Joel L. Schwartz

Transcriptomic dissection of tongue squamous cell carcinoma

BackgroundThe head and neck/oral squamous cell carcinoma (HNOSCC) is a diverse group of cancers, which develop from many different anatomic sites and are associated with different risk factors and genetic characteristics. The oral tongue squamous cell carcinoma (OTSCC) is one of the most common types of HNOSCC. It is significantly more aggressive than other forms of HNOSCC, in terms of local invasion and spread. In this study, we aim to identify specific transcriptomic signatures that associated with OTSCC.ResultsGenome-wide transcriptomic profiles were obtained for 53 primary OTSCCs and 22 matching normal tissues. Genes that exhibit statistically significant differences in expression between OTSCCs and normal were identified. These include up-regulated genes (MMP1, MMP10, MMP3, MMP12, PTHLH, INHBA, LAMC2, IL8, KRT17, COL1A2, IFI6, ISG15, PLAU, GREM1, MMP9, IFI44, CXCL1), and down-regulated genes (KRT4, MAL, CRNN, SCEL, CRISP3, SPINK5, CLCA4, ADH1B, P11, TGM3, RHCG, PPP1R3C, CEACAM7, HPGD, CFD, ABCA8, CLU, CYP3A5). The expressional difference of IL8 and MMP9 were further validated by real-time quantitative RT-PCR and immunohistochemistry. The Gene Ontology analysis suggested a number of altered biological processes in OTSCCs, including enhancements in phosphate transport, collagen catabolism, I-kappaB kinase/NF-kappaB signaling cascade, extracellular matrix organization and biogenesis, chemotaxis, as well as suppressions of superoxide release, hydrogen peroxide metabolism, cellular response to hydrogen peroxide, keratinization, and keratinocyte differentiation in OTSCCs.ConclusionIn summary, our study provided a transcriptomic signature for OTSCC that may lead to a diagnosis or screen tool and provide the foundation for further functional validation of these specific candidate genes for OTSCC.

In vivo Imaging of Oral Mucositis in an Animal Model Using Optical Coherence Tomography and Optical Doppler Tomography

Purpose: To assess noninvasive optical coherence tomography (OCT) and optical Doppler tomography (ODT) for early detection and evaluation of chemotherapy-induced oral mucositis. Experimental Design: Cheek pouches of 10 Syrian golden hamsters were imaged using OCT/ODT during development of chemotherapy-induced mucositis. I.p. injections of 5-fluorouracil and mechanical irritation induced oral lesions. At 2, 4, 7, and 11 days, one hamster was sacrificed and processed for histopathology. OCT images were visually examined; ODT results were semiquantified. Imaging data were compared with histologic findings. Results: During the development of mucositis, OCT/ODT identified the following events: (a) change in epithelial thickness (beginning on day 2), (b) loss of surface keratinized layer continuity (beginning on day 4), (c) loss of epithelial (day 4 onwards) and submucosal integrity (day 7 onwards), (d) changes in axial blood flow velocity (increased on days 2 and 4; decreased on day 7), and (e) changes in blood vessel size (diameter doubled on day 2; quadrupled on day 4; unchanged on day 7). The semiquantitative imaging-based scoring system identified the severity of mucositis as defined by histopathology. The combination of imaging criteria used allowed for the detection of early, intermediate, and late mucositic changes. Imaging data gave higher scores compared with clinical scores early on, suggesting that the imaging-based diagnostic scoring was more sensitive to early mucositic change than the clinical scoring system. Once mucositis was established, imaging and clinical scores converged. Conclusion: OCT/ODT identified chemotherapy-induced oral changes before their clinical manifestation, and the proposed scoring system for oral mucositis was validated for the semiquantification of mucositic change.

RNA from brush oral cytology to measure squamous cell carcinoma gene expression

BACKGROUND RNA expression analysis of oral keratinocytes can be used to detect early stages of disease such as oral cancer or to monitor on-going treatment responses of the same or other oral diseases. A limitation is the inability to obtain high quality RNA from oral tissue without using biopsies. While oral cytology cell samples can be obtained from patients in a minimally invasive manner they have not been validated for quantitative analysis of RNA expression. METHODS As a starting point in the analysis of tumor markers in oral squamous cell carcinoma (OSCC), we examined RNA in brush cytology samples from hamsters treated with dibenzo[a,l]pyrene to induce oral carcinoma. Three separate samples from each animal were assessed for expression of candidate marker genes and control genes measured with real-time RT-PCR. RESULTS Brush oral cytology samples from normal mucosa were shown to consist almost exclusively of epithelial cells. Remarkably, ss-2 microglobulin and cytochrome p450, 1B1 (CYP1B1) RNA showed potential utility as markers of OSCC in samples obtained in this rapid and non-surgical manner. CONCLUSION Brush oral cytology may prove useful as a source of RNA for gene expression analysis during the progression of diseases of the oral epithelium such as OSCC.

