Which putatively pre‐malignant oral lesions become oral cancers?

Oral squamous cell carcinomas continue to be a group of diseases with high mortality and increasing incidence rates, particularly among young individuals. This is a paradox finding, since most oral cancers are preceded - even by several years - by readily detectable mucosal changes, most often white or red patches (leukoplakias and erythroplakias, respectively). However, only a small fraction of leukoplakias or erythroplakias are related to cancer development, and the challenge has been to identify the high-risk lesions. From the vast literature on molecular markers in oral pre-cancer, no reliable prognostic marker for risk assessment in putatively pre-malignant lesions has emerged. For this reason, a wait-and-see approach has been adopted for this group of lesions. Recently published data point to gross genomic aberrations (DNA aneuploidy) as a tool for targeting patients at particular risk for future cancerous lesions in the oral cavity. Thus, DNA aneuploidy signalled a very high risk for subsequent development of carcinomas in a wide range of lesions from the oral mucous membrane, ranging from oral leukoplakias to oral erythroplakias and even including lesions that had been explicitly defined as being without a malignant potential by a group of trained pathologists. As an extension of research in oral pre-malignancies, the enzyme cyclooxygenase-2 (COX-2) seems to be enhanced specifically in high-risk oral lesions, as defined by the aberrant DNA content of their lesions. These data strongly indicate that COX-2 inhibitors (coxibs) should be investigated as chemopreventive agents in patients identified to be at high risk of developing oral cancer.     

Urban legends series: oral leukoplakia

To date, the term oral leukoplakia (OL) should be used to recognize ‘predominantly white plaques of questionable risk, having excluded (other) known diseases or disorders that carry no increased risk of cancer’. In this review, we addressed four controversial topics regarding oral leukoplakias (OLs): (i) Do tobacco and alcohol cause OLs?, (ii) What percentage of OLs transform into oral squamous cell carcinoma (OSCC)?, (iii) Can we distinguish between premalignant and innocent OLs?, and (iv) Is proliferative verrucous leukoplakia (PVL) a specific entity or just a form of multifocal leukoplakia? Results of extensive literature search suggest that (i) no definitive evidence for direct causal relationship between smoked tobacco and alcohol as causative factors of OLs, (ii and iii) the vast majority of OLs follow a benign course and do not progress into a cancer, and no widely accepted and/or validated clinical and/or biological factors can predict malignant transformation, and (iv) the distinction between multifocal/multiple leukoplakias and PVL in their early presentation is impossible; the temporal clinical progression and the high rate of recurrences and development of cancer of PVL are the most reliable features for diagnosis.     

DNA analysis of oral leukoplakia by flow cytometry.

DNA ploidy of 19 oral leukoplakias with and without epithelial dysplasia was investigated and the results were compared with 11 normal gingival biopsies, 14 oral benign tumours and 50 oral squamous cell carcinomas. The results suggest a possible relationship between DNA aneuploidy and oral leukoplakias or squamous cell carcinomas, as 32% of the oral leukoplakias and 48% of the oral squamous cell carcinomas were aneuploid although all the normal gingival biopsies and the benign oral tumours examined were diploid. No significant relationship was observed, however, between DNA ploidy and epithelial dysplasia in the leukoplakias.     

Meta‐analysis of the predictive value of DNA aneuploidy in malignant transformation of oral potentially malignant disorders

DNA aneuploidy is an imbalance of chromosomal DNA content that has been highlighted as a predictor of biological behavior and risk of malignant transformation. To date, DNA aneuploidy in oral potentially malignant diseases (OPMD ) has been shown to correlate strongly with severe dysplasia and high‐risk lesions that appeared non‐dysplastic can be identified by ploidy analysis. Nevertheless, the prognostic value of DNA aneuploidy in predicting malignant transformation of OPMD remains to be validated. The aim of this meta‐analysis was to assess the role of DNA aneuploidy in predicting malignant transformation in OPMD . The questions addressed were (i) Is DNA aneuploidy a useful marker to predict malignant transformation in OPMD ? (ii) Is DNA diploidy a useful negative marker of malignant transformation in OPMD ? These questions were addressed using the PECO method. Five studies assessing aneuploidy as a risk marker of malignant change were pooled into the meta‐analysis. Aneuploidy was found to be associated with a 3.12‐fold increased risk to progress into cancer (RR =3.12, 95% CI 1.86‐5.24). Based on the five studies meta‐analyzed, “no malignant progression” was more likely to occur in DNA diploid OPMD by 82% when compared to aneuploidy (RR =0.18, 95% CI 0.08‐0.41). In conclusion, aneuploidy is a useful marker of malignant transformation in OPMD , although a diploid result should be interpreted with caution.     

