A Non-Interventional Study on Vismodegib for Basal Cell Carcinoma in Swedish Patients.

INTRODUCTION: Real-life data on vismodegib in advanced basal cell carcinoma (aBCC) are limited. Optimal treatment duration is left to the discretion of the physician. OBJECTIVES: To assess the effectiveness, safety and treatment pattern for vismodegib in aBCC in clinical practice. METHODS: In this multicenter, non-interventional, prospective study, 49 Swedish patients planned for vismodegib treatment were included. The treatment pattern observed was treatment until remission, allowing unlimited discontinuations/pauses. RESULTS: The majority of patients (93.8%), discontinued at least once during the study. Compared to earlier studies there was a decrease of more than 2 months with actual drug intake, reducing the patients burden and costs, at the same time as a high number of responses were seen (87.8%). Median progression-free-survival was 16.7 months, and 90% of the patients were alive at 13.3 months. Ten patients were re-challenged with vismodegib at recurrence or progression, resulting in five partial remissions and three complete remissions. CONCLUSIONS: Clinical response rates with vismodegib for aBCC were comparable to those of similar trials despite a shorter and more intermittent treatment duration. The majority of re-challenges lead to partial or complete remissions.

Pharmacokinetic and biodistribution study following systemic administration of Fospeg®--a Pegylated liposomal mTHPC formulation in a murine model.

Fospeg® is a newly developed photosensitizer formulation based on meso-tetra(hydroxyphenyl)chlorin (mTHPC), with hydrophilic liposomes to carry the hydrophobic photosensitizer to the target tissue. In this study the pharmacokinetics and biodistribution of Fospeg® were investigated by high performance liquid chromatography at various times (0.5-18 hours) following systemic i.v. administration. As a model an experimental HT29 colon tumor in NMRI nu/nu mice was employed. Our study indicates a higher plasma peak concentration, a longer circulation time and a better tumor-to-skin ratio than those of Foslip®, another liposomal mTHPC formulation. Data from ex vivo tissue fluorescence and reflectance imaging exhibit good correlation with chemical extraction. Our results have shown that optical imaging provides the potential for fluorophore quantification in biological tissues.

Design and validation of a fiber optic point probe instrument for therapy guidance and monitoring.

Optical techniques for tissue diagnostics currently are experiencing tremendous growth in biomedical applications, mainly due to their noninvasive, inexpensive, and real-time functionality. Here, we demonstrate a hand-held fiber optic probe instrument based on fluorescence/reflectance spectroscopy for precise tumor delineation. It is mainly aimed for brain tumor resection guidance with clinical adaptation to minimize the disruption of the standard surgical workflow and is meant as a complement to the state-of-the-art fluorescence surgical microscopy technique. Multiple light sources with fast pulse modulation and detection enable precise quantification of protoporphyrin IX (PpIX), tissue optical properties, and ambient light suppression. Laboratory measurements show the system is insensitive to strong ambient light. Validation measurements of tissue phantoms using nonlinear least squares support vector machines (LS-SVM) regression analysis demonstrate an error of <5% for PpIX concentration ranging from 400 to 1000 nM, even in the presence of large variations in phantom optical properties. The mean error is 3% for reduced scattering coefficient and 5% for blood concentration. Diagnostic precision of 100% was obtained by LS-SVM classification for in vivo skin tumors with topically applied 5-aminolevulinic acid during photodynamic therapy. The probe could easily be generalized to other tissue types and fluorophores for therapy guidance and monitoring.

In vivo measurements of diffuse reflectance and time-resolved autofluorescence emission spectra of basal cell carcinomas.

