Ken Berean

Use of Allelic Loss to Predict Malignant Risk for Low-grade Oral Epithelial Dysplasia

One of the best approaches to identifying genetic changes critical to oral cancer progression is to compare progressing and nonprogressing oral premalignant lesions. However, such samples are rare, and they require long-term follow-up. The current study used the large archive network and clinical database in British Columbia to study loss of heterozygosity (LOH) in cases of early oral premalignancies, comparing those with a history of progression to carcinoma in situ or invasive cancer and those without a history of progression (referred to as nonprogressing cases). Each of 116 cases was analyzed for LOH at 19 microsatellite loci on seven chromosome arms (3p, 4q, 8p, 9p, 11q, 13q, and 17p). The progressing and nonprogressing cases showed dramatically different LOH patterns of multiple allelic losses. An essential step for progression seems to involve LOH at 3p and/or 9p because virtually all progressing cases showed such loss. However, LOH at 3p and/or 9p also occurred in nonprogressing cases. Individuals with LOH at 3p and/or 9p but at no other arms exhibit only a slight increase of 3.8-fold in relative risk for developing cancer. In contrast, individuals with additional losses (on 4q, 8p, 11q, or 17p), which appeared uncommon in nonprogressing cases, showed 33-fold increases in relative cancer risk. In conclusion, analysis of LOH at 3p and 9p could serve as an initial screening for cancer risk of early premalignancies. Follow-up investigation for additional losses would be essential for predicting cancer progression.

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.

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.

Loss of Heterozygosity (LOH) Profiles—Validated Risk Predictors for Progression to Oral Cancer

A major barrier to oral cancer prevention has been the lack of validated risk predictors for oral premalignant lesions (OPL). In 2000, we proposed a loss of heterozygosity (LOH) risk model in a retrospective study. This paper validated the previously reported LOH profiles as risk predictors and developed refined models via the largest longitudinal study to date of low-grade OPLs from a population-based patient group. Analysis involved a prospective cohort of 296 patients with primary mild/moderate oral dysplasia enrolled in the Oral Cancer Prediction Longitudinal Study. LOH status was determined in these OPLs. Patients were classified into high-risk or low-risk profiles to validate the 2000 model. Risk models were refined using recursive partitioning and Cox regression analyses. The prospective cohort validated that the high-risk lesions (3p and/or 9p LOH) had a 22.6-fold increase in risk (P = 0.002) compared with low-risk lesions (3p and 9p retention). Addition of another 2 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 with the low-risk group, intermediate- and high-risk groups had 11.6-fold and 52.1-fold increase in risk (P < 0.001). LOH profiles as risk predictors in the refined model were validated in the retrospective cohort. Multicovariate analysis with clinical features showed LOH models to be the most significant predictors of progression. LOH profiles can reliably differentiate progression risk for OPLs. Potential uses include increasing surveillance for patients with elevated risk, improving target intervention for high-risk patients while sparing a large number of low-risk patients from needless screening and treatment. Cancer Prev Res; 5(9); 1081–9. ©2012 AACR.

Cytogenetic findings in seven lacrimal gland neoplasms

We have undertaken cytogenetic investigation of seven benign and malignant lacrimal gland neoplasms. This study showed recurrent chromosomal abnormalities involving chromosomes 3, 8, 9, and 12. These features are similar to those found in benign and malignant salivary gland tumors, which suggests possible common mechanisms involved in the neoplastic proliferation of these histologically related tumors.

Complex karyotypic alterations in an endometrial stromal sarcoma

Cytogenetic investigation of a low-grade endometrial stromal sarcoma (ESS) revealed structural rearrangements of chromosomes 3, 6, and 7. The karyotypic findings in the few cases of ESS reported reveal recurrent involvement of both homologues of chromosome 7, with less consistent changes affecting chromosomes 6 and 17. The cytogenetic and histologic features of these uterine stromal tumors require further study.

Loss of desmoglein 1 expression associated with worse prognosis in head and neck squamous cell carcinoma patients

Aims: Mucosal squamous cell carcinomas are the most common head and neck malignancies. We hypothesised that over‐expression of intracellular signalling proteins and decreased expression of desmoglein molecules would be associated with aggressive tumour behaviour in patients with head and neck squamous cell carcinoma. Methods: Seventy‐eight cases of head and neck squamous cell carcinoma were immunohistochemically stained for desmoglein 1, desmoglein 2, desmoglein 3, p53, bcl‐2, vimentin, cyclin D1, p16, p21, p27, E‐cadherin, and E2F‐1 in paraffin‐embedded tissue blocks in a microarray. Results: The disease‐specific survival was 56% at 5 years and 49% at 10 years. Expression of the desmoglein isotypes correlated positively with each other except for desmoglein 2 and desmoglein 3, which did not show a significant correlation. Desmoglein 1 and E‐cadherin expression also correlated. On univariate analysis, only expression of desmoglein 1 correlated with patient outcome; lack of expression of desmoglein 1 was associated with a significantly worse disease‐specific survival (p = 0.035). Hierarchical clustering analysis identified a subgroup of three patients with an immunophenotype distinct from the other tumours, characterised by co‐expression of p16, p27, E2F‐1 and bcl‐2. Further statistical analysis of the prognostic significance of this small subgroup was not possible, but these three patients are alive and well. Conclusions: Decreased expression of desmoglein 1 is associated with a worse prognosis in head and neck squamous cell carcinoma patients. Examination of an extended panel of immunomarkers revealed a rare subtype of squamous cell carcinoma characterised by the expression of multiple proliferation‐associated markers and the anti‐apoptotic protein, bcl‐2; determination of the prognostic significance of this subgroup will require study of a larger case series.

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.