Correlation between Autofluorescence Intensity and Histopathological Features in Non-Melanoma Skin Cancer: An Ex Vivo Study

Abstract

Non-melanoma skin cancer (NMSC) is the most common malignant tumor affecting fair-skinned people. Increasing incidence rates of NMSC have been reported worldwide, which is an important challenge in terms of public health management. Surgical excision with pre-operatively identified margins is one of the most common and effective treatment strategies. Incomplete tumor removal is associated with a very high risk of recurrence and re-excision. Biological tissues can absorb and re-emit specific light wave-lengths, detectable through spectrophotometric devices. Such a phenomenon is known as autofluorescence (AF). AF spectroscopy has been widely explored for non-invasive, early detection of NMSC as well as for evaluation of surgical margins before excision. Fluorescence-aided diagnosis is based on differences in spectral characteristics between healthy and neoplastic skin. Understanding the biological basis of such differences and correlating AF intensity to histological features could improve the diagnostic accuracy of skin fluorescence spectroscopy. The primary objective of the present pre-clinical ex vivo study is to investigate the correlation between the intensity of cutaneous AF and the histopathological features of NMSC. Ninety-eight lesions suggestive for NMSCs were radically excised from 75 patients (46 M; 29 F; mean age: 79 years). After removal, 115 specific reference points on lesions (“cases”; 59 on BBC, 53 on SCC and 3 on other lesions) and on peri-lesional healthy skin (controls; 115 healthy skin) were identified and marked through suture stitches. Such reference points were irradiated at 400–430 nm wavelength, and resulting emission AF spectra were acquired through spectrophotometry. For each case, AFIR (autofluorescence intensity ratio) was measured as the ratio between the number of photons emitted at a wavelength ranging between 450 and 700 nm (peak: 500 nm) in the healthy skin and that was captured in the pathological tissue. At the histological level, hyperkeratosis, neoangiogenesis, cellular atypia, epithelial thickening, fibrosis and elastosis were quantified by light microscopy and were assessed through a previously validated grading system. Statistical correlation between histologic variables and AFIR was calculated through linear regression. Spectrometric evaluation was performed on 230 (115 cases + 115 controls) reference points. The mean AFIR for BCC group was 4.5, while the mean AFIR for SCC group was 4.4 and the fluorescence peaks at 500 nm were approximately 4 times lower (hypo-fluorescent) in BCCs and in SCCs than in healthy skin. Histological variables significantly associated with alteration of AFIR were fibrosis and elastosis (p < 0.05), neoangiogenesis, hyperkeratosis and epithelial thickening. Cellular atypia was not significantly associated with alteration of AFIR. The intensity of fluorescence emission in neoplastic tissues was approximately 4 times lower than that in healthy tissues. Histopathological features such as hyperkeratosis, neoangiogenesis, fibrosis and elastosis are statistically associated with the decrease in AFIR. We hypothesize that such tissue alterations are among the possible biophysical and biochemical bases of difference in emission AF between neoplastic and healthy tissue. The results of the present evaluation highlighted the possible usefulness of autofluorescence as diagnostic, non-invasive and real-time tool for NMSCs.

Comparative study of in‐vitro autofluorescence of normal versus non‐melanoma‐skin‐cancer cells at different excitation wavelengths

“…It is important to notice that all contributions are strongly depressed in the cancerous cells. This is in general agreement with observations also made in vivo [6,13,20,21]. FAD and NAD(P)H are metabolic coenzymes involved in the electron transport chain which are very sensitive to neoplastic alterations [22].…”

“…AF spectroscopy would provide a powerful and fast diagnostic tool to guide the correct early treatment of the affected areas of the skin [12]. It was recently demonstrated by some of us that AF spectroscopy can be used as an optical biopsy tool, for the early detection of NMSC ex vivo [13]. It was found that the overall intensity of the fluorescence emission in NMSC tissues reduced sizably compared to tissues in healthy conditions.…”

“…Our group already demonstrated the ex‐vivo hypofluorescent aspect of cutaneous neoplasms [13]. However, several well‐known dermal and epidermal chromophores could give reason of such findings (e.g., hemoglobin, collagen, keratins of the stratum corneum); therefore, whether cancerous cells specifically could be (or, at least, partially) responsible for NMSC hypofluorescence remained to be clarified [5, 17].…”

The Immunohistochemical Assessment of Neoangiogenesis Factors in Squamous Cell Carcinomas and Their Precursors in the Skin

“…Measurements have shown a decrease in autofluorescence (after irradiation with a wavelength of 400–430 nm) in cancerous lesions such as BCC and cSCC, especially in relation to histological alterations such as elastosis, fibrosis, severe hyperkeratosis, and angiogenesis. However, cell atypia did not play a significant role in the difference in autofluorescence emission [ 45 ].…”

Introduction to the Special Issue on “Keratinocyte Carcinomas: Biology and Evolving Non-Invasive Management Paradigms”

“…Giovannacci, I. et al [ 5 ] assessed whether specific histological features may account for changes in autofluorescence (AF). In this ex vivo study, AF spectroscopy followed by histopathology was performed on lesional and surrounding non-lesional tissue from 98 surgical excisions of suspected NMSC.…”