Mark R. Stringer

Fluorescence Photobleaching of ALA‐induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect

The photobleaching of 5‐aminolaevulinic acid (ALA)‐induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HRt hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT‐induced dam‐age were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photo‐bleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photo‐bleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50–150 mW/cm2, 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT‐induced dam‐age. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photo‐bleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.

The place of bronchoscopic photodynamic therapy in advanced unresectable lung cancer: experience of 100 cases

OBJECTIVES The objectives of the study were: (1) to evaluate effectiveness of photodynamic therapy (PDT) for symptom palliation in patients with inoperable lung cancer; (2) to determine survival benefit in a subset of patients. METHODS One hundred patients, 68 male, 32 female, aged 44-81 years (mean 62.5) with advanced inoperable bronchogenic cancer and endobronchial luminal obstruction were prospectively studied. Eighty-two percent had previous chemo/radiotherapy. The pre-treatment protocol consisted of: clinical, radiological and bronchoscopic examination, pulmonary function testing, assessment of WHO performance status and clinical staging. Treatment protocol was: intravenous injection of 2 mg/kg body weight of photofrin/polyhaematoporphyrin and interstitial illumination using 630 nm laser light 24-72 h later. Follow-up was at 6-8 weeks for 1 year. Then every 3-6 months if applicable. Repeat PDT as necessary. RESULTS All patients were stage IIIa-IV. The histology of the tumour was: non small cell in 90 and small cell in 10. There was no treatment related mortality. Mean endoluminal obstruction fell from 85.8% to 17.5%, mean forced vital capacity (FVC) and forced expiratory volume in 1s (FEVI) improvement was 430 ml and 280 ml, respectively. Ninety patients died from 6 weeks to 37 months, mean and median survival: 9 months and 5 months, respectively. Ten patients are alive from 13 to 72 months, mean 36 months, median 29 months. Overall 2-year survival was 19%. Multivariant analysis indicated that age, sex, histology and stage of disease did not influence survival significantly but performance status did. Patients with WHO < 2 had mean and median survival of 17.8 and 14 months versus WHO > 2, 6.9 mean and 4 months median survival (log-rank P < 0.0001). CONCLUSIONS (1) PDT is effective in palliation of inoperable advanced lung cancer. (2) Subset of patients with a better performance status have added survival benefit.

Improved response of plaque psoriasis after multiple treatments with topical 5-aminolaevulinic acid photodynamic therapy.

We investigated the clinical response of 10 patients with plaque psoriasis to multiple treatments with photodynamic therapy, using topical application of 5-aminolaevulinic acid followed by exposure to broad-band visible radiation. Treatment was performed up to 3 times per week, with a maximum of 12 treatments, using a light dose of 8 Jcm(-2) delivered at a dose-rate of 15 mW cm(-2). Eight patients showed a clinical response. Out of 19 treated sites, 4 cleared, 10 responded but did not clear and 5 showed no improvement. Of the 4 sites that cleared only 1 did so fully, after 7 treatments, 45 days after the start of therapy. Of the 10 sites that responded partially, the greatest reduction in scale, erythema and induration index occurred after a minimum of 3 and a maximum of 8 treatments. The intensity of 5-aminolaevulinic acid-induced protoporphyrin IX fluorescence, recorded prior to the first treatment, varied between sites on the same patient as well as between patients. There was also a variation in fluorescence intensity recorded from the same site immediately prior to subsequent treatments, although the pretreatment levels generally decreased as the study progressed and then increased as psoriasis relapsed. Biopsies confirmed that fluorescence was localized throughout the epidermis and stratum corneum, but the level was not consistent between sections taken within the same biopsy. We also observed fluorescence at sites distant from the ones that received 5-aminolaevulinic acid, which was not present prior to the start of the treatment programme, but found no evidence of elevated levels of plasma porphyrins. The level of discomfort associated with this therapy increased with increasing values of the calculated photodynamic dose, defined as the product of the initial photosensitizer concentration and the percentage reduction in fluorescence following irradiation. Therefore, although clinical efficacy improved with multiple treatments, unpredictable response and patient discomfort make ALA-PDT unsuitable for the treatment of psoriasis.