We present a clinical investigation of diffuse reflectance and time-resolved autofluorescence spectra of skin cancer with an emphasis on basal cell carcinoma. A total of 25 patients were measured using a compact steady-state diffuse reflectance/fluorescence spectrometer and a fibre-optic-coupled multispectral time-resolved spectrofluorometer. Measurements were performed in vivo prior to surgical excision of the investigated region. Singular value decomposition was used to reduce the dimensionality of steady state diffuse reflectance and fluorescence spectra. Linear discriminant analysis was then applied to the measurements of basal cell carcinomas (BCCs) and used to predict the tissue disease state with a leave-one-out methodology. This approach was able to correctly diagnose 87% of the BCCs. With 445 nm excitation a decrease in the spectrally averaged fluorescence lifetime was observed between normal tissue and BCC lesions with a mean value of 886 ps. Furthermore, the fluorescence lifetime for BCCs was lower than that of the surrounding healthy tissue in all cases and statistical analysis of the data revealed that this decrease was significant (p = 0.002).

Drug quantification in turbid media by fluorescence imaging combined with light-absorption correction using white Monte Carlo simulations.

Accurate quantification of photosensitizers is in many cases a critical issue in photodynamic therapy. As a noninvasive and sensitive tool, fluorescence imaging has attracted particular interest for quantification in pre-clinical research. However, due to the absorption of excitation and emission light by turbid media, such as biological tissue, the detected fluorescence signal does not have a simple and unique dependence on the fluorophore concentration for different tissues, but depends in a complex way on other parameters as well. For this reason, little has been done on drug quantification in vivo by the fluorescence imaging technique. In this paper we present a novel approach to compensate for the light absorption in homogeneous turbid media both for the excitation and emission light, utilizing time-resolved fluorescence white Monte Carlo simulations combined with the Beer-Lambert law. This method shows that the corrected fluorescence intensity is almost proportional to the absolute fluorophore concentration. The results on controllable tissue phantoms and murine tissues are presented and show good correlations between the evaluated fluorescence intensities after the light-absorption correction and absolute fluorophore concentrations. These results suggest that the technique potentially provides the means to quantify the fluorophore concentration from fluorescence images.

Photodynamic therapy: superficial and interstitial illumination.

Photodynamic therapy (PDT) is reviewed using the treatment of skin tumors as an example of superficial lesions and prostate cancer as an example of deep-lying lesions requiring interstitial intervention. These two applications are among the most commonly studied in oncological PDT, and illustrate well the different challenges facing the two modalities of PDT-superficial and interstitial. They thus serve as good examples to illustrate the entire field of PDT in oncology. PDT is discussed based on the Lund University group's over 20 yr of experience in the field. In particular, the interplay between optical diagnostics and dosimetry and the delivery of the therapeutic light dose are highlighted. An interactive multiple-fiber interstitial procedure to deliver the required therapeutic dose based on the assessment of light fluence rate and sensitizer concentration and oxygen level throughout the tumor is presented.

Multimodal imaging to study the morphochemistry of basal cell carcinoma.

Basal cell carcinoma is the most abundant malignant neoplasm in humans, the pathology of which is characterized by an abnormal proliferation of basal cells. Basal cell carcinoma can show a variety of different morphologies, which are based on different cellular biology. Furthermore, the carcinoma often grows invisibly to the eye imbedded in the surrounding skin. Therefore, in some cases its clinical detection is challenging. Thus, our work aims at establishing an unsupervised tissue classification method based on multimodal imaging and the application of chemometrics to discriminate basal cell carcinoma from non-diseased tissue. A case study applying multimodal imaging to ex-vivo sections of basal cell carcinoma is presented. In doing so, we apply a combination of various linear and non-linear imaging modalities, i.e. fluorescence, Raman and second-harmonic generation microscopy, to study the morphochemistry of basal cell carcinoma. The joint information content obtained by such multimodal approach in studying various aspects of the malignant tissue alterations associated with basal cell carcinoma is discussed.

Tumor selectivity at short times following systemic administration of a liposomal temoporfin formulation in a murine tumor model.