hMLH1 immunoexpression is related to the degree of epithelial dysplasia in oral leukoplakia

J Oral Pathol Med (2011) 40 : 153–159 Background: hMLH1 is a protein of the mammalian mismatch repair system responsible for genomic stability during repeated duplication. Relation between its altered expression linked to microsatellite instability has also been observed in oral leukoplakias (OL) and squamous cell carcinomas pointing to a possible role of hMLH1 in oral carcinogenesis. To our knowledge, this is the first study evaluating the immunoexpression of hMLH1 in OLs regarding their different degrees of epithelial dysplasia. Methods: Sixty‐two specimens of OL were classified in four groups: 17 without dysplasia, 19 with mild dysplasia, 16 with moderate dysplasia, and 10 with severe dysplasia. Immunohistochemistry for hMLH1 was performed, and percentage of positive cells was assessed. In the statistical analysis, P values <0.005 were considered significant. Results: hMLH1 immunoexpression showed decreasing indexes from lesions with lower degrees of dysplasia to lesions with more severe dysplasia. Statistical difference was found mainly between suprabasal layers and total indexes. Conclusions: hMLH1 immunoexpression was inversely related to the OL degree of dysplasia. The total epithelial hMLH1 index seems to be of more clinical relevance than the evaluation stratified by layers. Our findings also suggest a role of such alterations in this pathway of DNA repair as an early event in oral carcinogenesis.     

The relationship of genetic aberrations detected by comparative genomic hybridization to DNA ploidy and tumor size in human oral squamous cell carcinomas

We have examined genetic alterations in 11 surgically removed oral squamous cell carcinomas (OSCCs) using comparative genomic hybridization (CGH) and laser scanning cytometry (LSC), which allow quantitative analysis of chromosomal abnormalities. CGH analysis revealed gains and/or losses of DNA sequence copy number in all tumors. Gains in DNA sequence copy number were detected frequently for chromosome arms 3q25-28 (6/11), 5p (6/11) and 8q (5/11), and losses in chromosome arms 18q (4/11), 19q (4/11), 17p (3/11), and 19p (3/11). Amplification of 5p was observed in two tumors. LSC detected DNA aneuploidy with DNA indices ranging from 1.30 to 1.82 in 6 of 11 tumors. The number of chromosomal aberrations was higher in DNA aneuploid tumors than in diploid tumors (8.17 vs 3.60/tumor, P<0.05). Furthermore, the average number of chromosomal aberrations was significantly higher in stage T2 tumors and larger tumors than in stage T1 tumors (7.71 vs 3.25/tumor, P<0.05). Our results suggest that DNA aneuploidy and large tumor size reflect an underlying chromosomal instability.     

FCM与AgNORs在Wistar大鼠腭黏膜癌变过程中的诊断价值

目的：研究流式细胞术（FlowCytometry,FCM）和银染核仁组织区（AgNORs）2种方法在诊断Wistar大鼠腭黏膜癌变过程中的作用。方法：通过化学诱导法建立大鼠口腔癌模型,采用流式细胞术和银染核仁组织区法对7例正常黏膜和63例不同癌变发展阶段黏膜细胞的DNA含量和细胞增殖状态进行检测,并对结果做相关性分析。结果：DNA指数（DI）、异倍体率和AgNOR颗粒计数随着组织病理学分级增加而逐渐增加,异常增生、原位癌、浸润鳞癌与正常黏膜相比差异有统计学意义（P〈0．05）。Spearman线性相关分析显示DNA指数和AgNOR颗粒计数呈正相关（r-0．713,P〈0．05）。结论：流式细胞仪检测和银染核仁组织区两种方法可以作为口腔癌诊断的辅助检测手段,2种方法相结合可以提高口腔黏膜病变的检出率。     

Tobacco use increase the number of aneuploid nuclei in the clinically healthy oral epithelium

J Oral Pathol Med (2010) 39 : 605–610 Background: The most important risk factor linked to the development of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) is tobacco use. Tobacco contains carcinogens that influence the DNA repair, cell cycle control and may produce chromosomal aberrations. The loss or acquisition of one or more chromosomes is defined as aneuploidy. Methods: Aneuploidy was determined by means of the DNA‐content included in cells obtained by exfoliative cytology and Feulgen’s staining. The cells were collected from the clinically healthy lateral margin of the tongue of non‐smokers without oral lesions, smokers without oral lesions, smokers with OL, and smokers with OSCC, using the CytoBrush^®. Each group was composed of 20 individuals. A Carl Zeiss image analyzer system and the KS300 software were used. Statistical analysis was performed with BioEstat^® software. Results: The mean percentage of aneuploid nuclei was statistically higher in the smokers (79.65%), smokers with OL (68.4%), and smokers with OSCC (93.65%), as compared to non‐smokers (39.3%) (P < 0.05). A trend toward an increase in the aneuploidy of the smokers with OSCC group (P = 0.02), as compared to the non‐smoker group, could be observed. No significant difference could be observed as regards the mean percentage of aneuploid nuclei in relation to duration of tobacco use or the number of the cigarettes smoked. Conclusions: Tobacco use is responsible for an increased number of aneuploid nuclei in the oral epithelium.     

