Detection of sentinel lymph node using contrast-enhanced agent, Sonazoid™, and evaluation of its metastasis with superb microvascular imaging in oral and oropharyngeal cancers: a preliminary clinical study.

BACKGROUND: In sentinel lymph node (SLN) biopsy for head and neck cancers, the radioisotope method has been the gold standard. However, this method has several problems, such as unavoidable radiation exposure and requirements of expensive equipment. AIMS/OBJECTIVES: To overcome these problems, we evaluated the contrast-enhanced ultrasonography (CEUS)-guided SLN-detection method, and predicted the SLN metastatic status using novel ultrasound technology, superb microvascular imaging (SMI). METHODS: Ten patients (6 with oral and 4 with oropharyngeal cancers) without neck lymph node metastasis were enrolled in this study. Ultrasound contrast agent, Sonazoid™, was infiltrated into the mucosa at the primary site to observe the lymphatic ducts and SLNs in the neck field. The detected SLNs were examined for blood flow using SMI to categorize the SLNs metastases-positive or negative. RESULTS: SLNs were successfully detected in 8 out of 10 cases. In 7 out of the 8 cases, in whom SLNs were successfully detected, the metastatic status of SLNs was correctly diagnosed with SMI. CONCLUSIONS AND SIGNIFICANCE: Although more clinical data are needed based on a larger cohort, establishing the CEUS-guided SLN-detection and criteria for the accurate diagnosis of SLN-metastases using SMI would be valuable as an alternative to radioisotope method, in oral and oropharyngeal cancers.

Downregulation of the α1- and ß1-subunit of sGC in Arterial Smooth Muscle Cells of OPSCC Is HPV-Independent.

The nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an α and ß subunit. NO binds to heme of the ß1-subunit of sGC, activates the enzyme in the reduced heme iron state in vascular smooth muscle cells (VSMCs), and generates cGMP-inducing vasodilatation and suppression of VSMC proliferation. In the complex tumor milieu with higher levels of reactive oxygen species (ROS), sGC heme iron may become oxidized and insensitive to NO. To change sGC from an NO-insensitive to NO-sensitive state or NO-independent manner, protein expression of sGC in VSMC is required. Whether sGCα1ß1 exists at the protein level in arterial VSMCs of oropharyngeal squamous cell carcinoma (OPSCC) is unknown. In addition, whether differences in the genetic profile between human papillomavirus (HPV)-positive and HPV-negative OPSCC contributes to the regulation of sGCα1ß1 is unclear. Therefore, we compared the effects of HPV-positive and HPV-negative OPSCC on the expression of sGCα1ß1 in arterial VSMCs from tumor-free and tumor-containing regions of human tissue sections using quantitative immunohistochemistry. In comparison to the tumor-free region, we found a decrease in expression of both α1- and ß1-subunits in the arterial VSMC layer of the tumor-containing areas. The OPSCC-induced significant downregulation of the α1- and ß1-subunits of sGC in arterial VSMC was HPV-independent. We conclude that the response of sGC to NO in tumor arterial VSMCs may be impaired by oxidation of the heme of the ß1-subunit, and thus, α1- and ß1-subunits of sGC could be targeted to degradation under oxidative stress in OPSCC in an HPV-independent manner. The degradation of sGCα1ß1 in VSMCs may result in increased proliferation of VSMCs, promoting tumor arteriogenesis in OPSCC. This can be interrupted by preserving the active heterodimer sGCα1ß1 in arterial VSMCs.

Diagnosis of the extent of advanced oropharyngeal and hypopharyngeal cancers by narrow band imaging with magnifying endoscopy.

