Exploring the microbiome of oral epithelial dysplasia as a predictor of malignant progression.

A growing body of research associates the oral microbiome and oral cancer. Well-characterized clinical samples with outcome data are required to establish relevant associations between the microbiota and disease. The objective of this study was to characterize the community variations and the functional implications of the microbiome in low-grade oral epithelial dysplasia (OED) using 16S rRNA gene sequencing from annotated archival swabs in progressing (P) and non-progressing (NP) OED. We characterised the microbial community in 90 OED samples - 30 swabs from low-grade OED that progressed to cancer (cases) and 60 swabs from low-grade OED that did not progress after a minimum of 5 years of follow up (matched control subjects). There were small but significant differences between P and NP samples in terms of alpha diversity as well as beta diversity in conjunction with other clinical factors such as age and smoking status for both taxa and functional predictions. Across all samples, the most abundant genus was Streptococcus, followed by Haemophilus, Rothia, and Neisseria. Taxa and predicted functions were identified that were significantly differentially abundant with progression status (all Ps and NPs), when samples were grouped broadly by the number of years between sampling and progression or in specific time to progression for Ps only. However, these differentially abundant features were typically present only at low abundances. For example, Campylobacter was present in slightly higher abundance in Ps (1.72%) than NPs (1.41%) and this difference was significant when Ps were grouped by time to progression. Furthermore, several of the significantly differentially abundant functions were linked to the Campylobacteraceae family in Ps and may justify further investigation. Larger cohort studies to further explore the microbiome as a potential biomarker of risk in OED are warranted.

Influence of fluorescence on screening decisions for oral mucosal lesions in community dental practices.

BACKGROUND: Quality of oral screening examinations is dependent upon the experience of the clinician and can vary widely. Deciding when a patient needs to be referred is a critical and difficult decision for general practice clinicians. A device to aid in this decision would be beneficial. The objective of this study was to to examine the utility of direct fluorescence visualization (FV) by dental practitioners as an aid in decision-making during screening for cancer and other oral lesions. METHODS: Dentists were trained to use a stepwise protocol for evaluation of the oral mucosa: medical history, head, neck and oral exam, and fluorescent visualization exam. They were asked to use clinical features to categorize lesions as low (LR), intermediate (IR), or high (HR) risk and then to determine FV status of these lesions. Clinicians made the decision of which lesions to reassess in 3 weeks and based on this reassessment, to refer forward. RESULTS: Of 2404 patients screened over 11 months, 357 initially had lesions with 325 (15%) identified as LR, 16 (4.5%) IR, and 16 (4.5%) HR. Lesions assessed initially as IR and HR had a 2.7-fold increased risk of FV loss persisting to the reassessment appointment versus the LR lesions. The most predictive model for lesion persistence included both FV status and lesion risk assessment. CONCLUSION: A protocol for screening (assess risk, reassess, and refer) is recommended for the screening of abnormal intraoral lesions. Integrating FV into a process of assessing and reassessing lesions significantly improved this model.

Gender- and ethnicity-specific survival trends of oral cavity and oropharyngeal cancers in British Columbia.

INTRODUCTION: A shift in etiology of oral cancers has been associated with a rise in incidence for oropharyngeal cancers (OPC) and decrease for oral cavity cancers (OCC); however, there is limited information about population-based survival trends. We report epidemiological transitions in survival for both OPC and OCC from a population-based cancer registry, focusing upon gender and ethnic differences. METHODS: All primary oral cancers diagnosed between 1980 and 2005 were identified from the British Columbia Cancer Registry and regrouped into OPC and OCC by topographical subsites, time periods (1980-1993 and 1994-2005), stage at diagnosis, and ethnicity. Cases were then followed up to December 2009. Using gender-based analysis, actuarial life tables were used to calculate survival rates, which were compared using Kaplan-Meier curves and log-rank tests. RESULTS: For OPC, survival improved, significant for tonsil and base of tongue in men and marginally significant at base of tongue in women. This improvement occurred in spite of an increase in late-stage diagnosis for OPC in both genders. Interestingly, there was no difference in survival for early- and late-stage disease for OPC in men. For OCC, there was a decrease in survival for floor of mouth cancers in both genders although significant in women only. South Asians had the poorest survival for OCC in both genders. CONCLUSION: Survival for OPC improved, more dramatically in men than women, in spite of late-stage diagnosis and increasing nodal involvement. Given the poor survival rates and need for early detection, targeted OCC screening programs are required for South Asians.

