Patients

In our study 148 patients with squamous cell carcinoma of the head and neck were included prospectively, all diagnosed at the Department of Otolaryngology of the Medical University of Vienna. The inclusion criterion was the presence of a previously untreated, histologically proven squamous cell carcinoma of the oropharynx, hypopharynx, oral cavity or larynx. Exclusion criteria were infectious diseases, immunosuppression and malignancies other than squamous cell carcinoma of the upper respiratory tract.

All patients underwent a physical examination with special attention to the head and neck region, a panendoscopy, a complete blood count, a biochemical analysis of liver and kidney function and electrolytes, an electrocardiogram, chest x-rays, an abdominal ultrasound, a computed tomography scan of the head and neck and the medical history were obtained.

The tumor samples were taken during diagnostic panendoscopy. Blood samples were taken at the time of diagnosis, 3 months and 12 months after the end of therapy, respectively. In addition, serum of 31 healthy volunteers who were selected to approximate the age range in patient samples served as control.

Tumor samples from 100 patients were available for immunohistochemical analysis. Tumor and blood samples were taken from 31 patients and used to compare immunohistochemistry to Elisa results. The median observation period was 32 months (range 25-40 months). Of the 148 patients included, 66 were primarily operated, 79 received primary radiotherapy and 3 refused treatment.

The patients were primarily Caucasian and male. There were 31 female patients and 117 male patients. Of the 79 patients whose serums were analyzed 11 were women and 68 were men. The carcinomas which were included were distributed as follows: 30 Hypopharynx carcinomas, 24 carcinomas of the oral cavity, 34 carcinomas of the tongue, 25 carcinomas of the Larynx, 35 Oropharynx carcinomas including 18 carcinomas of the tonsils. The patients primarily had advanced tumor stages. 50 patients had a T1/T2 carcinoma, 98 had a T3/T4.

Radiotherapy was performed using external beam irradiation with a final dose of 72 Gy in fractions of 1.8 Gy. Chemotherapy consisted of Cisplatin 100 mg/m²/day on weeks 1 and 3.

All samples were obtained after informed consent and collected using protocols approved by the Institutional Review Board.

Tissue microarray

Before analysis, hematoxylin-eosin-stained sections from each tumor sample were reevaluated and the suitability of inclusion in the study was determined. Sections of 2 to 3 μm were used for the analysis. The suitability of the tissue was evaluated using a number of inclusion criteria such as the size (1-2 mm in depth and at least 5 mm in length and width), as well as other features, such as appropriate fixation, absence of significant electrosurgical device lesions, signs of acidic decalcifying agents, and the presence of usable tissue in each block. Each hematoxylin-eosin-stained slide was reevaluated and mapped to identify the specific areas for tissue acquisition to build the tissue microarrays using a manual tissue arrayer (MTA-1; Beecher Instruments, Sun Prairie, WI). Core diameter was 0.6 mm. 3 cores were used per patient. We constructed tumor tissue microarrays from 100 paraffin embedded tumor samples.

Immunohistochemistry

Before analyzing the samples the optimal dilution ratio and best choice of retrieval buffer was determined. To determine the expression of ADAM8 we stained the tissue microarrays with a commercially available antibody specific for ADAM8.

Paraffin embedding was removed from the microarray slides and after rehydration they were subjected to antigen retrieval in a microwave oven (600 W) employing Tris-EDTA buffer followed by 3 wash cycles with TBS buffer for 5 min. Unspecific binding was avoided by adding 5% TBS/BSA for 1 h at room temperature. Then the primary antibody (ADAM8, 1:100, mouse IgM) (MBL, USA) was applied and incubated overnight at 4° in a wet chamber. A sample without primary antibody was used as negative control. The next day the secondary biotinylated antibody (1:200, Multilink, Dako, DK) in 1% TBS/BSA was applied and incubated for 1 h at room temperature. After another washing cycle alkaline phosphatase conjugated Streptavidin-AP/TBS/BSA (1:250, Dako, DK) was applied for 1 h at room temperature. Visualization was performed by fast red (Sigma, Missouri, USA) and counterstained by haemalaun. Samples were analyzed using an Olympus BH-2 microscope.