OBJECTIVES/HYPOTHESIS: Narrow band imaging combined with magnifying endoscopy (NBI-ME) is useful for the detection of superficial cancer in the oropharynx, hypopharynx, and esophagus. We used NBI-ME to evaluate the frequency of superficial cancer spread (SCS) contiguous with advanced oropharyngeal and hypopharyngeal cancers and esophageal cancers. STUDY DESIGN: Retrospective. METHODS: We retrospectively studied 45 patients with oropharyngeal and hypopharyngeal cancer and 44 with esophageal cancer who underwent NBI-ME from October 2006 through April 2009. The following variables were evaluated: 1) the frequency of SCS contiguous with advanced oropharyngeal and hypopharyngeal cancer and esophageal cancer, and 2) the influence of SCS contiguous with advanced oropharyngeal and hypopharyngeal cancer on clinical T category and clinical stage. RESULTS: SCS contiguous with the primary tumor was found in 49% (22/45) of the patients with advanced oropharyngeal and hypopharyngeal cancer and in 52% (23/44) of those with advanced esophageal cancer. When SCS contiguous with the primary tumor was included in the evaluation of tumor size in advanced oropharyngeal and hypopharyngeal cancer, the clinical T category and clinical stage were revised in 20% (9/45) and 4% (2/45) of patients, respectively; SCS was ≤ 2 cm in 64% of cases (14/22) and between >2 cm and ≤ 4 cm in 36% (8/22). CONCLUSIONS: NBI-ME should be included in the pretreatment diagnostic work-up to evaluate lesion extent and decide optimal surgical margins and radiation fields in patients with advanced oropharyngeal and hypopharyngeal cancer.

Confocal endomicroscopy: a novel application for imaging of oral and oropharyngeal mucosa in human.

Confocal endomicroscopy is an emerging technique for intravital visualization of neoplastic lesions, but its use has so far been limited to the gastrointestinal (GI) tract. This study was designed to assess the feasibility of in vivo confocal endomicroscopy of different regions of the oropharyngeal mucosa and to evaluate different contrast agents. We examined five different regions of the human oropharynx in vivo, and images were collected in real time by using a confocal laser endoscope as formerly described for the GI tract. Additionally ex vivo specimens were examined using a topical contrast agent. Confocal scanning was performed at 488-nm illumination for excitation of exogenously applied fluorophores (topical acriflavine and intravenous fluorescein). Confocal endomicroscopy allowed for visualization of cellular and subcellular structures of the anterior human oropharyngeal region. Fluorescein staining yielded architectural details of the surface epithelium and also subepithelial layers. Images taken at increasing depth beneath the epithelium showed the mucosal capillary network. Acriflavine strongly contrasted the cell nuclei of the surface epithelium. The findings correlated well with the histology of biopsy specimens. This is the first report showing that the use of fluorescence confocal endomicroscopy represents a promising method to examine cellular details in vivo in different oropharyngeal regions in human.

Radiotherapy and concomitant intra-arterial docetaxel combined with systemic 5-fluorouracil and cisplatin for oropharyngeal cancer: a preliminary report--improvement of locoregional control of oropharyngeal cancer.

PURPOSE: To confirm the advantage of chemoradiotherapy using intra-arterial docetaxel with intravenous cisplatin and 5-fluorouracil. PATIENTS AND METHODS: A total of 26 oropharyngeal cancer patients (1, 2, 2, and 21 patients had Stage I, II, III, and IVa-IVc, respectively) were treated with two sessions of this chemoradiotherapy regimen. External beam radiotherapy was delivered using large portals that included the primary site and the regional lymph nodes initially (range, 40-41.4 Gy) and the metastatic lymph nodes later (60 or 72 Gy). All tumor-supplying branches of the carotid arteries were cannulated, and 40 mg/m(2) docetaxel was individually infused on Day 1. The other systemic chemotherapy agents included 60 mg/m(2) cisplatin on Day 2 and 500 mg/m(2) 5-fluorouracil on Days 2-6. RESULTS: The primary response of the tumor was complete in 21 (81%), partial in 4 (15%), and progressive in 1 patient. Grade 4 mucositis, leukopenia, and dermatitis was observed in 3, 2, and 1 patients, respectively. During a median follow-up of 10 months, the disease recurred at the primary site and at a distant organ in 2 (8%) and 3 (12%) patients, respectively. Three patients died because of cancer progression. Two patients (8%) with a partial response were compromised by lethal bleeding from the tumor bed or chemotherapeutic toxicity. The 3-year locoregional control rate and the 3-year overall survival rate was 73% and 77%, respectively. CONCLUSION: This method resulted in an excellent primary tumor response rate (96%) and moderate acute toxicity. Additional follow-up is required to ascertain the usefulness of this modality.

Outcome prediction after surgery and chemoradiation of squamous cell carcinoma in the oral cavity, oropharynx, and hypopharynx: use of baseline perfusion CT microcirculatory parameters vs. tumor volume.

