Snezana Andrejevic-Blant

Prognostic value of arginase‐II expression and regulatory T‐cell infiltration in head and neck squamous cell carcinoma

Tumor‐infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3+ T cells, and relatively high levels of BDCA2+ and FOXP3+ cells in stromal (peripheral) regions of the tumors. Tumor‐infiltrating (intraepithelial) FOXP3+ T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase‐II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase‐2 by 43%, and B‐cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c+ myeloid dendritic cells, and high numbers of FOXP3+ T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease‐free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.

Blue-violet excited autofluorescence spectroscopy and imaging of normal and cancerous human bronchial tissue after formalin fixation

Autofluorescence (AF) imaging is a powerful tool for the detection of (pre‐)neoplastic lesions in the bronchi. Several endoscopic imaging systems exploit the spectral and intensity contrast of AF between healthy and (pre‐)neoplastic bronchial tissues, yet, the mechanisms underlying these contrasts are poorly understood. In this report, the effect of formalin fixation on the human bronchi AF, hence on the contrast, was studied by spectrofluorometric point measurements and DAFE (Diagnostic AutoFluorescence Endoscopy) broad field imaging. Generally, formalin‐fixed samples have higher AF intensity than in vivo, whereas the emission spectral shape is similar. Additionally, the spectrofluorometric data showed a moderate decrease of the AF intensity on (pre‐)neoplastic lesions relative to the healthy bronchial samples. However, this decrease was lower than that reported from in vivo measurements. Neither spectral measurements nor imaging revealed spectral contrast between healthy bronchial tissue and (pre‐)neoplastic lesions in formalin. These results indicate that epithelial thickening and blood supply in the adjacent lamina propria are likely to play a key role in the generation of the AF contrast in bronchial tissues. Our results show that the AF contrast in bronchial tissues was significantly affected by standard, 10% buffered, formalin fixation. Therefore, these samples are not suited to AF contrast studies.

Photodynamic Therapy Selectively Enhances Liposomal Doxorubicin Uptake in Sarcoma Tumors to Rodent Lungs

In specific conditions, photodynamic therapy (PDT) can enhance the distribution of macromolecules across the endothelial barrier in solid tumors. It was recently postulated that tumor neovessels were more responsive to PDT than the normal vasculature. We hypothesized that Visudyne®‐mediated PDT could selectively increase liposomal doxorubicin (Liporubicin™) uptake in sarcoma tumors to rodent lungs while sparing the normal surrounding tissue.

Distribution of Free and Liposomal Doxorubicin After Isolated Lung Perfusion in a Sarcoma Model

BACKGROUND Isolated lung perfusion (ILP) with free and a novel liposomal-encapsulated doxorubicin (Liporubicin, CT Sciences SA, Lausanne, Switzerland) was compared with respect to drug uptake and distribution in rat lungs bearing a sarcomatous tumor. METHODS A single sarcomatous tumor was generated in the left lung of 39 Fischer rats, followed 10 days later by left-sided ILP (n = 36) with free and equimolar-dosed liposomal doxorubicin at doses of 100 microg (n = 9) and 400 microg (n = 9) for each doxorubicin formulation. In each perfused lung, the drug concentration and distribution were assessed in the tumor and in three areas of normal lung parenchyma by high-performance liquid chromatography (n = 6) and fluorescence microscopy (n = 3). Histologic assessment and immunostaining with von Willebrand factor was performed in 3 animals with untreated tumors. RESULTS The sarcomatous tumors in controls were well vascularized with fine branching capillaries present throughout the tumors. Isolated lung perfusion resulted in a heterogeneous drug distribution within the perfused lung and a consistently lower drug uptake in tumors than in lung parenchyma for both doxorubicin formulations and both drug doses applied. Isolated lung perfusion with free doxorubicin resulted in a significantly higher drug uptake than Liporubicin in both the tumor and lung tissue for both drug doses applied (p < 0.01). However, the tumor/normal tissue drug ratio was lower for free than for liposomal doxorubicin at a drug dose of 100 microg (0.27 +/- 0.1 vs 0.53 +/- 0.5; p = 0.225) and similar for both doxorubicin formulations at a drug dose of 400 microg (0.67 +/- 0.2 vs 0.54 +/- 0.2; p = 0.335). Both doxorubicin formulations resulted in fluorescence signaling emerging from all tissue compartments of normal lung parenchyma but only in weak and sporadic signaling from the tumors confined to the tumor periphery and vessels situated within the tumor for both drug doses assessed. CONCLUSIONS Isolated lung perfusion with free and liposomal doxorubicin resulted in a heterogeneous drug distribution within the perfused lung and in a lower drug uptake in tumors than in lung tissue for both doxorubicin formulations and drug doses applied.

Clinicobiological progression and prognosis of oral squamous cell carcinoma in relation to the tumor invasive front: impact on prognosis.

