleted in azoospermia (DAZ) sequence - a human Y chromosome quantitative polymorphism.

The evolution of the deleted in azoospermia (DAZ) gene family supports prevalent theories on the origin and development of sex chromosomes and sexual dimorphism. The ancestral DAZL gene in human chromosome 3 is known to be involved in germline development of both males and females. The available phylogenetic data suggest that some time after the divergence of the New World and Old World monkey lineages, the DAZL gene, which is found in all mammals, was copied to the Y chromosome of an ancestor to the Old World monkeys, but not New World monkeys. In modern man, the Y-linked DAZ gene complex is located on the distal part of the q arm. It is thought that after being copied to the Y chromosome, and after the divergence of the human and great ape lineages, the DAZ gene in the former underwent internal rearrangements. This included tandem duplications as well as a T > C transition altering an MboI restriction enzyme site in a duplicated sequence. In this study, we report on the ratios of MboI-/MboI+ variant sequences in individuals from seven worldwide human populations (Basque, Benin, Egypt, Formosa, Kungurtug, Oman and Rwanda) in the DAZ complex. The ratio of PCR MboI- and MboI+ amplicons can be used to characterize individuals and populations. Our results show a nonrandom distribution of MboI-/MboI+ sequence ratios in all populations examined, as well as significant differences in ratios between populations when compared pairwise. The multiple ratios imply that there have been more than one recent reorganization events at this locus. Considering the dynamic nature of this locus and its involvement in male fertility, we investigated the extent and distribution of this polymorphism.