PURPOSE: To assess whether pretreatment perfusion computed tomography (PCT) may predict outcome in chemoradiated patients with oral cavity, oropharynx, and hypopharynx squamous cell carcinoma (SCCA) after surgical excision. MATERIALS AND METHODS: Twenty-one patients with SCCA were examined before treatment. The primary site was oral cavity in 6, oropharynx in 7, and hypopharynx in 8 patients; there were 11 T2, 6 T3, and 4 T4 tumors. PCT was performed at the level of largest tumor diameter based on standard neck CT. The data were processed to obtain blood flow (BF), blood volume (BV), mean transit time (MTT), and permeability surface area product (PS). Regions of interest were free-hand positioned on the lesions to obtain PCT measurements. Tumor volume was also calculated. Follow-up was performed with positron emission tomography (PET)/CT and endoscopy. Pearson correlation coefficient was used for comparison between the subgroups. A regression model was constructed to predict recurrence based on the following predictors: age, gender, tumor (T) and nodal (N) stage, tumor volume, and PCT parameters. RESULTS: BF(mean), BF(max), BV(mean), BV(max), MTT(mean), PS(mean), and PS(max) were significantly different between patients with and without tumor recurrence (0.0001, p < 0.04). T stage, tumor volume, N stage, BF(max), BV(max), MTT(mean), and radiation dose (p < 0.001) were independent predictors for recurrence. Cox proportional hazards model for tumor recurrence revealed significantly increased risk with high tumor volume (p = 0.00001, relative risk [RR] 7.4), low PS(mean) (p = 0.0001, RR 14.3), and low BF(max) (p = 0.002, RR 5.9). CONCLUSIONS: Our data suggest that PCT parameters have a prognostic role in patients with SCCA.

Quantitative measurements of perfusion and permeability of oropharyngeal and oral cavity cancer, recurrent disease, and associated lymph nodes using first-pass contrast-enhanced computed tomography studies.

OBJECTIVES: We sought to evaluate the routine clinical use of perfusion computed tomography in the detection and differentiation of primary and recurrent oropharynx and oral cavity tumors as well as of nodal disease. MATERIALS AND METHODS: A total of 77 patients with primary cancer as well as suspected recurrent disease and lymph nodes were evaluated. A dynamic acquisition (4 x 6-mm slices) of the largest axial tumor surface was performed and the tumor blood flow (BF), blood volume (BV), and mean transit time (MTT) were calculated by using a modified deconvolution-based analysis taking into account the extravasation of the contrast agent for permeability surface area product imaging (PS). Tumor volume was calculated and region of interest analysis was performed on the pathologic and normal tissue. RESULTS: The mean BF, BV, and PS values in the primary tumors (77.48 mL/min/100 g tissue; 5.29 mL/min; 13.33 mL/min/100 g tissue, respectively) were highly significantly different (P < 0.01) than those obtained in the normal structures. Mean MTT values (9.01 seconds) also were significantly lowered in the tumors compared with normal tissue (P < 0.05). There was no statistical difference in the perfusion values between the primary and the recurrent tumors. Recurrent disease could be differentiated on the basis of BF (P < 0.05) from tissue changes after chemo-radiation-treatment (mean BF: 69.71 versus 45.31 mL/min/100 g tissue, respectively). Differentiation of the lymph nodes was not possible by means of perfusion values. Tumor volume did not significantly correlate with any perfusion parameter. CONCLUSIONS: Perfusion CT of oropharyngeal and oral cavity cancer in clinical routine is feasible and helps outlining the malignant tissue as well as differentiating recurrent disease from nonspecific post-therapeutic changes.

Endoglin expression is associated with poor oncologic outcome in oral and oropharyngeal carcinoma.

CONCLUSIONS: The present preliminary results suggest that endoglin (CD105)-assessed micro-vessel density (MVD) in primary oral and oropharyngeal squamous cell carcinomas (SCCs) may identify patients at risk of disease recurrence or poor oncological outcome after treatment. OBJECTIVES: MVD is an independent prognostic indicator in several human malignancies. Endoglin antibodies have shown a greater specificity for tumour vasculature in comparison with pan-endothelial markers. The present explorative study evaluated endoglin expression and its prognostic role in oral and oropharyngeal SCCs. PATIENTS AND METHODS: The study considered 13 consecutive cases of oral and oropharyngeal SCC with lymph node metastases (pN+) and 13 consecutive pN0 cases. CD105-assessed MVD was calculated at 400x magnification. RESULTS: The mean MVDs were 3.6 and 3.1 in pN+ and pN0 groups, respectively (p>0.05). The mean CD105-assessed MVDs were 4.7 in the group with locoregional recurrence and 2.9 in the group without locoregional recurrence or post-treatment diagnosis of distant metastasis (p=0.01). The mean CD105-assessed MVD in primary oral and oropharyngeal SCCs with poor oncological outcome (recurrence of disease or occurrence of distant metastasis) was 4.3. The mean MVD in primary oral and oropharyngeal SCCs with good outcome was 2.9. Statistical analysis showed a significant difference between CD105-assessed MVD in poor and good outcome groups (p=0.02).