Canadian Optically-guided approach for Oral Lesions Surgical (COOLS) trial: study protocol for a randomized controlled trial.

BACKGROUND: Oral cancer is a major health problem worldwide. The 5-year survival rate ranges from 30-60%, and has remained unchanged in the past few decades. This is mainly due to late diagnosis and high recurrence of the disease. Of the patients who receive treatment, up to one third suffer from a recurrence or a second primary tumor. It is apparent that one major cause of disease recurrence is clinically unrecognized field changes which extend beyond the visible tumor boundary. We have previously developed an approach using fluorescence visualization (FV) technology to improve the recognition of the field at risk surrounding a visible oral cancer that needs to be removed and preliminary results have shown a significant reduction in recurrence rates. METHOD/DESIGN: This paper describes the study design of a randomized, multi-centre, double blind, controlled surgical trial, the COOLS trial. Nine institutions across Canada will recruit a total of 400 patients with oral severe dysplasia or carcinoma in situ (N = 160) and invasive squamous cell carcinoma (N = 240). Patients will be stratified by participating institution and histology grade and randomized equally into FV-guided surgery (experimental arm) or white light-guided surgery (control arm). The primary endpoint is a composite of recurrence at or 1 cm within the previous surgery site with 1) the same or higher grade histology compared to the initial diagnosis (i.e., the diagnosis used for randomization); or 2) further treatment due to the presence of severe dysplasia or higher degree of change at follow-up. This is the first randomized, multi-centre trial to validate the effectiveness of the FV-guided surgery. DISCUSSION: In this paper we described the strategies, novelty, and challenges of this unique trial involving a surgical approach guided by the FV technology. The success of the trial requires training, coordination, and quality assurance across multiple sites within Canada. The COOLS trial, an example of translational research, may result in reduced recurrence rates following surgical treatment of early-stage oral cancer with significant impacts on survival, morbidity, patients' quality of life and the cost to the health care system. TRIAL REGISTRATION: Clinicaltrials.gov NCT01039298.

Predicting Progression of Low-Grade Oral Dysplasia Using Brushing-Based DNA Ploidy and Chromatin Organization Analysis.

Most oral cancers arise from oral potentially malignant lesions, which show varying grades of dysplasia. Risk of progression increases with increasing grade of dysplasia; however, risk prediction among oral low-grade dysplasia (LGD), that is, mild and moderate dysplasia can be challenging as only 5%-15% transform. Moreover, grading of dysplasia is subjective and varies with the area of the lesion being biopsied. To date, no biomarkers or tools are used clinically to triage oral LGDs. This study uses a combination of DNA ploidy and chromatin organization (CO) scores from cells obtained from lesion brushings to identify oral LGDs at high-risk of progression. A total of 130 lesion brushings from patients with oral LGDs were selected of which 16 (12.3%) lesions progressed to severe dysplasia or cancer. DNA ploidy and CO scores were analyzed from nuclear features measured by our in-house DNA image cytometry (DNA-ICM) system and used to classify brushings into low-risk and high-risk. A total of 57 samples were classified as high-risk of which 13 were progressors. High-risk DNA brushing was significant for progression (P = 0.001) and grade of dysplasia (P = 0.004). Multivariate analysis showed high-risk DNA brushing showed 5.1- to 8-fold increased risk of progression, a stronger predictor than dysplasia grading and lesion clinical features. DNA-ICM can serve as a non-invasive, high-throughput tool to identify high-risk lesions several years before transformation. This will help clinicians focus on such lesions whereas low-risk lesions may be spared from unnecessary biopsies.Prevention Relevance: DNA ploidy and chromatin organization of cells collected from oral potentially malignant lesions (OPMLs) can identify lesions at high-risk of progression several years prior. This non-invasive test would enable clinicians to triage high-risk (OPMLs) for closer follow-up while low-risk lesions can undergo less frequent biopsies reducing burden on healthcare resources.

Intraoral Photography Recommendations for Remote Risk Assessment and Monitoring of Oral Mucosal Lesions.

