David H. Crean

Fluorescence detection of tumors : early diagnosis of microscopic lesions in preclinical studies

Background. The growth of microscopic tumor lesions at or beyond treatment field margins poses a major problem in the diagnosis and treatment of cancer. Early detection techniques that clearly define the location or field spread of disease may improve the planning of disease treatment.

Evaluation of porfimer sodium fluorescence for measuring tissue transformation

Background. Neoplastic tissue can be detected by its increased fluorescence compared with surrounding normal tissue after the injection of the tumor‐localizing compound porfimer sodium (Photofrin; Quadra Logic Technologies, Vancouver, BC, Canada). In vivo fluorescence photometry is a nonimaging photodetector technique that detects specific 690 nm fluorescence of the porphyrin by subtracting nonspecific 612 nm excitation from 630 nm excitation. The technique was applied in the developmental stages of the 9,10 dimethyl‐1,2‐benzanthracene (DMBA)‐induced hamster buccal cheek pouch carcinoma model to (1) quantitate and characterize porfimer sodium fluorescence and uptake as it relates to lesion progression and biochemical changes and (2) determine whether porfimer sodium‐induced fluorescence will vary with promotional and inhibitory stimuli.

Localization and treatment of transformed tissues using the photodynamic sensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a

Photofrin is the photosensitizer currently used in most clinical trials examining the efficacy of photodynamic therapy (PDT) for the treatment and/or palliation of neoplasia. Although this drug has been shown to be efficacious in many of these trials, it possesses less than ideal qualities for use in a systematically administered photosensitizer. A new photosensitizer, 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a (HPPH), was developed for PDT. HPPH possesses more rapid clearance from skin and greater cytotoxicity per drug dose than Photofrin. The aims of this study were to: (1) examine the uptake and retention of HPPH in tissues undergoing malignant transformation using laser‐induced fluorescence, and (2) evaluate the efficacy of HPPH and 665 nm light in treating carcinogen‐induced tumors of the hamster buccal cheek pouch.

Fluorescence detection of tumors: studies on the early diagnosis of microscopic lesions in preclinical and clinical studies

The growth of microscopic tumor lesions at or beyond treatment field lesions poses major problems in the diagnosis and curative treatment of numerous cancers. Early detection techniques which clearly define the extent of condemned or field spread of disease may improve the primary treatment of the disease. In vivo fluorescence photometry is a non-imaging technique which digitally displays relative fluorescence values in volts. The sensitivity of the instrument has allowed the detection of micrometastases in both pre-clinical and clinical studies using drug doses that are 80-90 lower than those used therapeutically. This technique is now being applied in preliminary experiments to the hamster cheek pouch models to (1) discern varying grades of dysplasia; (2) levels of uptake of the drug in normal growing and quiescent tumors. Results will be shown in two models in which this technique has shown to be efficacious preclinically in the Pollard rat adenocarcinoma model in which micrometastases in the lymph node have been detected, and preliminary studies involving the hamster cheek pouch model in which the pouch is painted with 9, 10 dimethyl-1, 2-benzanthracene (DMBA) for initiation and promotion of tumors. Clinically results will be shown in which fluorescence detection, confirmed by biopsy and histopathological examination, was capable of detecting the existence of micrometastatic involvement of less than 100 cells.

Fluorescence detection of premalignant, malignant, and micrometastatic disease using hexylpyropheophorbide

We investigated the in vivo fluorescence detection of premalignant and malignant lesions in carcinogen-induced tumors of the hamster buccal cheek pouch and micrometastases in rat lymph nodes using hexylpyropheophorbide-a (HPPH) at 0.5 mg/kg and He-Ne laser-induced fluorescence photometry. Time course studies of HPPH in hamster tumor model showed maximal relative fluorescence readings at 48 hours after i.p. injection in each stage (dysplasia: 0.35, papilloma: 0.58, squamous cell carcinoma: 1.04). Squamous cell carcinoma showed significantly greater fluorescence than papillomas and dysplasias at all time points (p < 0.01). Metastatic lymph nodes were significantly greater than normal lymph nodes at all time points (p < 0.01). Maximal fluorescence levels of normal lymph nodes were observed at 6 hours after i.v. injection. Metastatic lymph nodes still showed high fluorescence levels at 72 hours. Micrometastases showed fluorescence levels between the levels of metastatic and normal lymph nodes (normal: 0.40 < micrometastasis: 0.62 < metastasis: 0.75 at 48 hours after i.v. injection). The results demonstrated good correlation between fluorescence levels and histopathological developments at all time points. Therefore, HPPH may be a promising fluorophore for the detection of developing malignancies and occult disease.