Antiangiogenic effects of radiotherapy but not initial microvessel density predict survival in inoperable oropharyngeal squamous cell carcinoma.

PURPOSE: To evaluate the predictive value of intratumoral microvessel density (MVD) in patients with locally advanced, primarily inoperable oropharyngeal cancer (OPCC) treated with irradiation alone. PATIENTS AND METHODS: Tumor samples of 35 patients with OPCC subjected to radiotherapy were taken before and after 20 Gy irradiation. Histological grade, mitotic activity index and MVD of OPCC were determined. Correlations with response and survival were analyzed. RESULTS: Stage and posttreatment MVD decrease showed significant correlation with objective response. Postirradiation MVD decrease correlated significantly with response to irradiation and overall survival, and a trend could be seen for progression-free survival. CONCLUSION: This study has shown that the change in MVD after irradiation can be an indicator of the success of radiotherapy in locally advanced OPCC, and may serve as a predictive factor thus helping to select patients who could benefit from more aggressive treatment.

Correlation between initial and early follow-up CT perfusion parameters with endoscopic tumor response in patients with advanced squamous cell carcinomas of the oropharynx treated with organ-preservation therapy.

BACKGROUND AND PURPOSE: Current organ-preservation regimens for upper aerodigestive tract squamous cell carcinoma (SCCA) require endoscopic procedures under general anesthesia to evaluate the tumor response. The purpose of our study was to determine whether CT perfusion (CTP) parameters correlate with response to induction chemotherapy as assessed by endoscopy under general anesthesia. METHODS: Nine patients with advanced (stage 3 or 4) SCCA of the oropharynx were enrolled in a nested phase 2 prospective trial in which induction chemotherapy was used to assess the tumor response. Patients underwent direct laryngoscopy and CTP before and 3 weeks after one cycle of induction chemotherapy. The outcome variables were the surgeon's estimate of tumor volume during endoscopy with biopsy under anesthesia and CTP parameters (capillary permeability (CP), blood volume (BV), blood flow (BF), and mean transit time (MTT)). Wilcoxon rank sum analysis was used to correlate the baseline values of BF and BV with response to induction chemotherapy. Comparison of agreement between the reduction in tumor volume and change in CTP parameters was performed by using kappa estimates. RESULTS: Seven of 9 patients demonstrated > or =50% tumor volume reduction, representing positive response to induction chemotherapy. In the responder group, the following changes in mean pre- and postinduction chemotherapy values were noted: mean BF, 114.2 mL/100 g /min (preinduction) to 45.1 mL/100 g/min (postinduction); mean BV, 5.11 mL/100 g to 3.1 mL/100 g; mean CP, 25.6 mL/100 g /min (preinduction) to 18.3 mL/100 g / min (postinduction); mean MTT, 4.9 seconds (preinduction) to 8.0 seconds (postinduction). In the nonresponder group, the following changes were noted: mean BF, 56.9 mL/100 g/min to 75.9 mL/100 g/min; mean, BV 2.7 mL/100 g to 4.71 mL/100 g; mean CP, 24.1 mL/100 g/min to 23.7 mL/100 g/min; mean MTT, 4.3 seconds to 5.34 seconds. Higher baseline (pretherapy) values of BV showed significant correlation with endoscopic tumor response (P < .05). Reduction in the BV (by >/=20%) on follow-up studies also showed substantial agreement with clinical response as assessed with endoscopy (kappa = 0.73). The agreement between decreased BF, decreased CP, and increased MTT and clinical response was fair (kappa = 0.37). CONCLUSION: These preliminary results show that deconvolution-based CTP technique offers potential for noninvasive monitoring of response to induction chemotherapy in patients with oropharyngeal cancers. Percentage reduction of BV is significantly correlated to endoscopic response to induction chemotherapy, though we acknowledge that the data correspond to short-term outcomes and long-term durability of response cannot be established. Nevertheless, validation of the use of deconvolution CTP parameters as predictors of tumor response may permit replacement of an invasive diagnostic procedure conducted under anesthesia currently used to assess response with noninvasive perfusion CT imaging.