Oral cancer is a global health issue with substantial morbidity and a high mortality rate mainly because of late-stage diagnosis. Cancerous lesions are often preceded by potentially malignant lesions that may be detected during routine dental examinations. Not only is the oral cavity easily accessible for screening, but the clinical risk factors of the disease are also known. However, patients may not always be able to access screening services or receive follow-up for diagnosed lesions. In these circumstances, intraoral photos are crucial for timely triage, risk assessment, and monitoring of oral lesions. Further, photos form an integral part of a patient's records, facilitate patient education and communication between health care providers, and provide important information during the referral process. To ensure that intraoral photos are of good quality and standardised there is a need to establish recommendations regarding intraoral photography in oral mucosal screening. This article recommends methods to help health professionals and patients obtain interpretable intraoral photographs. Suggestions to achieve ideal lighting, mirror placement, camera angle, and retraction have been discussed. These recommendations are adaptable to easily available smartphone or point-and-shoot cameras and may be further used to develop future teledentistry platforms.

Basement membrane degeneration is common in lichenoid mucositis with dysplasia.

Background: Two subtypes of lichenoid mucositis (LM) with oral epithelial dysplasia have been proposed, with differing risks of malignant transformation. However, no research has been done to authenticate this hypothesis. The study objective was to determine whether there are 2 subcategories within this entity, one with primary lichenoid and secondary dysplastic features (L1D2), and the other with primary dysplastic and secondary lichenoid features (D1L2), and to compare the proportion of malignant progression in these groups. Methods: Patients with a diagnosis of lichenoid mucositis with low-grade (mild/moderate) oral epithelial dysplasia, no history of head and neck cancer, and who had at least 5 years of follow-up were eligible to participate in this nested case-control study. Cases (n = 10) were defined as lesions that progressed to severe dysplasia, carcinoma in situ or squamous cell carcinoma; controls (n = 32) were defined as those that did not progress. Immunohistochemistry was performed to assess for basement membrane (BM) degeneration using collagen IV-an integral BM protein. Results: Lesions that progressed to cancer exhibited a similar proportion of BM degeneration at baseline (70%) compared to non-progressors (78%), with no statistically significant difference between groups (p = 0.69). Conclusion: BM degeneration is frequently seen in LM with dysplasia and alone does not appear to be a predictor of malignant progression in lesions with both lichenoid and low-grade dysplastic features. Dysplasia should not be discounted in the presence of LM. Lesions that display any degree of dysplasia warrant clinical follow-up and continued monitoring.

Contexte: Deux sous-types de mucosites lichénoïdes (ML) avec dysplasie épithéliale buccale ont été proposés, avec des risques différents de transformation maligne. Cependant, aucune recherche n'a été faite pour valider cette hypothèse. L'objectif de l'étude était de déterminer s'il y a 2 sous-catégories au sein de cette entité, la première avec des caractéristiques lichénoïdes primaires et dysplasiques secondaires (L1D2), et l'autre avec des caractéristiques dysplasiques primaires et lichénoïdes secondaires (D1L2), et de comparer la proportion de progression maligne dans ces groupes. Méthodologie: Les patients ayant reçu un diagnostic de mucosite lichénoïde avec une dysplasie épithéliale buccale de faible intensité (faible/modérée), qui n'avaient aucun antécédent de cancer de la tête et du cou, et qui avaient eu au moins 5 ans de suivi, étaient admissible à participer à cette étude de cas-témoins emboîtés. Les cas (n = 10) étaient définis comme des lésions qui ont progressé à la dysplasie sévère, un carcinome in situ ou un carcinome squameux; les contrôles (n = 32) étaient définis comme ceux qui n'ont pas progressé. L'immunohistochimie a été effectuée pour évaluer s'il y avait eu une dégénérescence de la membrane basale (MB) en utilisant du collagène IV, une protéine MB intrinsèque. Résultats: Les lésions qui ont évolué en cancer ont présenté une proportion semblable de dégénérescence de MB au début (70 %) par rapport aux non-progresseurs (78 %), et aucune différence statistiquement significative entre les groupes (p = 0,69). Conclusion: La dégénérescence des MB est fréquemment constatée dans les ML avec dysplasie et seule, ne paraît pas être une variable explicative de l'évolution maligne dans les lésions à caractéristiques à la fois lichénoïdes et dysplasiques de faible intensité. Il ne faut pas sous-estimer la dysplasie en présence de ML. Les lésions qui présentent de la dysplasie, peu importe son étendue, exigent un suivi clinique et une surveillance continue.

Effect of Fluorescence Visualization-Guided Surgery on Local Recurrence of Oral Squamous Cell Carcinoma: A Randomized Clinical Trial.

