Denise M. Laronde

Toluidine Blue Staining Identifies High-Risk Primary Oral Premalignant Lesions with Poor Outcome

There is a pressing need for the development of visual aids that will facilitate the detection of oral premalignant lesions (OPLs) with a high-risk of progression. Preliminary data suggest that toluidine blue stain may be preferentially retained by OPLs with high-risk molecular clones. In this study, we monitored OPLs from 100 patients without any history of oral cancer for an average of 44 months in order to evaluate the association of toluidine blue status with clinicopathologic risk factors, molecular patterns (microsatellite analysis on seven chromosome arms: 3p, 9p, 4q, 8p, 11q, 13q, and 17p) and outcome. Toluidine blue-positive staining correlated with clinicopathologic risk factors and high-risk molecular risk patterns. Significantly, a >6-fold elevation in cancer risk was observed for toluidine blue-positive lesions, with positive retention of the dye present in 12 of the 15 lesions that later progressed to cancer (P = 0.0008). This association of toluidine blue status with risk factors and outcome was evident even when the analysis was restricted to OPLs with low-grade or no dysplasia. Our results suggest the potential use of toluidine blue in identifying high-risk OPLs.

DIRECT FLUORESCENCE VISUALIZATION OF CLINICALLY OCCULT HIGH-RISK ORAL PREMALIGNANT DISEASE USING A SIMPLE HAND-HELD DEVICE

A considerable proportion of oral cancer and precancer is not clinically apparent and could contribute significantly to the late diagnosis and high mortality of oral cancer. A simple method to identify such occult change is needed.

Squamous cell carcinoma and precursor lesions: diagnosis and screening in a technical era.

Oral cancer is a substantial, though often unrecognized issue globally, with close to 300,000 new cases reported annually (18). The disease presents a management conundrum. It occurs at a site that is easily accessible for examination, yet, regrettably, is often diagnosed at an advanced stage when functional impairment due to treatment and mortality rates are high. Late stage identification is associated with high mortality with 5-year survival rates ranging from 30- 60%, depending on the global locale (64; 76). It is also associated with high rates of second oral malignancies, with up to a third of such patients suffering a recurrence of the tumor or the development of a second primary despite intensive follow-up (11; 36; 56). These figures have shown little improvement over the past few decades. Despite these statistics, there is hope for change in the not-too-distant future. This hope is fueled by recent strides being made in our understanding of cancer and in the emergence of promising technologies that could aid clinicians in detection, risk assessment and management of this disease. Several alternative detection and diagnostic aids for examining the oral cavity are commercially available or in development. The current challenge is to recognize that these devices are still evolving, yet in many cases they already are moving into the clinical arena. There is a need for the clinician to better understand these new innovations: the biology underlying their function, their strengths and weaknesses, and the scenarios under which they may demonstrate clinical utility. The intent of this paper is to highlight parameters to be considered when utilizing such technology and to indicate where we are in the development and validation of such approaches and in their incorporation into clinical practice. The paper will explore more recent concepts with respect to tissue change that underlies evident disease, briefly summarize difficulties with the current clinicopathological approaches for detection and diagnostic management of disease and then present an overview of current and developing diagnostic and screening methods. There have been several recent reviews of some of these devices (16; 32; 33; 40; 41). Our intent is not to duplicate this activity but rather to supplement it by building a framework for technology assessment that integrates data summarized in these papers.

Abstract 3251: Assessing risk markers for oral cancer recurrence

Oral cancer has a poor 5-year survival rate, partly due to a high local recurrence (REC) rate. Clinical indicators such as size, color, and site have been found to be associated with progressing primary oral premalignant lesions (OPL), but have yet to be evaluated for OPL development at treated cancer sites. Similarly, recent LOH regions highly predictive of primary progression (Zhang et al., Cancer Prevention Research 2012), have not been evaluated as predictive markers of OPLs in oral cancer REC. Methods: The analysis involved 194 patients enrolled in the Oral Cancer Prevention Longitudinal (OCPL) study with early stage (Stage I & II) squamous cell carcinoma (SCC) or carcinoma-in-situ (CIS), treated with curative intent. Data was collected every 3 months post-treatment up to 2 years, then every 6 months. Demographic (age, gender, risk habits), tumor information (site, stage, treatment) and clinical data (presence of an OPL, size, color, texture and appearance) were collected. Biopsies taken during follow-up were tested for high-risk molecular patterns. REC was defined as development of a severe dysplasia, CIS or SCC within 3 cm of the primary tumor site. Results: 31(16%) patients suffered a REC at the former tumor site. Presence of an OPL in follow-up had an almost 7-fold increased risk of REC (P Conclusion: The presence of an OPL at a former tumor site is a critical predictor of oral cancer REC regardless of its characteristics, with nearly all cases preceded in time by its appearance. The issue is whether it is reactive or a true OPL with risk. Risk of REC increases if an OPL is present at each follow-up visit and/or is present within the first year following treatment. OPLs persisting during follow-up should be biopsied. Regardless of histology, the presence of high-risk molecular patterns in these samples offers an additional opportunity to assess risk of REC. Citation Format: Denise M. Laronde, Lewei Zhang, P. Michele Williams, Bertrand Chan, Jay H. Park, Catherine F. Poh, Miriam P. Rosin. Assessing risk markers for oral cancer recurrence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2014-3251

