Increased nitric oxide levels and iNOS over-expression in oral squamous cell carcinoma.

Inducible nitric oxide synthase (iNOS) is responsible for generating high levels of nitric oxide (NO) in tissues. Increased iNOS expression has been demonstrated in a number of carcinomas including head and neck squamous cell carcinoma (SCC). However, iNOS levels have not been evaluated specifically in oral cavity SCC, or in the precancerous lesions that progress to oral SCC. Also, NO levels have not been measured in oral precancerous or cancerous tissues. We therefore measured iNOS mRNA, iNOS protein and NO in oral SCC, oral dysplasias and normal oral epithelium. We used RT-PCR to quantify and compare iNOS mRNA levels in these oral tissue specimens. We found that iNOS mRNA was overexpressed in 41% of oral SCC but in only 8% of dysplasia specimens (P = 0.003). Immunohistochemistry was used to evaluate iNOS protein levels in oral SCC, oral dysplasias and normal oral epithelium. A significantly higher percentage of oral SCC specimens showed the highest level of iNOS staining relative to the oral dysplasias and normal oral epithelial samples (95% of oral SCC, 50% of dysplasias, and only 0% of normal epithelial controls, P < 0.0001). The positive staining for iNOS was limited to the SCC cells. Production of NO from iNOS was quantified using HPLC and found to be significantly higher in oral SCC (1.45 +/- 0.56 microg/ml) than normal epithelial controls (0.43 +/- 0.26 microg/ml) (P = 0.0013). We conclude that iNOS mRNA levels and NO production are significantly increased, in oral SCC compared to oral dysplasias and normal epithelial controls. These findings suggest that increased iNOS expression and the generation of high NO levels might have a role in oral SCC development.

Overexpression of matrix metalloproteinase-1 and -9 mRNA is associated with progression of oral dysplasia to cancer.

PURPOSE: Although an important risk factor for oral cancer is the presence of epithelial dysplasia, many lesions will not progress to malignancy. Matrix metalloproteinases (MMPs) are zinc-dependent proteinases capable of digesting various structural components of the extracellular matrix. Because MMPs are frequently overexpressed in oral squamous cell carcinoma (SCC), we hypothesized that they are also overexpressed in oral dysplasias; we also hypothesized that those dysplasias that progress to oral cancer express higher levels of MMPs than those lesions that do not progress. EXPERIMENTAL DESIGN: In this retrospective study, we examined changes in MMP-1, -2, and -9 mRNA expression using quantitative TaqMan reverse transcription-polymerase chain reaction in 34 routinely processed oral dysplasias and 15 SCCs obtained from 34 patients. After several years of close follow-up, 19 dysplasias progressed to oral SCC and 15 did not. RESULTS: Overall, MMP-1 mRNA was overexpressed (>2-fold) in 24 of 34 (71%) dysplasias and 13 of 15 (87%) oral SCCs. MMP-2 overexpression was seen in 11 of 34 (32%) dysplasias and 7 of 15 (47%) cancers; for MMP-9, overexpression was identified in 29 of 34 (85%) dysplasias and 15 of 15 (100%) cancers. MMP-1 and -9 levels were significantly higher in the SCCs compared with all oral dysplasias (P = 0.004 and P = 0.01, respectively). MMP-1 and -9 mRNA levels were significantly higher in the oral dysplasias that progressed to oral cancer compared with those that did not (P = 0.04 and P = 0.002, respectively). CONCLUSIONS: Levels of MMP-1 and -9 mRNA may be markers of malignant transformation of oral dysplasia to oral cancer.

Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression.

Although an important risk factor for oral cancer is the presence of epithelial dysplasia, most of these lesions will not progress to malignancy. Presently, for the individual patient with dysplasia, there are few reliable markers that may indicate the likelihood of progression to oral cancer. Cathepsin L is a lysosomal protease that degrades extracellular matrix material. Because cathepsin L is frequently overexpressed in oral squamous cell carcinoma (SCC) we hypothesized that it is also overexpressed in oral premalignancy and that premalignant lesions that progressed to oral cancer expressed higher levels of cathepsin L than those premalignant lesions that did not. In this retrospective pilot study we examined changes in cathepsin L expression at the mRNA level using quantitative TaqMan RT-PCR and at the protein level by immunohistochemistry in 33 routinely processed oral dysplastic lesions and 14 SCCs obtained from 33 patients. Sixteen of the dysplastic lesions progressed to oral SCC and 17 did not after several years of follow-up. Cathepsin L mRNA was overexpressed in 16/33 (48%) dysplastic lesions and in 9/14 (64%) oral SCC. Cathepsin L protein was also overexpressed in a large proportion of dysplasias and cancers. Overexpression was independent of dysplasia grade and identified in both those patients who progressed to oral SCC and in those who did not. Levels of cathepsin L mRNA and protein did not differ significantly in the progressing versus non-progressing dysplasias (P=0.27). However, cathepsin L mRNA and protein were significantly lower in the non-progressing dysplasias when compared to the oral cancers (P=0.03) but not in the progressing dysplasias suggesting a trend for dysplasias with overexpressed cathepsin L to be more likely to progress to oral cancer.