Analysis of RNA from brush cytology detects changes in B2M, CYP1B1 and KRT17 levels with OSCC in tobacco users

RNA expression analysis of oral keratinocytes can be used to detect early oral cancer, but a limitation is the inability to obtain high quality RNA from oral tissue without using biopsies. While oral cytology cell samples can be obtained from patients in a minimally invasive manner, they have not been validated for quantitative analysis of RNA expression. Earlier we showed RNA from brush cytology of hamster Oral Squamous Cell Carcinoma (OSCC) demonstrated differential expression of B2M and CYP1B1 using real time RT-PCR in a dibenz[a,I]pyrene, tobacco carcinogen, induced model of this disease. Here we show reproducibility of this approach to measuring gene expression in humans. Cytology brush samples from 12 tobacco and betel related OSCC and 17 nonmalignant oral lesions revealed B2M mRNA was enriched in tumor samples while CYP1B1 mRNA was reduced, similar to what was seen in the model system. Additionally, we showed that KRT17 mRNA, a gene linked to OSCC in another brush cytology study, was also enriched in OSCC versus nonmalignant lesions, again supporting the promise of using RNA from brush oral cytology to reproducibly monitor oral gene expression.

Similar Squamous Cell Carcinoma Epithelium microRNA Expression in Never Smokers and Ever Smokers

The incidence of oral tumors in patients who never used mutagenic agents such as tobacco is increasing. In an effort to better understand these tumors we studied microRNA (miRNA) expression in tumor epithelium of never tobacco users, tumor epithelium of ever tobacco users, and nonpathological control oral epithelium. A comparison of levels among 372 miRNAs in 12 never tobacco users with oral squamous cell carcinoma (OSCC) versus 10 healthy controls was made using the reverse transcription quantitative polymerase chain reaction. A similar analysis was done with 8 ever tobacco users with OSCC. These comparisons revealed miR-10b-5p, miR-196a-5p, and miR-31-5p as enriched in the tumor epithelium in OSCC of both never and ever tobacco users. Examination of The Cancer Genome Atlas (TCGA) project miRNA data on 305 OSCCs and 30 controls revealed 100% of those miRNAs enriched in never smoker OSCCs in this patient group were also enriched in ever smoker OSCCs. Nonsupervised clustering of TCGA OSCCs was suggestive of two or four subgroups of tumors based on miRNA levels with limited evidence for differences in tobacco exposure among the groups. Results from both patient groups together stress the importance of miR196a-5p in OSCC malignancy in both never and ever smokers, and emphasize the overall similarity of miRNA expression in OSCCs in these two risk groups. It implies that there may be great similarity in etiology of OSCC in never and ever smokers and that classifying OSCC based on tobacco exposure may not be helpful in the clinic.

microRNA from brush biopsy to characterize oral squamous cell carcinoma epithelium

Few cancers are diagnosed based on RNA expression signatures. Oral squamous cell carcinoma (OSCC) is no exception; it is currently diagnosed by scalpel biopsy followed by histopathology. This study sought to identify oral tumor epithelial microRNA (miRNA) expression changes to determine if these changes could be used to diagnose the disease noninvasively. Analysis of miRNA profiles from surgically obtained OSCC tissue, collected under highly standardized conditions for The Cancer Genome Atlas, was done to determine the potential accuracy in differentiating tumor from normal mucosal tissue. Even when using small 20 subject datasets, classification based on miRNA was 90 to 100% accurate. To develop a noninvasive classifier for OSSC, analysis of brush biopsy miRNA was done and showed 87% accuracy in differentiating tumor from normal epithelium when using RT‐qPCR or miRNAseq to measure miRNAs. An extensive overlap was seen in differentially expressed miRNAs in oral squamous cell carcinoma epithelium obtained using brush biopsy and those reported in saliva and serum of oral squamous cell carcinoma patients in several studies. This suggested that nonselective release of these miRNAs into body fluids from tumor epithelium was largely responsible for the changes in levels in these fluids seen with this disease. Using a variation in mirRPath we identified the KEGG pathway of neurotrophin signaling as a target of these miRNAs disregulated in tumor epithelium. This highlights the utility of brush biopsy of oral mucosa to allow simple acquisition of cancer relevant miRNA information from tumor epithelium.