Abstract 3567: Loss of heterozygosity (LOH) profiles predict oral cancer recurrence in the Oral Cancer Recurrence Prediction Cohort

Oral squamous cell carcinoma (SCC) has a poor prognosis with local recurrence occurring in up to 30% of cases. Oral mucosal changes are common at previously treated cancer sites; however, despite close follow-up, they often develop into recurrent tumor. LOH analysis has been shown to predict cancer recurrence in lesions at former tumour sites in retrospective studies. Objective: To determine the value of specific LOH markers in predicting oral cancer recurrence in samples from an ongoing longitudinal study. Design: Data were collected from 365 oral cancer patients enrolled in the Oral Cancer Recurrence Prediction Cohort. Methods: The study selection criteria included: 1) Patients originally diagnosed with carcinoma in situ (CIS) or SCC and treated with a curative intent (mostly Stage 0/I/II); 2) Appearance of a lesion at the tumor site during follow-up with biopsy histologically diagnosed as hyperplasia or low-grade (mild/moderate dysplasia) (higher diagnoses went to treatment); 3) Continued follow-up of patient; 4) Sufficient tissue in biopsy for LOH analysis. 71 patients met these criteria. Recurrence outcome was defined as development of severe dysplasia, CIS or SCC at the target site. Timeline was calculated from target date to outcome for recurring group or last follow-up date for non-recurring group. Target biopsies were analyzed using 19 microsatellite loci on 7 chromosome arms (3p, 4q, 8p, 9p, 11q, 13q, and 17p). Univariant Cox analysis was used to determine the prognostic value of clinical and microsatellite markers. Recursive partitioning trees and Kaplan Meier curves were constructed to create molecular models for both 9p and 3p. Results: Recurrence occurred in 26 or the 71 patients (36.6%) with 18 months median follow-up time (compared with 33 months in non-recurring patients). There were no differences between recurrent and non-recurrent groups for gender, ethnicity, tobacco habit, original diagnosis before treatment and histology of target biopsy (P>0.05). RR was significantly increased for cases with LOH at 9p, 3p, 8p, 13q and 17p (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3567. doi:1538-7445.AM2012-3567

Abstract CN02-02: Malignant risk prediction for patients with oral premalignant lesions