[Local chemotherapy for squamous cell cancer of the oral cavity and oropharynx]. / Lokale Chemotherapie oraler und oropharyngealer Plattenepithelkarzinome.

BACKGROUND: Modern treatment concepts for patients suffering from oral and oropharyngeal cancer include more and more adjuvant therapeutic options. Local chemotherapy offers the possibility to apply an extremely high drug concentration at the tumor site while minimizing possible side effects by systemic neutralization at the same time. PATIENTS AND METHODS: A total of 289 patients with histologically proven carcinoma of the oral cavity and the oropharynx underwent neoadjuvant intra-arterial chemotherapy with high-dosage cisplatin within a multimodal therapeutic setting. Concerning the TNM classification, more than 70% of the patients were classified as stages III and IV. The mean age at the time of intervention was 60 years, and 71% of the patients were male. RESULTS: After the first cycle 19.3% of the patients presented with complete remission (grade I); 35.4% and 41.5% showed partial remissions (grade II) or stable disease (grade III), respectively. The mean observation time after treatment was 28 months (median: 24.2 months). Of the 137 patients who completed the full multimodal therapeutic scheme, 11% developed local recurrence, and 12.4% developed lymph node or distant metastasis. At the time of evaluation, 72.5% of these patients were still alive. CONCLUSION: Intra-arterial chemotherapy is a safe and highly effective procedure that should be considered as an important option in multimodal therapeutic concepts for oral and oropharyngeal cancer.

Narrow band imaging: a new diagnostic approach to visualize angiogenesis in superficial neoplasia.

Although numerous gastrointestinal endoscopes pass through the oropharynx and the hypopharynx, it is extremely difficult to detect an early cancer in these sites during routine endoscopic examination. Most patients with cancer in these sites are usually diagnosed in advanced stages. If effective screening methods can detect an earlier stage, such as carcinoma in situ, it would obviously be of great benefit. Narrow band imaging is an innovative optical technology that can clearly visualize the microvascular structure of the organ surface. Herein, we demonstrate that narrow band imaging combined with magnifying endoscopy can identify a carcinoma in situ in oropharyngeal and hypopharyngeal mucosal lesions. Scattered irregular foci of microvascular proliferation projecting to the dysplastic squamous epithelium are the typical features. These results indicate that an approach to visualize angiogenesis or morphologic changes of microvessels in the superficial neoplasm can be a new diagnostic method not only for the head and neck region but also for other sites in the gastrointestinal tract.

[Radiation-induced alterations in microvessel density as predictor of treatment efficiency in oropharyngeal cancer]. / A sugárkezelés hatására létrejött érdenzitás-változás a terápia eredményességének prediktora mesopharynxrák esetében.

In this prospective study we have evaluated the predictive role of intratumoral microvessel density (MVD) for the therapeutic response and progression of inoperable oropharyngeal cancer (OPCC) treated with radiotherapy. Thirty-five OPCC patients were enrolled in this study. Biopsies from the primary tumor were taken before and after 20 Gy irradiation. Pathological factors such as histological grade, mitotic activity index and MVD were determined in both samples. Correlations of these factors with response to therapy, progression-free and overall survival were analyzed after a follow-up period of a minimum of 50 months. Objective response and survival was independent of the pretreatment MVD of OPCC. On the other hand, objective response was significantly affected by stage and low posttreatment MVD. Response to irradiation, and therapy-induced low postirradiation MVD were significant indicators of better overall and progression-free survival. We have shown in this small exploratory study that the anti-angiogenic effect of irradiation has a predictive value of the success of radiotherapy in locally advanced OPCC and can be used to select a radioresistant patient population which might require a more aggressive protocol.

[Interest of PET/CT scan fusion to assess mandible involvement in oral cavity and oro pharyngeal carcinomas]. / Intérêt de la fusion TEP/TDM dans l'exploration de l'envahissement mandibulaire des cancers de la cavité orale et de l'oropharynx.