Importance: High local recurrence rates with aggressive disease remain the main concern in oral cancer survival. Use of a translational device using fluorescence visualization (FV) approved by the US Food and Drug Administration and Health Canada, has shown a marked reduction in the 3-year local recurrence rate of high-grade oral lesions in a single-center observational study. Objective: To determine whether FV- guided surgery can improve local control rates in the treatment of in situ or T1 to T2 category oral squamous cell carcinoma (OSCC). Design, Setting, and Participants: A multicenter randomized clinical trial was conducted in a surgical setting. A total of 457 patients were enrolled between January 18, 2010, and April 30, 2015. Data analysis of the intention-to-treat population was performed from April 3, 2019, to March 20, 2020. Patients with histologically confirmed high-grade dysplasia/carcinoma in situ or T1 to T2 category OSCC were randomized to receive traditional peroral surgery or FV-guided surgery. Intervention: Fluorescence visualization during surgery. Main Outcomes and Measures: The primary outcome was local recurrence of OSCC. Secondary outcomes were failure of the first-pass margin, defined as a histologically confirmed positive margin for severe dysplasia or greater histologic change of the main specimen (ie, not the margins taken from the resection bed), regional or distant metastasis, and death due to disease. Results: Of the 457 patients enrolled in the study, 443 patients (264 [59.6%] men; mean [SD] age, 61.5 [13.3] years) completed the randomized treatment: 227 FV-guided and 216 non-FV guided surgery. The median follow-up was 52 (range, 0.29-90.8) months. In total, 45 patients (10.2%) experienced local recurrence. The 3-year local recurrence rate was 9.4% in the FV-guided group and 7.2% in the non-FV group (difference, 2.2%; 95% CI, -3.2% to 7.4%). Other similarities between the FV vs non-FV groups included failure of first-pass margin (68/227 [30.0%]) vs 65/216 [30.1%]), regional failure (39/227 [17.2%] vs 37/216 [17.1%]), disease-specific survival (23/227 [10.1%] vs 19/26 [8.8%]), and overall survival (41/227 [18.1%] vs 38/216 [17.6%]) were also similar between groups. No adverse events were judged to be related to the intervention. Conclusions and Relevance: In this randomized clinical trial, FV-guided surgery did not improve local control rates in the treatment of patients with in situ or T1 to T2 category oral cancer. Under a controlled environment, FV-guided surgery did not have an evident effect in reduction of local recurrence for localized OSCC. This result suggests that attention be directed to strategies other than improving definitions of nonapparent disease at clinical margins to identify the sources of local recurrence. Trial Registration: ClinicalTrial.gov Identifier: NCT01039298.

Genomic imbalances in precancerous tissues signal oral cancer risk.

Oral cancer develops through a series of histopathological stages: through mild (low grade), moderate, and severe (high grade) dysplasia to carcinoma in situ and then invasive disease. Early detection of those oral premalignant lesions (OPLs) that will develop into invasive tumors is necessary to improve the poor prognosis of oral cancer. Because no tools exist for delineating progression risk in low grade oral lesions, we cannot determine which of these cases require aggressive intervention. We undertook whole genome analysis by tiling-path array comparative genomic hybridization for a rare panel of early and late stage OPLs (n = 62), all of which had extensive longitudinal follow up (>10 years). Genome profiles for oral squamous cell carcinomas (n = 24) were generated for comparison. Parallel analysis of genome alterations and clinical parameters was performed to identify features associated with disease progression. Genome alterations in low grade dysplasias progressing to invasive disease more closely resembled those observed for later stage disease than they did those observed for non-progressing low grade dysplasias. This was despite the histopathological similarity between progressing and non-progressing cases. Strikingly, unbiased computational analysis of genomic alteration data correctly classified nearly all progressing low grade dysplasia cases. Our data demonstrate that high resolution genomic analysis can be used to evaluate progression risk in low grade OPLs, a marked improvement over present histopathological approaches which cannot delineate progression risk. Taken together, our data suggest that whole genome technologies could be used in management strategies for patients presenting with precancerous oral lesions.

Multiple pathways in the FGF signaling network are frequently deregulated by gene amplification in oral dysplasias.