Utility of chemiluminescence (ViziLite™) in the detection of oral potentially malignant disorders and benign keratoses

J Oral Pathol Med (2011) 40 : 541–544 Background: Oral potentially malignant disorders (OPMD) are known to precede the development of oral cancer. Detection of OPMD allows delivery of interventions that may reduce the evolution of these disorders to malignancy. Following oral examinations, the accuracy of detection of OPMD by chemiluminescence was evaluated using a commercially available detection kit – ViziLite. Data derived were compared in relation to conventional oral examination and surgical biopsy. Methods: A total of 126 patients, 70 men and 56 women (mean age 58.5 ± 11.9 years) attending Oral Medicine Clinics at King’s and Guy’s Hospitals, London, with oral white, red, and mixed white and red patches were enrolled. Sixty‐one patients were current smokers, 28 were ex‐smokers, while 92 were alcohol users. In a detailed investigation, these patients underwent ViziLite examination followed by surgical biopsy. Results: Based on the clinical diagnosis, 70 patients had oral leukoplakia/erythroplakia, 32 had oral lichen planus, nine had chronic hyperplastic candidiasis, and rest had frictional keratosis (13) or oral submucous fibrosis (2). Of 126 lesions, 95 (75.4%) showed aceto‐whitening. Most oral leukoplakias had enhanced visibility and sharpness of the lesion when viewed with the ViziLite system. Following biopsy, 44 had oral epithelial dysplasia (29 mild, eight moderate, and seven severe). The sensitivity (se) and specificity (sp) of chemiluminescence for the detection of a dysplastic lesion were 77.3% and 27.8%, respectively. Conclusion: While ViziLite has the ability to detect OPMD, it does not accurately delineate dysplastic lesions. The device can be used as a general oral mucosal examination system and may in particular improve the visualization of leukoplakias.     

Oral leukoplakias with different degrees of dysplasia: comparative study of hMLH1, p53, and AgNOR

J Oral Pathol Med (2011) 40 : 305–311 Background: hMLH1 is one of the major proteins of the mammalian mismatch repair system. It has been suggested that the mismatch repair machinery could be linked to p53, a tumor suppressor protein. The AgNOR technique is used to assess cell proliferation. The aim was to compare the immunoexpression of hMLH1 and p53, and AgNOR number in oral leukoplakias with different degrees of dysplasia. Methods: Sixty‐two samples were evaluated by immunohistochemistry for hMLH1 and p53, and AgNOR technique, being 17 without dysplasia, 19 with mild dysplasia, 16 with moderate dysplasia, and 10 with severe dysplasia. Results: hMLH1 immunoexpression showed decreasing indexes, while p53 and AgNOR showed increasing indexes from lesions with lower degrees of dysplasia to lesions with more severe dysplasia. An inverse correlation between hMLH1 and both p53 and AgNOR, and a direct correlation between p53 and AgNOR were observed. Conclusions: Alterations in the immunoexpression pattern of hMLH1 and p53 seemed to be early events in oral carcinogenesis. During acquisition of a more dysplastic phenotype, keratinocytes may show diminished capacity of DNA repair and tumor suppression, as well as higher cellular proliferation, and these pathways can be somehow interconnected.     

Podoplanin expression as a predictive marker of dysplasia in oral leukoplakia

Purpose

Recent studies have emphasized the role of podoplanin in oral lesions at risk of malignant transformation. We investigated a group of oral leukoplakias (OLs) to determine a possible relation between altered podoplanin expression and dysplasia, and to compare the results with those obtained by other, widely used biomarkers.