Meso-tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is one of the most potent photodynamically active substances in clinical use. Treatment protocols for Temoporfin-mediated photodynamic therapy often rely on drug-light intervals of several days in order for the photosensitizer to accumulate within the target tissue, though tumor selectivity is limited. Here, the mTHPC localization was studied at 2-8 h following systemic administration of a liposomal Temoporfin formulation (0.15 mg kg(-1) b.w.) in HT29 human colon adenocarcinoma in NMRI nu/nu mice. Photosensitizer distribution within tumor and internal organs was investigated by means of high performance liquid chromatography following chemical extraction, as well as in situ fluorescence imaging and point-monitoring fluorescence spectroscopy. For tumor tissue, the Temoporfin concentrations at 4 h (0.16+/-0.024 ng mg(-1)) and 8 h (0.18+/-0.064 ng mg(-1)) were significantly higher than at 2 h (0.08+/-0.026 ng mg(-1)). The average tumor-to-muscle and the tumor-to-skin selectivity were 6.6 and 2, respectively, and did not vary significantly with time after photosensitizer injection. In plasma, the Temoporfin concentration was low (0.07+/-0.07 ng mg(-1)) and showed no significant variation with time. Our results indicate a rapid biodistribution and clearance from the bloodstream. Within the same type of organ, data from both fluorescence methods generally exhibited a significant correlation with the extraction results.

Fluorescence monitoring of a topically applied liposomal Temoporfin formulation and photodynamic therapy of nonpigmented skin malignancies.

Meso-tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is a potent photodynamically active substance in clinical use today. Usually, the substance is given systemically and a known drawback with this administration route is a prolonged skin light sensitization. For the first time to our knowledge, a liposomal Temoporfin gel formulation for topical application was studied in connection with photodynamic therapy (PDT) of nonpigmented skin malignancies in humans. Intervals of 4 hr between drug administration and light irradiation were used. Sensitizer distribution within tumor and surrounding normal skin was investigated by means of point monitoring and imaging fluorescence spectroscopy before, during, and after PDT, showing high tumor selectivity. Furthermore, the bleaching of Temoporfin was studied during the PDT procedure by monitoring the fluorescence following excitation by using a therapeutic light. A 30-35% light-induced photometabolization was shown. No pain occurred during or after treatment. It was also observed that the treated area did not show any swollen tissue or reddening, as is often seen in PDT using topical delta-aminolevulinic acid. On controlling the patients one week after treatment, healing progress was observed in several patients and no complications were registered.

Fluorescence and absorption assessment of a lipid mTHPC formulation following topical application in a non-melanotic skin tumor model.

Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [+/- standard deviation (SD)] mTHPC concentration within lesions of 6.0(+/-3.1) ngmg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.

In vivo measurement of parameters of dosimetric importance during interstitial photodynamic therapy of thick skin tumors.

A system for interstitial photodynamic therapy is used in the treatment of thick skin tumors. The system allows simultaneous measurements of light fluence rate, sensitizer fluorescence, and tissue oxygen saturation by using the same fibers as for therapeutic light delivery. Results from ten tumor treatments using delta-aminolevulinic acid (ALA)-induced protoporphyrin IX show a significant, treatment-induced increase in tissue absorption at the therapeutic wavelength, and rapid sensitizer photobleaching. The changes in oxy- and deoxyhemoglobin content are monitored by means of near-infrared spectroscopy, revealing a varying tissue oxygenation and significant changes in blood volume during treatment. These changes are consistent with the temporal profiles of the light fluence rate at the therapeutic wavelength actually measured. We therefore propose the observed absorption increase to be due to treatment-induced deoxygenation in combination with changes in blood concentration within the treated volume. A higher rate of initial photobleaching is found to correlate with a less pronounced increase in tissue absorption. Based on the measured signals, we propose how real-time treatment supervision and feedback can be implemented. Simultaneous study of the fluence rate, sensitizer fluorescence, and local tissue oxygen saturation level may contribute to the understanding of the threshold dose for photodynamic therapy.

Methods for detailed histopathological investigation and localization of biopsies from cervix uteri to improve the interpretation of autofluorescence data.