Abstract Conclusion: There are several factors that influence the final outcome when treating oral squamous cell carcinoma (OSCC). Invasive front phenomena and more importantly their clinicopathological translation can have a direct impact on survival, and subsequently on the decision for an adjuvant treatment. Objectives: In recent years, the concept of tumor–host interaction has been the subject of substantial efforts in cancer research. Tumoral behavior may be better understood when studying the changes occurring at the tumor–host interface. This study evaluated the influence of several clinicopathological features on the outcome of OSCCs. Methods: The clinical records and pathology specimens of 54 patients with OSCC treated by primary resection were reviewed retrospectively. The pathologic features reviewed were: invasive front grading (IFG), stromal reaction, lymphovascular invasion (LVI), perineural invasion (PNI), margin status, and depth of invasion. Results: High IFGs had a significant relationship with pT status and pN status. High IFGs were strongly correlated with nodal metastases (odds ratio (OR) = 4.77; confidence interaval (CI) = 1.37–16.64). Concerning survival, IFG had a strong impact on disease-free survival in patients treated unimodally, as did the depth of invasion in the same group. Lymphovascular involvement was found to have a negative impact on overall survival in patients treated multimodally.

Interstitial photodynamic therapy with tetra(m-hydroxyphenyl)chlorin: tumor versus striated muscle damage

PURPOSE The present study was initiated to determine the conditions under which a single photodynamic treatment would induce maximal damage to a tumor with no or at least minimal reversible damage to a normal striated muscle. METHODS AND MATERIALS The technique of interstitial light delivery was used after prior 0.5 mg/kg tetra(m-hydroxyphenyl)chlorin administration in a hamster model. After having estimated the threshold light doses required for minimal muscle damage, the same light doses were applied to squamous cell carcinomas to evaluate the efficiency of interstitial photodynamic therapy. Sixteen and 96 h after the injection, irradiation at 650 nm was performed on the thigh muscle of the left hind leg. The applied light doses ranged between 0.3-15 J and were delivered at an intensity of 44 mW per cm of diffuser length. RESULTS The threshold of muscle damage was obtained using light doses of 1.5-3 J at two drug-light intervals of 16 and 96 h, respectively. More than 85% of the tumor mass was destroyed when lesions were illuminated using these threshold conditions. In terms of immediate short-term tumor response, this means that for the given irradiation conditions, a relatively low threshold energy of only 1.5 or 3 J, depending on the drug-light interval, is sufficient to induce massive tumor destruction with minimal muscle damage. CONCLUSION These results have implications for evaluating interstitial PDT for squamous cell cancers in unfavorable localization in the oral cavity or pharynx, such as at the base of the tongue.

Comparison of the influence of a water-soluble polymer carrier on the tumor localization and biodistribution of mesotetrametahydroxyphenylchlorin (mTHPC) in two animal models

Mesotetrametahydroxyphenylchlorin (mTHPC) is compared to mTHPC derivatized with one or more polyethyleneglycol (PEG) chains of about 6000 amu. The latter compound is called mTHPC-PEG hereafter. In particular the uptake and removal of these two compounds is studied in tumor and other tissues after injection as a function of the time. In a first model this is done with nude mice bearing human colorectal xenografts (LS174T); in a second model it is studied in a Chinese hamster with a 7,12 dimethylbenzanthracene (DMBA) induced early squamous cell carcinoma in the cheekpouch mucosa. In the nude mice with tumors grown to between 70 and 130 mg, tumor to muscle dye ratios are found to attain a maximum value of 4.8, eight hours after injection of mTHPC. With mTHPC-PEG the tumor to muscle ratio attained a value as high as 18.6, forty-eight hours after injection. In the hamster model the maximum tumor to normal mucosa ratio for mTHPC was about 3.5 at times up to 2 hours after injection. For the mTHPC-PEG this ratio is always near unity in this model.

Pharmacokinetics of meso-(tetrahydroxyphenyl)chlorin (m-THPC) studied by fluorescence spectroscopy on early cancer of the cheek pouch mucosa of Golden Syrian hamsters

Golden Syrian hamsters are evaluated as an animal model for phototherapy of early squamous cell carcinomas of the mucosa of the upper aerodigestive tract, the esophagus and the tracheobronchial tree. Carcinomas of this type are induced on the hamster cheek pouch mucosa by the application of the carcinogen 7,12 DMBA. For phototherapeutic experiments on the animals we utilized meso- (tetrahydoxyphenyl)chlorin (mTHPC). The same drug is currently in phase I, II clinical trials for ENT patients with superficial squamous cell carcinomas. By means of light induced fluorescence (LIF) we measured in vivo the kinetics of the uptake and removal of mTHPC in the normal and tumoral cheek mucosa and in the skin. The photodynamic therapy (PDT) reaction of the tissue after excitation of the photosensitizer by laser light at 652 nm was studied. Both pharmacokinetics and PDT efficacy are compared between animal model and clinical results with special emphasis on selectivity between normal and tumoral mucosa. These first experiments show that this tumor model in the hamster cheek pouch seems to be suitable for tests of a number of PDT variables of new photosensitizers preceding their clinical application as well as for optimization of the multiple parameters of clinical phototherapy.