Comparative evaluation of genetic assays to identify oral pre‐cancerous fields

Background: Oral squamous cell carcinomas often develop in a pre‐cancerous field, defined as mucosal epithelium with cancer‐related genetic alterations, and which may appear as a clinically visible lesion. The test characteristics of three genetic assays that were developed to detect pre‐cancerous fields were investigated and compared to histology. Methods: In total, 10 pre‐cancerous fields that were not visible at clinical inspection and gave rise to malignant transformation based on an identical TP53 mutation in tumor and mucosal epithelium in the surgical margin, as well as 10 normal oral mucosa specimens were analyzed for numerical chromosomal changes with multiplex ligation‐dependent probe amplification (MLPA), for loss of heterozygosity (LOH), with microsatellite PCR and for DNA index alterations with DNA image analysis. Results: No alterations were detected in normal tissue by either of the assays. Both MLPA and LOH assays detected all pre‐cancerous fields. DNA cytometry identified aneuploidy in four of 10 pre‐cancerous fields, while the corresponding tumors that developed in these fields were shown to be aneuploid. Conclusions: Both the MLPA and LOH assay seem suitable for screening pre‐cancerous fields in subjects at high risk for oral cancer even in the absence of clinically abnormal appearing oral mucosa. Measurements of DNA index might be valuable to determine the time to progression.     

DNA content and PCNA immunoreactivity in oral precancerous and cancerous lesions

Thirty-three dysplastic lesions showing varying degrees of atypia located in the oral cavity and 83 squamous cell carcinomas located in the oral cavity and tongue (n = 56) or in the lips (n = 27) were analysed by means of proliferating cell nuclear antigen (PCNA) immunoreactivity and image cytometric DNA measurements. The results show that in dysplastic lesions increasing cellular atypia correlated to elevated proliferative activity and aneuploidy occurring in the basal cell layers. In highly differentiated squamous carcinomas increased PCNA immunoreactivity and aneuploidy was preferentially observed focally (grade 1 tumours) or in the invasive zones (grade 2 tumours). In contrast, more poorly differentiated carcinomas (grade 3 and 4 tumours) showed strongly elevated proliferative activity and aneuploidy throughout the entire tumour mass.     

High malignant transformation rate of widespread multiple oral leukoplakias

OBJECTIVE: This study was undertaken in order to investigate clinicopathologic characteristics and malignant potential of widespread multiple oral leukoplakias. PATIENTS AND METHODS: The present study includes 12 patients with widespread multiple leukoplakias (widespread patients) and 99 with localized lesions (localized patients), and all patients were followed for more than 6 months with the mean follow-up period of 4 years. RESULTS: Gingiva and tongue were the major affected sites of leukoplakias in the localized patients, whereas gingiva and buccal mucosa were predominantly affected in the widespread patients. The rate of developing carcinoma was significantly (P < 0.02) higher in the widespread patients (3/12) than in the localized patients (5/99), although there was no significant difference in the rate of dysplastic lesions between these groups. CONCLUSION: These results indicate that the widespread leukoplakias have a higher potential for the development of carcinoma than do the localized lesions.     

Ultrastructure of different clinical forms of oral leukoplakia*

Abstract Twelve cases of oral leukoplakia were studied at the light and electron microscopic levels. In clinical leukoplakia simplex and verrucosa, both light and electron microscopic investigations revealed evidence of increased keratinization. Clinically erosive and histologically dysplastic leukoplakias showed ultrastructural alterations characteristic of oral carcinomas in some instances.     

Aberrations of erbB-1 and erbB-2 oncogenes in non-dysplastic leukoplakias of the oral cavity

The purpose of this study was to analyse erbB-1 and erbB-2 oncogenes in non-dysplastic oral leukoplakia to see if we could pinpoint the first steps towards dysplasia and possible carcinogenesis. Fresh biopsy specimens of leukoplakia in 13 patients with no history of oral cancer were examined using the competitive-differential polymerase chain reaction. The mean gene copy numbers of erbB-1 and erbB-2 were calculated from the formula to compare the absolute quantities of reference gene and oncogene from 24 patients who did not have leukoplakia. Healthy mucosa was taken as controls. In eight patients with leukoplakias, the results indicated aberrations of the erbB-1 oncogene, and two patients had gene dosage changes of erbB-2. There were no signs of deletions or amplifications in the controls. These results suggest that aberrations of erbB-1 and erbB-2 are additional markers in premalignant oral lesions at the beginning of the carcinogenic process, and that genetic alterations in histologically non-dysplastic premalignant oral lesions are common.     