Brand specific responses to smokeless tobacco in a rat lip canal model

BACKGROUND Different compositions of smokeless tobacco (ST) are widely thought to cause oral carcinoma at different rates but there is little direct evidence for this hypothesis. METHODS We used a rat lip canal model to examine the mucosal changes induced by chronic daily exposure to four different brands of ST: Skoal, Copenhagen, Ettan Swedish Snus, and Stonewall, differing in measured levels of: tobacco specific nitrosamines (TSNAs), unprotonated nicotine, moisture, and pH. RESULTS Exposure to the lip canal for 12 months produced changes in the mucosa marked by increases in S phase and M phase cells for the Skoal and Copenhagen exposed rats. This correlated with the high level of TSNAs and nicotine in these products. All the tobacco products, to different degrees, induced sites of moderate to severe dysplasia some with extensive rete peg outgrowth from the oral mucosa not seen in the controls. Many of these sites showed a loss of p16 expression. CONCLUSIONS While all ST products caused dysplasia, the products with lower levels of TSNAs and unprotonated nicotine caused less, consistent with the model that tobacco with low levels of nitrosamines might potentially induce fewer carcinomas in human users.

Modulations of benzo[a]pyrene-induced DNA adduct, cyclin D1 and PCNA in oral tissue by 1,4-phenylenebis(methylene)selenocyanate

Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.

A prototype tobacco-associated oral squamous cell carcinoma classifier using RNA from brush cytology

BACKGROUND Oral cancer in the form of squamous cell carcinoma (OSCC) is typically detected in advanced stages when treatment is complex and may not be curative. The need for surgical biopsy may contribute to delays in diagnosis and impede early detection. Multiple studies of RNA from surgically obtained tumor samples have revealed many genes differentially expressed with this disease. We sought to determine whether the identified mRNAs could be used as markers by a non-invasive detection system for OSCC using RNA from brush cytology. METHODS Levels of mRNAs from 21 genes known to be differentially expressed in head and neck squamous cell carcinoma surgical samples, compared with controls, were shown to be quantifiable in oral brush cytology samples. These mRNAs were quantified in a training set of 14 tumor and 20 non-malignant brush cytology samples from tobacco/betel nut users. With the measurement of two additional mRNAs and analysis using support vector machines algorithm for class prediction of these cancers was produced. RESULTS This OSCC classifier based on the levels of 5 mRNAs in RNA from brush cytology initially showed 0.93 sensitivity and 0.91 specificity in differentiating OSCC from benign oral mucosal lesions based on leave-one-out cross-validation. When used on a test set of 19 samples from 6 OSCCs and 13 non-malignant oral lesions, we found misclassification of only one OSCC and one benign lesion. CONCLUSIONS This shows the promise of using RNA from brush cytology for early OSCC detection and the potential for clinical usage of this non-invasive classifier.

Gene expression based evidence of innate immune response activation in the epithelium with oral lichen planus

OBJECTIVE Oral lichen planus (OLP) is a disease of the oral mucosa of unknown cause producing lesions with an intense band-like inflammatory infiltrate of T cells to the subepithelium and keratinocyte cell death. We performed gene expression analysis of the oral epithelium of lesions in subjects with OLP and its sister disease, oral lichenoid reaction (OLR), in order to better understand the role of the keratinocytes in these diseases. DESIGN Fourteen patients with OLP or OLR were included in the study, along with a control group of 23 subjects with a variety of oral diseases and a normal group of 17 subjects with no clinically visible mucosal abnormalities. Various proteins have been associated with OLP, based on detection of secreted proteins or changes in RNA levels in tissue samples consisting of epithelium, stroma, and immune cells. The mRNA level of twelve of these genes expressed in the epithelium was tested in the three groups. RESULTS Four genes showed increased expression in the epithelium of OLP patients: CD14, CXCL1, IL8, and TLR1, and at least two of these proteins, TLR1 and CXCL1, were expressed at substantial levels in oral keratinocytes. CONCLUSIONS Because of the large accumulation of T cells in lesions of OLP it has long been thought to be an adaptive immunity malfunction. We provide evidence that there is increased expression of innate immune genes in the epithelium with this illness, suggesting a role for this process in the disease and a possible target for treatment.