Afterwards three independent investigators (M.B., B.E., S.S.) analyzed the staining intensity by assigning each to one of 3 levels. 0 is no staining, 1 is moderate staining, and 2 is strong staining. Observer bias was avoided by repeating the evaluation of protein expression at two different time points and without knowledge of patients' clinical data. Mean IS was calculated from the three samples per patient.

ELISA

Serum ADAM8 levels were determined in 79 head and neck cancer patients before treatment, in 35 patients after treatment and in 10 patients 1 year after therapy and in 31 controls by using a commercially available enzyme test kit (R&D System Inc., USA).

The manufacturer's instructions were strictly followed. As a control one well was used without antibody. In short: after coating a 96-well micro plate with a monoclonal antibody specific for ADAM8 and incubated over night, the sera diluted 2:1 with the reagent diluent was added and allowed to incubate for 2 h at room temperature. After washing off the unbound substances the detection antibody was added, incubated for 2 h. After another washing step Streptavidin-HRP was added and incubated for 20 min. At the end the substrate solution was added to the wells. The color-reaction was stopped after 20 min by adding 50 μl of 2 NH²SO4.

The color reaction in the different wells was measured by a microplate reader set to 450 nm with a wavelength correction of 540 nm.

Statistical analysis

The overall significance level was set to be 0,05. The remaining significance level was distributed to 5 hypothesis [12].

To calculate the prognostic survival of the immunohistochemistry results the "Logrank-Test"was used and then shown in a Kaplan-Meier curve. To compare the ADAM8 serum levels of the 4 different groups a Kruskal-Willis rank sum test was used.

To test for trend of ADAM8 serum levels with rising immunohistologic grade of tumor samples a Jonckheere-Terpstra test was carried out. The difference of ADAM8 serum concentration between early and advanced patients was determined by a Wilcoxon rank sum test with continuity correction. This test was also used to evaluate if there was a difference in the serum level of ADAM8 between the healthy control group and patients with HNSCC. To determine the prognostic value of ADAM8 serum levels a Wilcoxon rank sum test was used.

Clinical data

In total 148 patients were included in the study of which 27 were women and 121 were men. The median age was 58 years (range 21-88 years) for the patients and 57 years (range 39-81 years) for the control group.

At time of diagnosis blood samples were taken from 79 patients, after 3 months 35 patients were available for a second serum sampling and after 1 year there were 10 patients for a third serum sampling. The remaining patients could not be analyzed for the following reasons: 12 had died, 6 were lost for follow up and the remaining were not able to come to our department in time for blood sampling.

The statistical analysis showed that expression levels of ADAM8 in immunohistochemistry corresponded with survival (p-value = 0.011, JT = 4380). Examples of the immunohistochemistry staining can be found in Figure 1. Patients with tumors overexpressing ADAM8 have significantly worse survival (see Figure 2). ADAM8 serum levels in the ELISA correlated well with immunohistochemistry expression levels, (p-value = 0.019). The correlation of the results of immunohistochemistry and ELISA is shown in Figure 3. We, however, did not find a correlation between soluble ADAM8 levels and patients' survival (p-value = 0.128).

We could not demonstrate a significant difference between ADAM8 serum levels of patients with HNSCC at the time of diagnosis compared to the healthy controls.

(p-value = 0.325). The comparison of soluble ADAM8 in early (T1/2, N0) and advanced (T3/4, N+) tumor stages did also not reveal a significant difference.

There was a difference of ADAM8 serum levels at different stages of disease when comparing pre-therapeutic patients, patients 3 months after therapy and patients 12 months after therapy (p-value = 0.010). The healthy control group and patients 3 months after therapy had higher ADAM8 serum levels than patients at the time of diagnosis and 12 months after therapy (see Figure 4).

Immunohistochemistry results were divided as follows: 51 patients had no staining, 45 had moderate staining and 4 had strong staining.