Frank K.J. Leusink

Validation of a Gene Expression Signature for Assessment of Lymph Node Metastasis in Oral Squamous Cell Carcinoma

PURPOSE Current assessment of lymph node metastasis in patients with head and neck squamous cell carcinoma is not accurate enough to prevent overtreatment. The aim of this study was validation of a gene expression signature for distinguishing metastasizing (N+) from nonmetastasizing (N0) squamous cell carcinoma of the oral cavity (OSCC) and oropharynx (OPSCC) in a large multicenter cohort, using a diagnostic DNA microarray in a Clinical Laboratory Improvement Amendments/International Organization for Standardization-approved laboratory. METHODS A multigene signature, previously reported as predictive for the presence of lymph node metastases in OSCC and OPSCC, was first re-evaluated and trained on 94 samples using generic, whole-genome, DNA microarrays. Signature genes were then transferred to a dedicated diagnostic microarray using the same technology platform. Additional samples (n=222) were collected from all head and neck oncologic centers in the Netherlands and analyzed with the diagnostic microarray. Human papillomavirus status was determined by real-time quantitative polymerase chain reaction. RESULTS The negative predictive value (NPV) of the diagnostic signature on the entire validation cohort (n=222) was 72%. The signature performed well on the most relevant subset of early-stage (cT1-T2N0) OSCC (n=101), with an NPV of 89%. CONCLUSION Combining current clinical assessment with the expression signature would decrease the rate of undetected nodal metastases from 28% to 11% in early-stage OSCC. This should be sufficient to enable clinicians to refrain from elective neck treatment. A new clinical decision model that incorporates the expression signature is therefore proposed for testing in a prospective study, which could substantially improve treatment for this group of patients.

Novel diagnostic modalities for assessment of the clinically node-negative neck in oral squamous-cell carcinoma

Oral squamous-cell carcinomas arise in mucosal linings of the oral cavity and frequently metastasise to regional lymph nodes in the neck. The presence of nodal metastases is a determinant of prognosis and clinical management. The neck is staged by palpation and imaging, but accuracy of these techniques to detect small metastases is low. In general, 30-40% of patients will have occult nodal disease and will develop clinically detectable lymph-node metastases when the neck is left untreated. The choice at present is either elective treatment or careful observation followed by treatment of the neck in patients who develop manifest metastases. These unsatisfying therapeutic options have been the subject of debate for decades. Recent developments in staging of the neck, including expression profiling and sentinel lymph-node biopsy, will allow more personalised management of the neck.

Nodal metastasis and survival in oral cancer: Association with protein expression of SLPI, not with LCN2, TACSTD2, or THBS2

Gene expression profiling revealed a strong signature predicting lymph node metastases in oral squamous cell carcinoma (OSCC). Four of the most predictive genes are secretory leukocyte protease inhibitor (SLPI), lipocalin‐2 (LCN2), thrombospondin‐2 (THBS2), and tumor‐associated calcium signal transducer 2 (TACSTD2). This study correlates their protein expression with lymph node metastases, overall survival (OS), and disease‐specific survival (DSS).

Cannabinoid receptor-2 immunoreactivity is associated with survival in squamous cell carcinoma of the head and neck

The prediction of progression of individual tumours, prognosis, and survival in squamous cell carcinoma (SCC) of the head and neck is difficult. Cannabinoid-1 (CB1) and cannabinoid-2 (CB2) receptor expression is related to survival in several types of cancer, and the aim of this study was to find out whether the expression of CB1 and CB2 receptors is associated with survival in primary SCC of the head and neck. We made immunohistochemical analyses of the cannabinoid receptors on tissue arrays from 240 patients with the disease. Receptor immunoreactivity was classified as none, weak, moderate, or strong staining. Overall survival and disease-specific survival were plotted using Kaplan-Meier survival curves. A multivariate Cox proportional hazard model was created with all the relevant clinical and pathological features. Strong immunoreactivity of the CB2 receptor was significantly associated with reduced disease-specific survival (p=0.007). Cox-proportional hazard ratio (HR) showed that CB2 receptor immunoreactivity contributed to the prediction of survival (HR 3.6, 95% CI 1.5-8.7, p=0.004). Depth of invasion (HR 2.2, 95% CI 1.2-4.2, p=0.01) and vascular invasion (HR 2.5, 95% CI 1.4-4.5, p=0.001) were also associated with survival.

Downregulation of SERPINB13 expression in head and neck squamous cell carcinomas associates with poor clinical outcome.

Tumorigenesis of head and neck squamous cell carcinomas (HNSCC) is associated with various genetic changes such as loss of heterozygosity (LOH) on human chromosome 18q21. This chromosomal region maps a gene cluster coding for a family of intracellular serine protease inhibitors (serpins), including SERPINB13. As SERPINB13 expression in HNSCC has recently been shown to be downregulated both at the mRNA and protein levels, here we investigated if such a low SERPINB13 expression is associated with histopathological and clinical parameters of HNSCC tumors and patient survival. By generating specific antibodies followed by immunohistochemistry on a well‐defined cohort of 99 HNSCC of the oral cavity and oropharynx, SERPINB13 expression was found to be partially or totally downregulated in 75% of the HNSCC as compared with endogenous expression in non‐neoplastic epithelial cells. Downregulation of SERPINB13 protein expression in HNSCC was significantly associated with the presence of LOH at the SERPINB13 gene in the tumors (p = 0.006), a poor differentiation grade of the tumors (p = 0.001), the presence of a lymph node metastasis (p = 0.012), and a decreased disease‐free (p = 0.033) as well as overall (p = 0.018) survival of the patients. This is the first report demonstrating that downregulation of SERPINB13 protein expression in HNSCC is positively associated with poor clinical outcome. Therefore, SERPINB13 seems to act as an important protease inhibitor involved in the progression of HNSCC.

