Diseases that manifest in the oral cavity can have dramatic effects on quality of life, morbidity and mortality. One such disease, which accounts for nearly 94% of malignancies in the oral cavity, is oral squamous cell carcinoma (OSCC). While a number of known risk factors exist for development of OSCC, tobacco is the most significant and easily modifiable. Currently, OSCC 5-year survival rate is 60%, representing only a moderate improvement over previous decades despite improved surgical techniques and adjuvant therapies. This 5-year survival rate is negatively influenced by late diagnosis, with post-surgical recurrence rates of up to 30% in histologically tumor-free margins. While clinically evident changes are present in most precancerous lesions, it is unknown how many OSCC may arise from clinically normal oral mucosa.
Autofluorescence technology (VELscope) has been recommended to aid detection of disease and tumor margin delineation, thus allowing surgeons to modify intraoperative surgical margins. Viewing tumor and surrounding tissue with this portable handheld device reveals autofluorescent changes resulting from complex interactions of light and tissue. Healthy tissue is reported to fluoresce pale green (referred to as visual fluorescence retention - VFR), while diseased tissue loses this fluorescent property (visual fluorescence loss- VFL). In the context of tumor excision, extension of VFL beyond the clinical margins may better delineate the extent of diseased tissue. We attempted to add a molecular profile through loss of heterozygosity (LOH) analysis to tumor margins delineated and obtained with assistance from autofluorescence technology (VELscope). This process is termed “tumor mapping”. After tissue acquisition, margins were graded histologically and assessed for LOH using microsatellite markers 3p, 9p and 17p. Both traditional formalin-fixed (FF), laser capture microdissected (LCM) tissue and fresh frozen tissue were subject to molecular analysis to determine if one method performed optimally. The epithelium and stroma from fresh frozen tissue was separated using 3.8% ammonium thiocyanate (AT) through a process not previously described in mucosa.
When combining all locations, markers, and tissue separation methods, LOH was identified in 7 of 10 patients with a total of 13 allelic loss events. LOH was found more frequently in areas of fluorescence loss compared to areas of retention (10-VFL, 3-VFR), with no trends identified between histologic grading of tissue and occurrence of LOH. AT yielded more informative results, while FF identified more occurrences of LOH. Unexpectedly, stromal LOH (SLOH) was seen in 6 of 13 total loss events, while epithelial LOH (ELOH) totaled 7.
While not overwhelming, these LOH findings support tumor mapping as a useful technique for delineating surgical margins. Areas of VFL extension exhibiting LOH, revealed dysplastic epithelium in 9 of 10 samples, suggesting histologic and molecular risk. The findings of SLOH are uncertain but may have important biologic and therapeutic implications and thus warrant further investigation. Due to possible sampling limitations using fresh frozen tissue and AT, FF-LCM tissue is recommended for future studies.