Proliferation and invasion factors in HIV-associated dysplastic and nondysplastic oral warts and in oral squamous cell carcinoma: an immunohistochemical and RT-PCR evaluation.

OBJECTIVE: Oral warts arising in human immunodeficiency virus (HIV) infection occasionally show marked epithelial dysplasia. However, anecdotal evidence suggests that they do not progress to oral squamous cell carcinoma (SCC). Therefore, we evaluated lesions for expression of proteins (tenascin-C, beta6 integrin, and matrix metalloproteinase-1[MMP1]) that have been identified as important in the invasive phase of oral SCC. STUDY DESIGN: Twenty-two oral dysplastic warts from 22 patients and 5 oral SCCs were stained for human papillomavirus (HPV) antigen, proliferation protein Ki-67, tenascin-C, beta6, and MMP1 by immunohistochemical methods. For comparison, 5 nondysplastic warts each from HIV-positive and HIV-negative patients and 5 normal mucosa specimens were included. Sections were semiquantitatively assessed, and results were compared. Because MMP1 was the lowest or least expressed interface protein, MMP1 mRNA was quantitatively assessed from formalin-fixed paraffin-embedded tissue in selected cases with quantitative reverse transcription-polymerase chain reaction. RESULTS: Twenty of 22 dysplastic warts stained positive for human papillomavirus common antigen, and all warts showed high proliferative fractions similar to SCCs. Tenascin-C and beta6 were variably expressed by the dysplastic warts but were consistently expressed at high levels in the SCCs. MMP1 protein levels were negative or low in 20 of 22 in dysplastic warts, but were elevated in 4 of 5 SCCs. MMP1 mRNA analysis indicated that message was low in 4 dysplastic warts and also suggested that protein translation was incomplete in 3 of the warts. CONCLUSION: We conclude that invasion-associated proteins are underexpressed in oral dysplastic warts in HIV-positive men. However, until these patients are followed for extended periods, the risk of development of SCC from oral dysplastic warts remains unknown.

Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses.

Increasingly, there is the need to analyze gene expression in tumor tissues and correlate these findings with clinical outcome. Because there are few tissue banks containing enough frozen material suitable for large-scale genetic analyses, methods to isolate and quantify messenger RNA (mRNA) from formalin-fixed, paraffin-embedded tissue sections are needed. Recovery of RNA from routinely processed biopsies and quantification by the polymerase chain reaction (PCR) has been reported; however, the effects of formalin fixation have not been well studied. We used a proteinase K-salt precipitation RNA isolation protocol followed by TaqMan quantitative PCR to compare the effect of formalin fixation for 24, 48, and 72 hours and for 1 week in normal (2), oral epithelial dysplasia (3), and oral squamous cell carcinoma (4) specimens yielding 9 fresh and 36 formalin-fixed samples. We also compared mRNA and protein expression levels using immunohistochemistry for epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP)-1, p21, and vascular endothelial growth factor (VEGF) in 15 randomly selected and routinely processed oral carcinomas. We were able to extract RNA suitable for quantitative reverse transcription (RT) from all fresh (9/9) and formalin-fixed (36/36) specimens fixed for differing lengths of time and from all (15/15) randomly selected oral squamous cell carcinoma. We found that prolonged formalin fixation (>48 h) had a detrimental effect on quantitative RT polymerase chain reaction results that was most marked for MMP-1 and VEGF but less evident for p21 and EGFR. Comparisons of quantitative RT polymerase chain reaction and immunohistochemistry showed that for all markers, except p21, there was good correlation between mRNA and protein levels. p21 mRNA was overexpressed in only one case, but protein levels were elevated in all but one tumor, consistent with the established translational regulation of p21. These results show that RNA can be reliably isolated from formalin-fixed, paraffin-embedded tissue sections and can produce reliable quantitative RT-PCR data. However, results for some markers are adversely affected by prolonged formalin fixation times.