Biophysics, Invited Lecture


E. Borisova(1), E. Pavlova(2), P. Troyanova(2), B. Nikolova(3), I. Tsoneva(3)

1) Institute of Electronics, Bulgarian Academy of Sciences, 72, Tsarigradsko chaussee Blvd., 1784 Sofia, Bulgaria
2) University hospital “Queen Giovanna-ISUL”, 8 Bialo more str., 1527 Sofia, Bulgaria
3) Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria, Acad. G. Bonchev str., Bl. 21, 1110 Sofia, Bulgaria


Optical spectral modalities applied in recent clinical trials for early diagnosis of skin pathologies allow broadening of possibilities for development of cutaneous malignancies precise non-invasive detection technique. Light-induced autofluorescence spectroscopy (LIAFS) is a very attractive tool for early diagnosis of cancer due to its high sensitivity, easy-to-use methodology for measurements, lack of need for contrast agents’ application on the tissue under investigation, possibilities for real time measurements and noninvasive tumor detection [1, 2]. It allows differentiation on the base of differences in biochemical content and metabolic state of the pathology. However, when the lesion is highly pigmented the obtained fluorescence signal is too weak to be used for diagnostics [3]. Diffuse reflectance spectroscopy is applied for the melanin-pigmented cutaneous pathologies, including malignant melanoma, as well combination of two spectral techniques allow increasing of the diagnostic accuracy in general for all pathologies investigated.
Long years of investigations and significant improvement of the spectral detection technologies still do not introduce on the market easy-to-use system for cutaneous autofluorescence cancer detection and differentiation. One could ask himself, why such system, which in principle does not seems to be very complicated technically, still does not exist. Of course, there are some attempts to introduce such diagnostic systems into standard clinical practice, such as fiber-based fluorimeter - SkinScan system (JobinYvon, France), where fluorescence of endogenous amino-acids is used for cutaneous lesions’ investigations [4], or more recently developed DYADERM system (Biocam GmbH, Germany), which is applied for photodynamic diagnosis with exogenous photosensitizers [5]. However, up to our days there is no such universal clinical apparatus, based on autofluorescence detection of skin surface, which could be used as a general tool for early cancer detection and differentiation. The reasons for such instrument absence in the field of clinical equipment based on the autofluorescence detection of skin cancer are very complex.
Our investigation is a part of a clinical trial for introduction of optical biopsy spectral diagnostic system for skin cancer detection. We apply autofluorescence and diffuse reflectance spectroscopy to several different classes of malignant non-melanoma cutaneous lesions. Initially, patients were classified visually and dermatoscopically using ABCD criteria by experienced dermatologist. Second step was detection of their lesion’ and surrounding normal skin autofluorescence using different excitation wavelengths, namely 365, 385, and 405 nm. Reflectance spectroscopy is applied in broad spectral region – from 400 to 900 nm. In the end for every lesion histological examination is used as a “gold standard” for all our investigations.
Based on the results obtained during initial diagnosis we start additional task – to monitor the lesion treatment process, where the non-invasive character of the optical biopsy procedure is acclaimed by the patients. Using optical biopsy monitoring of tumors’ treatment we could make initial diagnosis, to detect early changes, to evaluate the therapeutic effectiveness and to propose changes in the treatment plan, if needed.
Autofluorescence detection is applied for monitoring of electrochemotherapy of tumours as well. The therapeutic procedure itself - electrochemotherapy (ECT) combines chemotherapy and electroporation to increase locally cytostatic drug delivery in the cancer cells. The electroporator is battery supplied, associated with isolated ECG signals amplifier, QRS detection and synchronization circuits. The injection of local anaesthetic (1% lidocain) and cytostatic drug (bleomycin) in very small concentration direct to the tumor lesion, which is followed by application of electrical pulses. Drug delivery conditions (electric field intensity) and dose of cytostatic drug are personal for every single case. To monitor the effects of application of the electrochemotherapy fluorescence spectra are taken from the lesion and surrounding healthy skin, prior to, immediately after treatment and at the control check-ups. Patients are followed up at the first week after treatment, the first month and third month. On the fig.4. are presented results from such therapeutic monitoring, using 365 nm excitation of BCC lesion and normal skin. BCC tumour has lower intensity than normal tissue. It is clearly observed immediate reaction after therapeutic procedure application – appearance of specific minima at 543 and 575 nm, related to increased hemoglobin absorption. One week later the fluorescence intensity of the lesion area is higher and approach to the “normal skin” spectral shape, which is indication for successful treatment of the tumour.
The spectra and dermatoscopic evaluations were obtained from more than 400 patients up to now. Spectral properties of variety of benign cutaneous lesions are also evaluated for development of more precise discrimination algorithms for diagnosis of cancer lesions. Spectra from normal skin are used for comparison and evaluation of alterations occurred in lesions investigated. A clinical trial is currently under implementation and with broadening of the database with fluorescence spectra of major skin benign and malignant pathologies we expect to receive objective tool for cancer detection and treatment monitoring.
Acknowledgements: This work is supported by the National Science Fund of Bulgarian Ministry of Education, Youth and Science under grant #DMU-03-46/2011 “Development and introduction of optical biopsy for early diagnostics of malignant tumors”.
[1] S. Svanberg, “Environmental and medical applications of photonic interactions”, Physica Scripta Vol. T110, 39-51 (2004).
[2] J. Bigio, J.R. Mourant, “Ultraviolet and visible spectroscopy for tissue diagnostics: fluorescence spectroscopy and elastic-scattering spectroscopy”, Phys. Med. Biol. Vol. 42, 803-812 (1997).
[3] L. Bachmann, D.Zezell, A.da Costa Ribeiro, L.Gomes, A.Ito, “Fluorescence spectroscopy of biological tissues – a review”, Appl. Spectr. Rev. Vol. 41, 575-593 (2006)
[4] B. Valeur, “From well-known to underrated applications of fluorescence”, in “Fluorescence of supermolecules, polymers and nanosystems”, Springer Series on Fluorescence, Vol. 4, Part A, p. 21-43, (2008)
[5] de Leeuw J, van der Beek N, Neugebauer WD, Bjerring P, Neumann HA., “Fluorescence detection and diagnosis of non-melanoma skin cancer at an early stage”, Las Surg Med, Vol. 41(2), p. 96-103 (2009)

Representing author


Dr. Ekaterina Georgieva Borisova

Institute of Electronics, Bulgarian Academy of Sciences, Associate Professor
Sofia, Bulgaria

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