Alterations in receptor-mediated kinases and phosphatases during carcinogenesis

Increased phosphorylation in cancers can stimulate growth and up-regulate certain receptors. To test whether the functional response of phosphatase receptors is up-regulated during carcinogenesis, we examined the effects of ligands on net phosphorylation in isolated membranes derived from hamster cheekpouch tissues undergoing malignant transformation. The buccal mucosa of groups of Syrian golden hamsters was exposed thrice weekly to 0.5% dimethylbenzanthracene (DMBA) in acetone for 2–12 weeks to produce premalignant and malignant tissues. Homogenates of these tissues were then incubated with [32P]ATP in the presence of epidermal growth factor (EGF), agonist of somatostatin analogue RC-160, luteinizing-hormone-releasing hormone (LH-RH) [d-Trp6]LH-RH, or combinations of EGF, RC-160, and [d-Trp6]LH-RH. Changes compared to controls in phosphorylation in response to ligands provided estimates of kinase or phosphatase activity. Phosphorylation increased continuously, from the first application of DMBA in a linear fashion, and independently of EGF stimulation. RC-160 and [d-Trp6]LH-RH reduced phosphorylation in vitro. This response occurred in premalignant (weeks 6–10 after DMBA application) as well as malignant tissues (week 12 after DMBA application), but was not significant in normal tissues. The results show a continuous augmentation in phosphatase activity prior to the appearance of cancers, but with a delay in expression following the primary event of increased kinase activity. Significantly less phosphorylation of substrates was induced by both RC-160 and [d-Trp6]LH-RH after in vitro activation by EGF than in the absence of EGF. This suggests that EGF activates latent systems of hormonal receptors. Collectively, these results support the hypothesis that the enhancement of the hormonally stimulated phosphatase in cancers occurs secondarily to the increased kinase activity.

Photodynamic therapy and hyperthermia as an adjuvant modality in preventing tumor recurrence

The objective of this study was to determine the relative efficacy in preventing tumor recurrence by photodynamic therapy (PDT), and by ablative CO2 laser surgery followed by PDT, compared to ablative surgery alone (negative control) or ablative surgery followed by a course of hyperthermia (positive control).

Studies on the absence of photodynamic mechanism in the normal pancreas

Extraction procedures to quantitate porfimer sodium concentration in tissues were correlated with fluorescence measurements made in vivo, on hamster and rat normal pancreas and intra-pancreatic tumors. The uptake of photosensitizer has been shown to be high in both normal and malignant pancreatic tissues, in both animal models studied. Photobleaching of the drug, as evidenced by both techniques within the pancreatic tumor, occurs in a typical manner during PDT, with resultant tissue destruction. In contrast, when the normal pancreas is exposed to PDT, a negligible photobleaching effect, as well as a lack of tissue response, is observed. The lack of observable response is corroborated by a lack of measurable physiological response. Both serum amylase and serum glucose show acute changes up to 12 hours post treatment but quickly return to normal. HPLC analysis shows that the drug extracted from both the normal pancreas and intrapancreatic tumor is essentially the same as that extracted from other tissues and similar to that which has been injected into the animal. Fluorescence microscopy has shown that at time points between 12-120 hours the drug is associated with lymphatic channels. This would not, however, necessarily preclude normal tissue destruction. Similar results have been found with other photosensitizers. Understanding the lack of response in the pancreas may lead to a deeper understanding of the diseased state which is normally refractory to all therapy as well as understanding the fundamental concepts of the mechanisms of PDT.

Point fluorescence measurements of transformed tissues using 2-[1-hexyloxyethel]-2-devinyl pyropheophorbide-a

Recently, a new photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-1 (HPPH) was developed for PDT which possesses more rapid clearance from skin and greater cytotoxicity per drug dose than Photofrin. The spectral characteristics of HPPH shows an absorption band at 665 nm (50,000 M-1cm(superscript -1 and peak emission at 680 nm. The aid of this study was to examine HPPH as a fluorescent diagnostic compound for the detection of transformed tissues using the in vivo fluorescence photodetector. The model of tissue transformation was the carcinogen (DMBA [9, 10-dimethyl-1, 2- benzanthracene])-induced premalignant and malignant lesions of the hamster buccal cheek pouch. The results demonstrated significant correlations between fluorescence levels and histological developments at all time points after injection. Time course studies of HPPH showed highest fluorescence readings at 48 hours after injection of 0.5 mg/kg HPPH (mild-moderate dysplasia, 0.35 +/- 0.17 volts; papillary disease with severe dysplasia, 0.58 +/- 0.33 volts; and squamous cell carcinoma, 1.04

Fluorescence photometric analysis of Photofrin uptake and the detection of precancerous and true epidermoid lesions

OM 0.32 volts). Therefore, it appears that HPPH may be a promising fluorophore for the detection of transformed tissues.