Fluorescence spectroscopy is one of many optical methods that are potentially clinically useful for noninvasive detection and characterization of disorders on the cervical part of uterus, including precancerous lesions. The cervix uteri exhibits a biologically complex tissue and the morphology of a biopsy is generally not homogenous. The standard histopathological protocol accounts only for the most severe condition found within the biopsy and no information is given on other constituents potentially influencing the recorded fluorescence spectra. Spectra are usually correlated, using multivariate techniques, to the histopathological diagnosis of the biopsies. Since the probe volume of fluorescence spectroscopy is considerably smaller than the extension of the biopsy, this can cause problems in the search for correlation between the fluorescence signals and the pathological structures. In addition, the orientation and location of the biopsies are normally not recorded. We now report on the first detailed histopathological protocol where numerous tissue parameters, such as thickness and type of the epithelium and the number of blood vessels, glands, and inflammatory cells, are tabulated and the orientation and location of the biopsy are recorded as precisely as possible. Hopefully, the use of this protocol together with sophisticated mathematical methods will increase the probability to classify cervical disorders of the uterus, including precancerous lesions, with high sensitivity and specificity.

Photodynamic therapy of nodular basal cell carcinoma with multifiber contact light delivery.

To overcome the limited treatment depth of superficial photodynamic therapy we investigate interstitial light delivery. In the present work the treatment light was delivered using a system in which three or six clear-cut fibers were placed in direct contact with the tumor area. This placement was thought to represent a step toward general purpose interstitial PDT. Twelve nodular basal cell carcinomas were treated employing delta-aminolevulinic acid and 635 nm laser irradiation. Fluorescence measurements were performed monitoring the buildup and subsequent bleaching of the produced sensitizer protoporphyrin IX. The treatment efficacy, judged at a 28-month follow-up, showed a 100% complete response. Two punch excisions at 7 months converted two partial responses to complete responses. One patient failed to appear at all follow-up sessions. The outcome of the treatments was comparable to superficial photodynamic therapy in terms of histological, clinical, and cosmetic results.

Clinical system for interstitial photodynamic therapy with combined on-line dosimetry measurements.

A system for interstitial photodynamic therapy with delta-aminolaevulinic acid and multiple optical fibers has been developed. The system enables photodynamic treatment of large embedded tumor volumes and utilizes real-time measurements to allow on-line dosimetry. Important parameters such as light fluence rate, sensitizer fluorescence intensity, and changes in local blood oxygen saturation are measured with the same fibers that deliver the therapeutic light. Data from the first clinical treatments on nodular basal cell carcinomas indicate a major treatment-induced light absorption increase, rapid sensitizer photobleaching, and a relatively constant global tissue oxygen saturation level during the treatment.

Effects of Kaposi's sarcoma-associated herpesvirus ORF K1 on differentiating endothelium in murine embryoid bodies and teratomas.

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is a prerequisite for Kaposi's sarcoma, a multicentric endothelial tumor of mixed blood vessel-lymphatic phenotype with a marked production of endothelial basement membrane proteins. The viral K1 gene encodes a unique transmembrane protein capable of transforming T lymphocytes experimentally. We studied the effects of K1 regulated by the endothelium-specific tie-1 promoter in the embryonic stem cell (ES)-derived endothelium of subcutaneous murine teratomas and in embryoid body cultures with and without supplemental basic fibroblast growth factor and vascular endothelial growth factor. K1 embryoid bodies showed no difference in endothelial BM protein expression, formation of basal lamina by electron microscopy or fractional area of endothelial growth in comparison with vector controls, but maturation into branching pseudovascular tubes was mildly impaired. Moreover, the marked increase in endothelial fraction induced by both growth factors in the early growth period was absent in K1 clones. Teratoma endothelium, which is partly derived from ES, showed increased proliferation and apoptosis in K1 tumors, coinciding with a possible increased tendency for microhemorrhages. K1 can thus modulate endothelial cell turnover and growth factor response, but does not alter the extracellular matrix of differentiating endothelial cells.

Human epidermal energy metabolism is functionally anaerobic.