Protoporphyrin IX Fluorescence Photobleaching during ALA-Mediated Photodynamic Therapy of UVB-Induced Tumors in Hairless Mouse Skin

Abstract— Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB‐induced tumor tissue in the SKH‐HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT‐induced damage were examined. Results show that the rate of PpIX photobleaching (i.e. fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB‐induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB‐induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.

Large patches of Bowen's disease treated by topical aminolaevulinic acid photodynamic therapy

Large patches of Bowens disease (intraepidermal carcinoma in situ) can be difficult to treat by conventional methods. Photodynamic therapy (PDT) uses the combination of a photosensitizer, which preferentially accumulates in malignant cells, and photoactivation by visible light to kill the malignant cells, 5‐aminolaevulinic acid (ALA) PDT uses excess exogenous ALA, which produces, via the haem synthesis pathway, a build up of the photosensitizer protoporphyrin IX. We describe the use of topical ALA PDT to treat three patients with three especially large patches of Bowens disease. Following two treatments all three lesions achieved a complete clinical and histological response with a good cosmetic result. ALA PDT is a simple, effective and well tolerated treatment for large patches of Bowens disease.

Photodynamic therapy (PDT) in early central lung cancer: a treatment option for patients ineligible for surgical resection

Objectives: To review the Yorkshire Laser Centre experience with bronchoscopic photodynamic therapy (PDT) in early central lung cancer in subjects not eligible for surgery and to discuss diagnostic problems and the indications for PDT in such cases. Methods: Of 200 patients undergoing bronchoscopic PDT, 21 had early central lung cancer and were entered into a prospective study. Patients underwent standard investigations including white light bronchoscopy in all and autofluorescence bronchoscopy in 12 of the most recent cases. Indications for bronchoscopic PDT were recurrence/metachronous endobronchial lesions following previous treatment with curative intent in 10 patients (11 lesions), ineligibility for surgery because of poor cardiorespiratory function in 8 patients (9 lesions) and declined consent to operation in 3 patients. PDT consisted of intravenous administration of Photofrin 2 mg/kg followed by bronchoscopic illumination 24–48 h later. Results: 29 treatments were performed in 21 patients (23 lesions). There was no procedure-related or 30 day mortality. One patient developed mild skin photosensitivity. All patients expressed satisfaction with the treatment and had a complete response of variable duration. Six patients died at 3–103 months (mean 39.3), three of which were not as a result of cancer. Fifteen patients were alive at 12–82 months. Conclusion: Bronchoscopic PDT in early central lung cancer can achieve long disease-free survival and should be considered as a treatment option in those ineligible for resection. Autofluorescence bronchoscopy is a valuable complementary investigation for identification of synchronous lesions and accurate illumination in bronchoscopic PDT.

The role of photodynamic therapy (PDT) in inoperable oesophageal cancer.

OBJECTIVE To evaluate the role of PDT in palliation of patients with inoperable oesophageal cancer and to identify subgroups in which this role is of particular significance. METHODS Sixty-five patients (37 male, 28 female) aged 42-89 (mean 65.6) with advanced and inoperable oesophageal cancer were the subjects of this study. Inoperability was due to advanced stage of the disease in 61 and because of general condition in 4. Fifty-eight (89%) had previous treatments, other than PDT. All patients had dysphagia of whom 20 could not swallow fluid. Pre-PDT clinical, radiological and endoscopic examinations were carried out. Performance status (PS) and clinical staging was assessed. PDT protocol consisted of: intravenous injection of 2 mg/kg; photofrin (or equivalent polyhaematoporphyrin) followed 24-72 h later by endoscopic illumination using 630 nm laser light. MAIN OUTCOME MEASUREMENTS (1) Relief of dysphagia generally and specifically in those with cervical and post-cricoid carcinoma who were previously treated by external beam radiotherapy (EBR) (n=6) and those with previous intubation or stent (n=9); (2) Survival. RESULTS There was no PDT related mortality. Three patients (4.6%) developed a mild skin photosensitivity reaction. Dysphagia was relieved in all patients. The mean and median survival of the 58 patients who have died was 7. 7 and 6 months respectively. Seven patients are alive from 2-30 months (mean 16). Survival was not significantly influenced by tumour histology, location in the oesophagus, severity of dysphagia on admission, or by previous therapy. Survival was significantly influenced by Performance Status prior to treatment (P=0.03 log rank, for PS < or =2 vs. PS=3), an most significantly by the stage of the disease (P=0.0001 log rank, for Stage III vs. Stage IV). CONCLUSIONS (1) PDT is safe and effective for palliation of dysphagia in inoperable oesophageal cancer. This is particularly important in post-cricoid and cervical oesophageal cancer previously treated by other methods and for patients with recurrent malignant obstruction who previously had intubation or stent placement. (2) Survival is influenced by better PS (< or =2) and in those with disease Stage III rather than patients in Stage IV. This study has not been able to determine the influence of complete tumour staging on survival because, apart from four patients, all others were Stages III and IV cancer.

