Molecular diagnosis of minimal residual head and neck cancer and field cancerization

promotiedatum: 21-5-2014 � prom-id: 975

Expression signature in peripheral blood cells for molecular diagnosis of head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) have a poor prognosis due to the development of locoregional recurrences, distant metastases, and second primary tumors. There is an urgent need for biomarkers that enable detection and monitoring of the disease to provide adequate therapeutic strategies. In this study, we have investigated markers in peripheral blood cells (PBC) of 28 HNSCC patients who underwent surgery by means of expression profiling. Our hypothesis is that nucleated blood cells circulate continuously, also pass the tumor, and change their expression profile in response to tumor cell factors. For comparison, we enrolled a control group of 11 patients who underwent surgery in the head and neck region for non-HNSCC reasons. A set of 2949 genes was found to be statistically different between the groups (P < 0.05, false discovery rate-corrected) and the most prominently different pathways were EIF2, EIF4, and mTOR signaling. These preliminary results are promising and warrant further studies on the definitive role of PBC gene expression as a biomarker for HNSCC detection and monitoring. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Molecular detection of minimal residual cancer in surgical margins of head and neck cancer patients

A great disappointment in head and neck cancer surgery is that 10-30% of head and neck squamous cell carcinoma (HNSCC) patients develop local recurrences despite histopathologically tumor-free surgical margins. These recurrences result from either minimal residual cancer (MRC) or preneoplastic lesions that remain behind after tumor resection. Distinguishing MRC from preneoplasic lesions is important to tailor postoperative radiotherapy more adequately. Here we investigated the suitability of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using human Ly-6D (hLy-6D) transcripts as molecular marker to detect MRC in surgical margins. Submucosal samples of deep surgical margins were collected from 18 non-cancer control patients and 67 HNSCC patients of whom eight had tumor-positive surgical margins. The samples were analyzed with hLy-6D qRT-PCR, and the data were analyzed in relation to the clinicohistological parameters. A significant difference was shown between the group of patients with histopathological tumor-positive surgical margins and the non-cancer control group (p<0.001), and the group of patients with histopathological tumor-free surgical margins (p=0.001). This study shows a novel approach for molecular analysis of deep surgical margins in head and neck cancer surgery. The preliminary data of this approach for detection of MRC in deep margins of HNSCC patients are promising

Screening for Oral Precancer with Noninvasive Genetic Cytology

Oral squamous cell carcinomas develop in precancerous fields consisting of genetically altered mucosal epithelial cells. These precancerous fields may appear as clinically visible lesions, in particular, oral leukoplakia, but the large majority remains clinically undetectable. The aim of this study was to assess the potential value of a noninvasive screening approach to detect precancerous fields. As a first step, we developed a suitable assay and investigated 25 leukoplakia patients and 20 noncancer control subjects. Exfoliated cells were removed by a brush from multiple small areas of the oral mucosa, including the leukoplakia. Brushed samples were investigated for allelic imbalance (AI) at chromosomes 3p, 9p, 11q, and 17p using microsatellite markers known to show frequent alterations in oral precancer. AI was absent in all (137) of the samples of the 20 control subjects, yielding a specificity of 100%. AI was detected in exfoliated cell samples of 40% (10 of 25) of the leukoplakia lesions studied. Genetic changes were also found outside the leukoplakia lesions. Most frequent was AI at 9p (9 of 10). The noninvasive assay was validated against the biopsy results of the leukoplakia lesions yielding an estimate of sensitivity of 78% (7 of 9) and a positive predictive value of 100% (7 of 7). Altogether, these results show the feasibility of a noninvasive genetic screening approach for the detection and monitoring of oral precancer. This assay could therefore contribute to the secondary prevention of oral squamous cell carcinoma. The assay also shows promise for the detection of precancerous changes that are not macroscopically visible

Differential proteomics identifies protein biomarkers that predict local relapse of head and neck squamous cell carcinomas

Purpose: The 5-year survival rates of head and neck squamous cell carcinomas (HNSCC) remain disappointing. HNSCCs develop in precursor fields of genetically altered cells that are often not completely resected when the tumor is excised, causing local relapse. These precursor fields are mostly recognized as dysplasia, but histologic grading cannot reliably predict malignant transformation. Our aim was to discover and validate protein biomarkers that can detect precursor fields and predict local relapse in HNSCC using immunostaining of surgical margins. Experimental Design: We compared paired and genetically characterized normal, precursor, and tumor tissues of eight patients by proteome analysis to identify differentially expressed proteins. The prognostic value of candidate protein biomarkers was evaluated by immunohistochemical analysis of 222 surgical margins of 46 HNSCC patients who developed local relapse or remained disease free. Significant associations were determined by Kaplan-Meier survival analysis and Cox-proportional hazards models. Results: Forty proteins showed significant differential expression (false discovery rate-corrected P < 0.05). Most discriminative markers suited for immunostaining were keratin 4 and cornulin. Low expression in the surgical margins of keratin 4 (hazard ratio, 3.8; P = 0.002), cornulin (hazard ratio, 2.7; P = 0.025), and their combination (hazard ratio, 8.8; P = 0.0005) showed a highly significant association with the development of local relapse. Dysplasia grading had no prognostic relevance. Conclusions: Immunohistochemical assessment of keratin 4 and cornulin expression in surgical margins of HNSCC patients outperforms histopathologic grading in predicting the risk for local relapse. These markers can be used to initiate more frequent and lifelong surveillance of patients at high risk of local relapse, and enable selection for adjuvant treatment or tertiary prevention trials

Molecular screening of oral precancer

Objectives Early detection and treatment of high risk premalignant mucosal changes of the oral cavity, will expectedly improve survival and reduce treatment-related morbidity. Aims of this study were to evaluate a non-invasive screening approach and to assess the value of molecular markers to identify patients at risk for oral cancer. Materials and Methods Exfoliated cells and biopsies were obtained from oral leukoplakia lesions of 43 patients, of whom six developed oral cancer. All samples were investigated for loss of heterozygosity (LOH) at chromosomes 3p, 9p, 11q and 17p using microsatellite markers. On the biopsy specimen additional immunohistochemical staining for p53, TP53 mutation analysis and histopathological grading were performed. Results The analytical sensitivity of the non-invasive assay using exfoliated cells to detect genetic changes present in the lesions was 45% (9 of 20), the specificity was 100% (19 of 19), and the positive predictive value was also 100% (9 of 9). LOH was present in 20 of 39 (51%) of the biopsies with uniformly LOH at 9p. Mutated TP53 and LOH at 9p in the biopsy, as single markers and in combination, were significant risk factors for malignant progression of leukoplakia to oral cancer (Kaplan-Meier analysis, p < 0.05). Conclusion A non-invasive genetic screening approach using LOH in exfoliated cells has limited value for monitoring patients with leukoplakia. However, LOH at 9p, but also mutated TP53 in biopsies of oral leukoplakia have a significant association with malignant transformation and are promising candidate biomarkers to predict the risk for malignant progression