UNLABELLED: The aim of the study is to assess mandible involvement in oral cavity and oropharyngeal carcinomas. OBJECTIVE: To evaluate interest of fusion of Positron Emission Tomography (PET) with Computed Tomography scan (CT scan). METHOD: Eight patients were included in this prospective study. Each patient underwent PET and CT scan of the head and neck before surgery including tumorectomy and mandibulectomy. We compared results of PET- CT fusion with histologic examinations. RESULTS: Oral cavity (6), oropharyngeal (2) carcinoma: Mandibular invasion was suspected by PET-CT in 3 cases, but was confirmed in histological examination in only 2 cases. In 5 cases, PET-CT did not find mandibular invasion; this was confirmed in histological examination in all cases. Sensibility of PET-CT fusion was 100%, specificity was 83%. Positive predictive value was 66% and negative predictive value was 100%. DISCUSSION: PET-CT fusion provided maximal sensitivity. Specificity was better than for MRI but less than CT-scan. There were no false negatives and the false positive rate was 33%. CONCLUSION: PET-CT fusion is interesting to predict mandible involvement. Further studies are necessary to confirm these preliminary results.

[Dynamic magnetic resonance tomography (dMRT). It's value in advanced head-neck tumors treated with radiochemotherapy]. / Dynamische Magnetresonanztomographie (dMRT). Wertigkeit bei fortgeschrittenen Kopf-Hals-Tumoren unter Radiochemotherapie.

PURPOSE: With the help of dynamic magnetic resonance tomography (dMRT) the status of tissue microcirculation can be visualized. METHODS: Dynamic MRI was performed in 13 patients with advanced, nonresectable oro- or hypopharynx carcinoma at the beginning and the end of therapy. Maximal signal intensity and exchange rate constant in the tissue of the tumor and the lymph node metastases were analyzed using a pharmacokinetic two-compartment model. RESULTS: In all six patients with clinical complete response (CR), the maximal signal intensity increased after therapy in the tissue of the primary tumor and the lymph node metastases. Furthermore, a high decrease in the parameter k(21) was associated with a better prognosis and could be observed especially after combined radiochemotherapy. CONCLUSION: Our first results indicate that contrast-enhanced dynamic MRI studies before and after radio- or combined radiochemotherapy offer important information about the changes of microcirculation in the tissue of the tumor and lymph node metastases. Furthermore, this information seems to be a promising predictor for clinical outcome of therapy.

Expression of matrix metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 9 in carcinoma of the head and neck.

BACKGROUND: Numerous reports have documented a direct involvement of matrix metalloproteinase (MMP) overexpression in the development and progression of head and neck squamous cell carcinoma (HNSCC). In this study, the authors examined whether the expression of MMPs in HNSCC is correlated with other steps involved in tumor growth and metastasis, like angiogenesis, activation the nitric oxide (NO) pathway, and alteration of the p53 tumor suppressor gene. METHODS: MMP-1, MMP-2, and MMP-9 expression levels were examined immunohistochemically in samples from 43 patients with HNSCC. Microvessel density (MVD) was determined by immunostaining of endothelial cells with anti-CD31 monoclonal antibody. Inducible nitric oxide synthase (iNOS) activity and cyclic guanosine monophosphatate (cGMP) levels were assessed in fresh tumor samples, whereas exons 5-9 of the p53 gene were analyzed by reverse transcriptase-polymerase chain reaction, single-strand conformation polymorphism analysis and were sequenced. RESULTS: MMP-1 overexpression (>10% of tumor cells) was identified in 32 tumors (74.5%), whereas elevated levels of MMP-2 and MMP-9 were detected in 17 tumors (39.5%) each. Tumors with MMP-9 overexpression were characterized by significantly higher MVD (P = 0.05) and significantly higher iNOS activity and cGMP levels (P = 0.005 and P = 0.02, respectively). Moreover, p53 mutation was associated strongly with MMP-9 overexpression (P = 0.004). Conversely, no correlation was found between MMP-1 and MMP-2 expression, angiogenesis, iNOS activity, cGMP levels, and p53 mutation in this series. CONCLUSIONS: This study documents the existence of a correlation between MMP-9 expression, activity of the iNOS pathway, p53 status, and angiogenesis in patients with HNSCC. This raises the possibility that p53 mutation, which frequently is present in HNSCC, may result in increased angiogenesis and invasiveness related to increased nitric oxide and MMP production by tumor cells, ultimately contributing to tumor progression.

Intratumoral microvessel density predicts local treatment failure of radically irradiated squamous cell cancer of the oropharynx.