Genetic alteration in oral premalignant lesions (OPLs), the precursors of oral squamous cell carcinomas (OSCCs), may represent key changes in disease initiation and development. We ask if DNA amplification occurs at this early stage of cancer development and which oncogenic pathways are disrupted in OPLs. Here, we evaluated 50 high-grade dysplasias and low-grade dysplasias that later progressed to cancer for gene dosage aberrations using tiling-path DNA microarrays. Early occurrences of DNA amplification and homozygous deletion were frequently detected, with 40% (20/50) of these early lesions exhibiting such features. Expression for 88 genes in 7 recurrent amplicons were evaluated in 5 independent head and neck cancer datasets, with 40 candidates found to be overexpressed relative to normal tissues. These genes were significantly enriched in the canonical ERK/MAPK, FGF, p53, PTEN and PI3K/AKT signaling pathways (p = 8.95 x 10(-3) to 3.18 x 10(-2)). These identified pathways share interactions in one signaling network, and amplification-mediated deregulation of this network was found in 30.0% of these preinvasive lesions. No such alterations were found in 14 low-grade dysplasias that did not progress, whereas 43.5% (10/23) of OSCCs were found to have altered genes within the pathways with DNA amplification. Multitarget FISH showed that amplification of EGFR and CCND1 can coexist in single cells of an oral dysplasia, suggesting the dependence on multiple oncogenes for OPL progression. Taken together, these findings identify a critical biological network that is frequently disrupted in high-risk OPLs, with different specific genes disrupted in different individuals.

Primary malignant melanoma of maxillary gingiva--a case report and review of the literature.

Dentists may encounter pigmented lesions in routine clinical practice. In most cases, the lesions are asymptomatic and benign in nature; however, rarely, a pigmented lesion can be a sign of malignancy. We report a case of malignant melanoma of the maxillary gingiva to highlight the importance of biopsy and periodic follow-up of patients with unusual focal pigmented lesions in the oral cavity.

Oral cancer: just the facts.

Oral cancer screening should be an integral part of a clinician's routine. This article reviews facts about oral cancer that are relevant to screening. The relevance of some issues in a particular dental practice will vary with the patient composition of the practice.

Oral submucous fibrosis, a clinically benign but potentially malignant disease: report of 3 cases and review of the literature.

Oral submucous fibrosis (OSF) is a premalignant condition mainly associated with the practice of chewing betel quid containing areca nut, a habit common among South Asian people. It is characterized by inflammation, increased deposition of submucosal collagen and formation of fibrotic bands in the oral and paraoral tissues, which increasingly limit mouth opening. Recently, OSF has been reported among South Asian immigrants in Canada, the United Kingdom and Germany. Dentists in western countries should enhance their knowledge of this disease as it seems to be increasing with population migration. In this paper, we review the literature on OSF and present 3 cases representing different stages of the disease to help dentists make an early diagnosis and reduce the morbidity and mortality associated with this condition.

Evaluation of a suspicious oral mucosal lesion.

Dentists who encounter a change in the oral mucosa of a patient must decide whether the abnormality requires further investigation. In this paper, we describe a systematic approach to the assessment of oral mucosal conditions that are thought likely to be premalignant or an early cancer. These steps, which include a comprehensive history, step-by-step clinical examination (including use of adjunctive visual tools), diagnostic testing and formulation of diagnosis, are routinely used in clinics affiliated with the British Columbia Oral Cancer Prevention Program (BC OCPP) and are recommended for consideration by dentists for use in daily practice.

Biopsy and histopathologic diagnosis of oral premalignant and malignant lesions.

Accurate diagnosis of premalignant or malignant oral lesions depends on the quality of the biopsy, adequate clinical information and correct interpretation of the biopsy results. The purpose of this paper is to review the procedures for obtaining appropriate biopsy samples, and the criteria for diagnosing and grading dysplasias. The World Health Organization's description of the architectural and cytologic epithelial changes that characterize dysplasia is detailed, and guidelines for following up patients with premalignant and malignant lesions are provided. The benefits of using the centralized services and expertise of the British Columbia Oral Biopsy Service are also reviewed.

New hope for an oral cancer solution: together we can make a difference.

Oral cancer is associated with high mortality and morbidity rates, largely as a result of late diagnosis. Although dental practitioners are trained to identify premalignant and malignant lesions, an organized system is needed to offer guidance and to improve access to experts in diagnosis and management of these lesions. In this article, we describe the many ways in which the British Columbia Oral Cancer Prevention Program (BC OCPP) is addressing this challenge: by linking community dental practices and referral centres, by creating partnerships between scientists and clinicians that already have resulted in new technologies to enhance early diagnosis, by involving a broad range of stakeholders to ensure population-based screening and by engaging in provincial, national and international outreach.