Urban legends series: oral manifestations of HIV infection

Human immunodeficiency virus‐related oral lesions (HIV‐OLs), such as oral candidiasis (OC) and oral hairy leukoplakia (OHL), have been recognized as indicators of immune suppression since the beginning of the global HIV epidemic. The diagnosis and management of HIV disease and spectrum of opportunistic infection has changed over the past 30 years as our understanding of the infection has evolved. We investigated the following controversial topics: (i) Are oral manifestations of HIV still relevant after the introduction of highly active antiretroviral therapy (HAART)? (ii) Can we nowadays still diagnose HIV infection through oral lesions? (iii) Is the actual classification of oral manifestations of HIV adequate or does it need to be reviewed and updated? (iv) Is there any novelty in the treatment of oral manifestations of HIV infection? Results from extensive literature review suggested the following: (i) While HAART has resulted in significant reductions in HIV‐OLs, many are still seen in patients with HIV infection, with OC remaining the most common lesion. While the relationship between oral warts and the immune reconstitution inflammatory syndrome is less clear, the malignant potential of oral human papillomavirus infection is gaining increasing attention. (ii) Effective antiretroviral therapy has transformed HIV from a fatal illness to a chronic manageable condition and as a result expanded screening policies for HIV are being advocated both in developed and in developing countries. Affordable, reliable, and easy‐to‐use diagnostic techniques have been recently introduced likely restricting the importance of HIV‐OLs in diagnosis. (iii) The 1993 EC‐Clearinghouse classification of HIV‐OLs is still globally used despite controversy on the relevance of periodontal diseases today. HIV‐OL case definitions were updated in 2009 to facilitate the accuracy of HIV‐OL diagnoses by non‐dental healthcare workers in large‐scale epidemiologic studies and clinical trials. (iv) Research over the last 6 years on novel modalities for the treatment of HIV‐OLs has been reported for OC and OHL.     

Sanguinaria-associated oral leukoplakia: comparison with other benign and dysplastic leukoplakic lesions

OBJECTIVE: This study was undertaken to compare and contrast biomarkers and ploidy data from maxillary gingiva leukoplakias associated with dentifrices and mouthrinses containing the herbal compound sanguinaria with other forms of oral benign and premalignant mucosal keratosis. STUDY DESIGN: Representative archived specimens of benign keratosis, sanguinaria-associated keratosis, and keratosis with dysplasia were used for computerized image analysis and biomarker immunohistochemical assays to assess ploidy, DNA content, and p53 and proliferating cell nuclear antigen immunoreactivity of nuclei. RESULTS: DNA content was significantly higher and higher numbers of cell populations with hyperploid nuclei were encountered in the dysplastic group than in the other two groups (P <.001). Sanguinaria-associated keratosis did not harbor significant numbers of p53-expressing nuclei, yet it showed a significant elevation in proliferating cell nuclear antigen-labeled nuclei in total, in the basal layer, and in the spinous layer in comparison with benign keratoses (P <.001). In addition, 1.5% of the sanguinaria-associated leukoplakia epithelial cell population was characterized by nuclei with a greater than 4-fold increase in DNA content. CONCLUSIONS: Sanguinaria-associated keratoses show some marker and image analysis profiles similar to those of non-sanguinaria dysplastic lesions of the lip and mucosa. Preparations containing sanguinaria should be avoided until the risk for malignant transformation is determined.     

Ultrastructure of different clinical forms of oral leukoplakia

Twelve cases of oral leukoplakia were studied at the light and electron microscopic levels. In clinical leukoplakia simplex and verrucosa, both light and electron microscopic investigations revealed evidence of increased keratinization. Clinically erosive and histiologically dysplastic leukoplakias showed ultrastructural alterations characteristic of oral carcinomas in some instances.     

Clinicopathologic factors associated with malignant transformation of oral leukoplakias: a retrospective cohort study

It is clinically challenging to identify oral leukoplakias that have a high risk of undergoing malignant transformation. The aim of this retrospective study was to elucidate the associations between malignant transformation of oral leukoplakias and various clinicopathologic factors. Patients with a diagnosis of clinical oral leukoplakia, verified through histopathologic examination and with access to digital images of the lesion, were retrospectively included for the period 2003–2013. Using the clinical images, all lesions were re-evaluated regarding diagnosis and clinical subtype. Of the 234 included patients, with a median follow-up of 9 years, 27 (11.5%) developed oral squamous cell carcinoma. Among the clinicopathologic factors investigated, non-homogeneous oral leukoplakia (OL), OL with dysplasia, and OL localized to the tongue showed statistically significant increased rates of malignant transformation in the multivariate Cox regression analysis. Non-homogeneous OL showed a 15.2-times higher transformation rate than homogenous OL (P < 0.001). Dysplastic leukoplakias developed into carcinomas 2.4-times more often than did non-dysplastic leukoplakias (P = 0.048). OL located on the tongue showed a 2.8-times higher malignant transformation rate than OLs at other oral locations (P = 0.018), when other locations were combined into one group. Non-homogeneous OL, OL with dysplasia, and OL localized to the tongue have higher transformation rates.