PURPOSE: To determine the predictive value of intratumoral microvessel density (IMD), and of the expression of p53, vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) for the radiocurability of patients with squamous cell cancer of the oropharynx. MATERIALS AND METHODS: 139 patients with squamous cell cancer of the oropharynx were radically irradiated (median dose, 74 Gy) between 1991 and 1997. Biopsies from 100 patients were processed for immunohistochemistry. IMD was determined in hot spot areas of tissue stained with anti-CD31 at a magnification of x200. Staining for p53 was considered positive if more than 10% of the cell nuclei overexpressed the protein. Immunostaining of VEGF and TSP-1 was assessed semiquantitatively. RESULTS: Increasing IMD (range, 54-282) was strongly correlated with incomplete remission of both the primary tumors (p = 0.01) and lymph node metastases (p = 0.02). Moreover, multivariate Cox regression analysis revealed local failure-free survival to decline with increasing IMD (IMD continuous: risk ratio = 1.01 per increase of 1 microvessel, p = 0. 0001; IMD categorical: 130: risk ratio = 13.01). Neither the expression of p53, nor that of VEGF or TSP-1 was associated with the treatment outcome or IMD, but VEGF and TSP-1 expression were positively correlated (p = 0.02). CONCLUSION: IMD represents a powerful and independent predictive factor for local treatment failure in radically irradiated patients with squamous cell cancer of the oropharynx.

Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas.

Tumour oxygenation and vasculature are determinants for radiation treatment outcome and prognosis in patients with squamous cell carcinomas of the head and neck. In this study we visualized and quantified these factors which may provide a predictive tool for new treatments. Twenty-one patients with stage III-IV squamous cell carcinomas of the head and neck were intravenously injected with pimonidazole, a bioreductive hypoxic marker. Tumour biopsies were taken 2 h later. Frozen tissue sections were stained for vessels and hypoxia by fluorescent immunohistochemistry. Twenty-two sections of biopsies of different head and neck sites were scanned and analysed with a computerized image analysis system. The hypoxic fractions varied from 0.02 to 0.29 and were independent from T- and N-classification, localization and differentiation grade. No significant correlation between hypoxic fraction and vascular density was observed. As a first attempt to categorize tumours based on their hypoxic profile, three different hypoxia patterns are described. The first category comprised tumours with large hypoxic, but viable, areas at distances even greater than 200 micrometer from the vessels. The second category showed a typical band-like distribution of hypoxia at an intermediate distance (50-200 micrometer) from the vessels with necrosis at greater distances. The third category demonstrated hypoxia already within 50 micrometer from the vessels, suggestive for acute hypoxia. This method of multiparameter analysis proved to be clinically feasible. The information on architectural patterns and the differences that exist between tumours can improve our understanding of the tumour micro-environment and may in the future be of assistance with the selection of (oxygenation modifying) treatment strategies.

Expression of platelet-derived endothelial cell growth factor in oral and oropharyngeal carcinoma.

Platelet-derived endothelial cell growth factor (PD-ECGF) was isolated as an endothelial cell mitogen from platelets. In this study, we investigated the expression of PD-ECGF and counted microvessels in 58 oral and oropharyngeal squamous cell carcinoma (SCC) specimens by an immunohistochemical technique to examine their prognostic significance and performed tumor in vitro sensitivity to 5-fluorouracil (5-FU) and cisplatin as determined by a bioluminescence assay of the ATP values of tumor cells after continuous exposure. The percentage of PD-ECGF-positive tumor cells (PD-ECGF score) was correlated with the frequency of the recurrence of disease (P=0.0043) but not with sex, tumor size, metastasis, or clinical stage. Overall survival of the high PD-ECGF expression group (>40% PD-ECGF score) was shorter than the low expression (<40%) group (P=0.0365). Vessel count was correlated with lymph node metastasis and clinical stage. The survival of patients with hypervascularity (more than the median of intratumor vessel counts, >82) was shorter than that of those with hypovascularity (vessel count <81, P=0.0446). However, there was no association between PD-ECGF expression and vessel count. Cox proportional multivariate analysis showed that PD-ECGF expression was the most significant independent prognostic indicator for overall survival. The susceptibility to 5-FU cytotoxicity in the extremely high PD-ECGF expression groups (>70% of PD-ECGF score) was significantly higher than that in the low group, whereas there was no difference in their sensitivity to cisplatin. These results showed that carcinoma cells with high PD-ECGF expression were sensitive to 5-FU in spite of poor prognosis. These data provide further information when deciding on adjuvant therapy for oral and oropharyngeal SCCs.