The Co-Expression of Kallikrein 5 and Kallikrein 7 Associates with Poor Survival in Non-HPV Oral Squamous-Cell Carcinoma

Objective: Oral squamous-cell carcinoma (OSCC) still has a poor prognosis. Lymph node metastasis (LNM) is a major determinant of treatment decisions and prognosis. Serine protease inhibitor Kazal-type 5 (SPINK5) is the inhibitor of kallikrein 5 (KLK5) and KLK7. SPINK5, KLK5 and KLK7 are three of the genes of a recently validated LNM-predicting gene expression profile in OSCC. This study evaluates their clinicopathological role and value as biomarkers in OSCC. Methods: Eighty-three patients with primary OSCC, treated surgically between 1996 and 2000, were included. Gene expression data were acquired from a previously reported study. Human papillomavirus (HPV) status was determined by an algorithm for HPV-16. Protein expression for KLK5, KLK7 and SPINK5 was semi-quantitatively determined in all 83 tumours by immunohistochemistry. All expression data were correlated with clinicopathological parameters. Results: Concurrent loss of KLK5 and KLK7 correlates with worse disease-specific and overall survival (DSS and OS). Multivariate analysis proved that co-expression is an independent prognostic factor for DSS (p = 0.029) and OS (p = 0.001). Conclusion: This report demonstrates that concurrent loss of KLK5 and KLK7 associates with a poor clinical outcome in OSCC and could therefore serve as prognostic marker in this disease.

Nieuwe onderzoeksmethoden om occulte halskliermetastasen van mondkanker op te sporen

Orale plaveiselcelcarcinomen (OPCC) ontstaan vanuit de slijmvliezen van de mond en metastaseren bij voorkeur naar de regionale lymfeklieren in de hals. De aanwezigheid van halskliermetastasen is een belangrijke prognostische factor en bepalend voor de klinische behandeling.

Accuracy of computer-assisted surgery in mandibular reconstruction: A systematic review

Computer-assisted surgery (CAS) for mandibular reconstruction was developed to improve conventional treatment methods. In the past years, many different software programs have entered the market, offering numerous approaches for preoperative planning and postoperative evaluation of the CAS process of mandibular reconstruction. In this systematic review, we reviewed planning and evaluation methods in studies that quantitatively assessed accuracy of mandibular reconstruction performed with CAS. We included 42 studies describing 413 mandibular reconstructions planned and evaluated using CAS. The commonest software was Proplan/Surgicase CMF (55%). In most cases, the postoperative virtual 3-dimensional model was compared to the preoperative 3-dimensional model, revised to the virtual plan (64%). The commonest landmark for accuracy measurements was the condyle (54%). Accuracy deviations ranged between 0 mm and 12.5 mm and between 0.9° and 17.5°. Because of a lack of uniformity in planning (e.g., image acquisition, mandibular resection size) and evaluation methodologies, the ability to compare postoperative outcomes was limited; meta-analysis was not performed. A practical and simple guideline for standardizing planning and evaluation methods needs to be considered to allow valid comparisons of postoperative results and facilitate meta-analysis in the future.

Abstract 4122: Multi-center validation of a lymph node metastasis gene-expression signature for head and neck squamous cell carcinomas

Background: The inability of current diagnostic tools to detect small lymph node metastases in patients with Head and Neck Squamous Cell Carcinoma (HNSCC) leads to a rate of undetected metastases that is too high to refrain from elective neck treatment, resulting in overtreatment. The aim of this study is validation of a gene expression signature for distinguishing metastasizing from non-metastasizing HNSCC on a large multi-center cohort. Material and methods: Samples of oral cavity and oropharyngeal cancer were collected from all head and neck oncological centers in the Netherlands. Gene expression was analyzed with a DNA microarray representing 696 previously reported predictive genes. The negative predictive value (NPV) was assessed on the whole cohort (n=222), on a subset including only clinically node negative (cN0) tumors (n=143) and on the most relevant subset that included T1 and T2, cN0 oral cavity tumors (n=101). Histological examination was used as gold standard for nodal status. Results: Overall, the NPV of the signature was 72 %. The signature performed better on the cN0 subset (NPV 85%) and very well in the clinically most relevant subset of T1 and T2, cN0, oral cavity SCC (NPV 89%). Conclusion: Combining the results of clinical and radiological examination with the gene expression signature decreases the rate of undetected nodal metastasis to 11 % in the relevant group of early stage cancers of the oral cavity. This result should be considered sufficient to refrain from elective neck treatment in these patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4122. doi:10.1158/1538-7445.AM2011-4122