Photofrin PDT for early stage oesophageal cancer: Long term results in 40 patients and literature review

BACKGROUND Yorkshire Laser Centre experience of PDT in early oesophageal cancer (EOCa) to determine long survival at 3 and 5 years (absolute) and factors which might influence outcome. MATERIAL/METHOD The records of patients who had PDT (1997-2009) for oesophageal cancer were reviewed and those with EOCa were studied and analysed. All patients had standard work up and staging. PDT was carried out using Photofrin 2 mg/kw bw, iv followed 24-72 h later by endoscopic illumination with 630 nm laser light. Results were assessed based on pathological response to treatment and survival at 3 and 5 years post-PDT. RESULTS There were 40 patients with EOCa amongst 144 who had PDT for oesophageal cancer. 30 male and 10 female (mean age 77, range 48-84). 35 had adenocarcinoma and 5 squamous cell carcinoma. 20 of the former had Barretts mucosa. There was no operative or 30-day mortality and no serious complications. Adverse effects were noted in 10 patients including 2 with skin photosensitivity and 3 with mild stricture requiring one dilatation. The median follow up was 76.1 (range 36-150 months). In this period 24 patients have died between 2 and 150 months (median 41 months). 16 patients are alive in between 36 and 110 months. 3 and >or=5 years or more survival (absolute) were 72.5% and 53.8%, respectively. CONCLUSION Endoscopic PDT should be considered as the treatment of choice in patients with EOCa who are ineligible for surgical resection. We suggest that a carefully designed study of a cohort of patients with EOCa comparing surgical resection with endoscopic PDT is warranted.

The analysis of human cortical bone by terahertz time-domain spectroscopy.

Samples of cortical bone, derived from human femur, have been studied using terahertz time-domain transmission spectroscopy. The relationship between the broadband THz parameters and the previously acquired values of Youngs modulus and x-ray attenuation (CT number), and the density of each bone sample, is investigated. The only significant correlation is that between THz transmission and sample density, suggesting that the potential use of THz radiation as a non-invasive probe of bone quality is limited. The spectra of absorption coefficient and refractive index are plotted over the frequency range 0.1-1.25 THz. There is evidence that the sample hydration state is a factor in the resultant THz parameters.

Photodiagnosis and fluorescence imaging in clinical practice.

For cancer diagnosis clinicians rely upon histo pathological preparations in their broadest sense and the characteristic microscopic features which represent malignant changes. Standard method of in-vivo sampling (biopsy) uses white light indicating abnormal tissue. The manner in which light interacts with a specific tissue type is dictated by the wavelength dependent scattering and absorbtion properties. In the UV and visible part of the spectrum the tissue optical properties are dominated by the endogenous chromophores which is different for normal/abnormal tissue. It follows that abnormal tissue, absorbs light and fluoresces differently to normal tissue at specific light wavelengths. Autofluorescence takes advantage of this principle. Enhanced fluorescence employs exogenous markers to produce better definition. Fluorescence imaging has become an important diagnostic tool to highlight cancer at an early stage of development and/or to guide biopsy from representative samples.