Using quantitative tissue phenotype to assess the margins of surgical samples from a pan-Canadian surgery study.

BACKGROUND: The purpose of this study was to use quantitative tissue phenotype (QTP) to assess the surgical margins to examine if a fluorescence visualization-guided surgical approach produces a shift in the surgical field by sparing normal tissue while catching high-risk tissue. METHODS: Using our QTP to calculate the degree of nuclear chromatin abnormalities, Nuclear Phenotypic Score (NPS), we analyzed 1290 biopsy specimens taken from surgical samples of 248 patients enrolled in the Efficacy of Optically-guided Surgery in the Management of Early-staged Oral Cancer (COOLS) trial. Multiple margin specimens were collected from each surgical specimen according to the presence of fluorescence visualization alterations and the distance to the surgical margins. RESULTS: The NPS in fluorescence visualization-altered (fluorescence visualization-positive) samples was significantly higher than that in fluorescence visualization-retained (fluorescence visualization-negative) samples. There was a constant trend of decreasing NPS of margin samples from non-adjacent-fluorescence visualization margins to adjacent-fluorescence visualization margins. CONCLUSION: Our results suggested that using fluorescence visualization to guide surgery has the potential to spare more normal tissue at surgical margins.

Visualization and other emerging technologies as change makers for oral cancer prevention.

The genomic era has fueled a rapid emergence of new information at the molecular level with a great potential for developing innovative approaches to detection, risk assessment, and management of oral cancers and premalignant disease. As yet, however, little research has been done on complementary approaches that would use different technology in conjunction with molecular approaches to create a rapid and cost-effective strategy for patient assessment and management. In our ongoing 8-year longitudinal study, a set of innovative technologies is being validated alone and in combination to best correlate with patient outcome. The plan is to use these devices in a step-by-step sequence to guide key clinicopathological decisions on patient risk and treatment. The devices include a hand-held visualization device that makes use of tissue autofluorescence to detect and delineate abnormal lesions and fields requiring follow-up, to be used in conjunction with optical contrast agents such as toluidine blue. In addition, two semi-automated high-resolution computer microscopy systems will be used to quantitate the protein expression phenotype of cell nuclei in tissue sections and exfoliated cell brushings. Previously identified risk-associated molecular changes are being used to validate these systems as well as to establish their place in a population-based triage program that will filter out high-risk cases in the community and funnel them to dysplasia clinics where higher-cost molecular tools will guide intervention. A critical development for the translation of this technology into community settings is the establishment of an effective methodology for education and training of health practitioners on the front lines.

Fluorescence visualization detection of field alterations in tumor margins of oral cancer patients.

PURPOSE: Genetically altered cells could become widespread across the epithelium of patients with oral cancer, often in clinically and histologically normal tissue, and contribute to recurrent disease. Molecular approaches have begun to yield information on cancer/risk fields; tissue optics could further extend our understanding of alteration to phenotype as a result of molecular change. EXPERIMENTAL DESIGN: We used a simple hand-held device in the operating room to directly visualize subclinical field changes around oral cancers, documenting alteration to fluorescence. A total of 122 oral mucosa biopsies were obtained from 20 surgical specimens with each biopsy being assessed for location, fluorescence visualization (FV) status, histology, and loss of heterozygosity (LOH; 10 markers on three regions: 3p14, 9p21, and 17p13). RESULTS: All tumors showed FV loss (FVL). For 19 of the 20 tumors, the loss extended in at least one direction beyond the clinically visible tumor, with the extension varying from 4 to 25 mm. Thirty-two of 36 FVL biopsies showed histologic change (including 7 squamous cell carcinoma/carcinomas in situ, 10 severe dysplasias, and 15 mild/moderate dysplasias) compared with 1 of the 66 FV retained (FVR) biopsies. Molecular analysis on margins with low-grade or no dysplasia showed a significant association of LOH in FVL biopsies, with LOH at 3p and/or 9p (previously associated with local tumor recurrence) present in 12 of 19 FVL biopsies compared with 3 of 13 FVR biopsies (P=0.04). CONCLUSIONS: These data have, for the first time, shown that direct FV can identify subclinical high-risk fields with cancerous and precancerous changes in the operating room setting.