We have reported that epidermal Langerhans cells possess an H(+)-extruding mechanism signalling their existence in an anaerobic environment. This study highlights the energy metabolism of human epidermis. In their habitual state the keratinocytes contain more lactate than do most other cell types. Their lactate production in vitro is vigorous and independent of oxygen and most of it is released to the medium. Autoincubation of the epidermis under starved conditions resulted in a 30% increase of lactate, indicating ongoing glycogenolysis. Iodoacetate inhibited lactate production by > 90%. Energy charge values were low, approximately 0.82, and comparable with those previously reported for smooth muscle. Moreover, the overwhelming majority of the keratinocytic mitochondria had an appearance markedly deviating from those in the Langerhans cells, melanocytes and fibroblasts, and, above all, were characterized by an enormous reduction of the inner membrane. This structure is in all probability incompatible with an effective oxidative metabolism of glucose. We conclude that epidermal energy metabolism is predominantly anaerobic in spite of the formal presence of mitochondria. The high production of lactate obviously demands extracellular transport pathways for rapid elimination of this organic acid. An extracellular space complying with such a demand emerges on electron microscopy when an isotonic glutaraldehyde-based fixative is used. The prevailing view regarding the size of the extracellular space is based on the common use of hypotonic fixatives, such as Karnovski's fixative, which causes gross cellular swelling and concomitant near total elimination of the extracellular space, leaving interstices with a diameter significantly smaller than that allowing fluid flow.

New mechanism for amino acid influx into human epidermal Langerhans cells: L-dopa/proton counter-transport system.

We have characterized a stereospecific transport mechanism for L-dopa into human epidermal Langerhans cells (LCs). It is different from any other amino acid transport system. It is highly concentrative, largely pH-independent, and independent of exogenous Na+, glucose and oxygen, and fuelled by a renewable intracellular energy source inhibited by iodoacetate but not by arsenate. We propose that the mechanism is a unidirectional L-dopa/proton counter-transport system. We have recently demonstrated anaerobic glycolysis in human epidermis, which substantiates the need of proton pumps for resident LCs. The findings prompt a re-evaluation of the profound changes LCs undergo when exposed to oxygen in aerobic culture. L-dopa is not metabolized by LCs but can rapidly be dislocated to the intercellular space by certain extracellular amino acids, i.e. LCs can profit by L-dopa in a dualistic way, altogether a remarkable biological phenomenon.

Characterization of a new amino acid transport system in human epidermal Langerhans cells: the L-dopa influx/efflux cycle.

Various aspects concerning the L-dopa uptake mechanism into human epidermal Langerhans cells (LC) and the energy metabolism of epidermis have been examined. The study is based on the discovery of a new amino acid transport system in human epidermal LC. The amino acid transport system has been characterised. This system is stereospecific and is unique for L-dopa and LC as it does not share any characteristics with other described transport systems of other amino acids in other cell types. The uptake assay is based on the Falck/Hillarp histofluorescence method, which enables studying uptake in individual LC in situ in their microenvironment. For the first time, it is shown that the human epidermis is capable of an aerobic lactate production, which is a strange phenomenon, as this means that epidermal cells, despite the formal presence of mitochondria, nevertheless produce lactate in excess. This probably depends on a disturbed Pasteur effect. The same phenomenon is found under normal conditions in smooth muscle cells, but has not been described in other human cells. Also, for the first time, it is shown that the human epidermis has a lower energy charge value than other body cells except for, again, smooth muscle cells. The increased lactate production in epidermis, which can be inhibited by iodoacetate, implies an increased intracellular proton production. The energetics of the L-dopa transport into the LC is probably a counter-transport (antiport) of protons due to the aerobic production of lactate. It is proved that L-dopa can be dislocated out of the LC by certain amino acids on the outside of the cell, some acting in a trans-stimulatory fashion and others probably by exchanging with intracellular L-dopa, hereby getting "useful" amino acids into the cell instead. This, now extracellular L-dopa, can then begin a new in- and out-transport cycle. For further characterisation of the L-dopa transport system, experiments have been carried out with an H+,K+-ATPase-inhibitor (omeprazole and a number of its analogues). Most of these are inhibitory of the L-dopa transport into LC. This is most probably not caused by an effect on a thiol group essential for this transport, but the actual mechanism is not known.