Oral cancers remain both a challenge and an opportunity for clinicians and scientists working with this disease. Worldwide, it represents a significant global challenge, with close to 300,000 new cases diagnosed each year. The disease is potentially preventable: we have knowledge of its risk factors; it is also at an easily accessible site and is often preceded by oral premalignant lesions (OPLs). The challenge has been to develop a framework that would let us both detect and better manage such lesions. Unfortunately, only a small proportion of OPLs will progress to a cancer and histology alone has not allowed clinicians to differentiate high- from low-risk lesions. This uncertainty has also meant that, even when OPLs are detected, there is no consensus on who to treat and how. In 1999, we established the Oral Cancer Prevention Program in British Columbia to develop an integrated knowledge translation approach to facilitate systematic change for prevention, detection and management of this disease across the continuum of care throughout the province. This effort has involved optimization of screening in community dental practices, creation of a triage pathway from a centralized oral biopsy service to oral dysplasia clinics where patients are assessed, and formation of linkages to the cancer agency and local hospitals to facilitate both treatment and follow-up. A new Canadian Partnership Against Cancer-funded Oral Dysplasia Surveillance System has been developed in BC to monitor natural history of the disease. This province-wide structure has facilitated the formation of the Oral Cancer Prediction Longitudinal (OCPL) study, a unique resource for development and validation of biomarkers of risk of malignant progression. To date, ∼ 450 low-grade (mild and moderate) dysplasia cases have been accrued to this study and are in follow-up. In September of this year, we published results from the first 296 patients accrued to the OCPL study (Zhang et al., Cancer Prevention Research). We focused on the validation of a LOH risk model proposed by our group in 2000 that utilized a set of microsatellite markers at key chromosomal loci to predict progression of low-grade dysplasia in a retrospective cohort (Rosin et al., Clin Cancer Res 2000; 6:357–62). In the new study, cases were classified into high- or low-risk profiles to validate the 2000 model. Risk models were further refined using recursive partitioning and Cox regression analyses. The study showed that the high-risk lesions (3p &/or 9p LOH) had a 22•6 -fold increase in risk (P = 0•002) compared to low-risk lesions (3p & 9p retention). Addition of another two markers (loci on 4q/17p) further improved the risk prediction, with five-year progression rates of 3•1%, 16•3%, and 63•1% for the low-, intermediate-, and high-risk lesions, respectively. Compared to the low-risk group, intermediate- and high-risk groups had 11•6-fold and 52•1-fold increase in risk (P < 0•001). The new LOH profiles in the refined model were validated as risk predictors by using the initial retrospective cohort from 2000. Multi-covariate analysis with clinical features showed LOH models to be the most significant predictors of progression. The importance of this study is that it has direct implication for standard of care in the community setting, providing a tool by which patients could be triaged to different levels of intervention. Patients with elevated risk would be guided towards increased surveillance with this risk providing a rationale for the targeting of intervention regimes, even if these were associated with some morbidity. Two of three patients with such high-risk profiles progressed to cancer in 5 years. In contrast, patients with low-risk profiles could be spared in from aggressive monitoring and intervention. In our study, low-risk patients represented close to half of those individuals that were accrued to the study. Future research should be aimed towards exploring such possibilities, as an initial step towards personalizing oral cancer prevention strategies. LOH is currently being used in two chemoprevention trials of patients with OPLs to guide patient accrual: the phase III Erlotinib Prevention of Oral Cancer study and the Phase II Cetuximab for Treatment of High-Risk Pre- Malignant Upper Aerodigestive Lesions trial. The results of these studies should provide even greater knowledge upon which to build new strategies for patient triage that could better focus risk reduction programs for this disease. (Supported by grants from the NIH and the National Institute of Dental and Craniofacial Research (R01DE13124 and R01DE17013). Citation Format: Miriam P. Rosin, Lewei Zhang, Catherine Poh, Michele Williams, Denise M. Laronde, Ken Berean, Pamela J. Gardner, Huijun Jiang, Lang Wu, J. Jack Lee. Malignant risk prediction for patients with oral premalignant lesions. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr CN02-02.

Abstract 2901: Visualization and delineation of high-risk fields in the oral cavity

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Oral cancer is a substantial, though often unrecognized issue globally, with close to 300,000 new cases reported annually. The disease represents a management conundrum: this is a cancer site that is easily examined; yet more that 40% of oral cancers are diagnosed at a late stage when the chance of death is high and treatment can be disfiguring and devastating. Visualization of high-risk fields can be improved by application of contrast agents, such as toluidine blue (TB) or through use of devices that measure alteration to tissue optics, such as fluorescence visualization (FV) both of which could facilitate assessment of abnormalities. This studys objective was to evaluate FV, within a high-risk clinic, to look for associations between loss of autofluorescence (FVL) and alterations to clinical, histological and molecular features and to determine its ability to detect high-risk oral premalignant fields and cancer. Methods: The study involved 170 patients, with 192 oral lesions (64 cancers, 28 severe dysplasia, 66 low-grade (mild/moderate) dysplasia and 34 nondysplasia), being followed in the ongoing Oral Cancer Prediction Longitudinal Study. Four categories of data were collected: 1) demographic and habit information (age, gender, ethnicity and tobacco habits); 2) lesion histology; 3) clinicopathological features at time of biopsy (lesion size, site, appearance, toluidine blue (TB) staining and FV status); and 4) molecular risk patterns of the lesions (loss of heterozygosity, LOH). Results: Demographics and smoking habit were not associated with FV status. Clinicopathological features of the lesion, appearance (P 2) arms (P=0.009). TB positivity was found to be highly associated with FVL (P<0.001). To date, 7 premalignant lesions have progressed to a high-grade lesion or SCC and all were FVL at time of low-grade dysplasia biopsy (FVL and progression, P=0.047). Six of the 7 progressing lesions were TB+. Conclusion: FV was found to be a very useful adjunctive tool when used by experienced clinicians in high-risk clinics. Integrating TB and FV results may aid in the detection of low-grade lesions at risk of progression. Further study of FV in low-grade and nondysplasia with a larger sample size is required. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2901.