title: Using Fourier transform infrared spectroscopy to evaluate biological effects induced by photodynamic therapy.
abstract: Vibrational spectroscopic methods associated with multivariate statistical techniques have been succeeded in discriminating skin lesions from normal tissues. However, there is no study exploring the potential of these techniques to assess the alterations promoted by photodynamic effect in tissue. The present study aims to demonstrate the ability of Fourier Transform Infrared (FTIR) spectroscopy on Attenuated total reflection (ATR) sampling mode associated with principal component-linear discriminant analysis (PC-LDA) to evaluate the biochemical changes caused by photodynamic therapy (PDT) in skin neoplastic tissue.
  
  
  
  Cutaneous neoplastic lesions, precursors of squamous cell carcinoma (SCC), were chemically induced in Swiss mice and submitted to a single session of 5-aminolevulinic acid (ALA)-mediated PDT. Tissue sections with 5 μm thickness were obtained from formalin-fixed paraffin-embedded (FFPE) and processed prior to the histopathological analysis and spectroscopic measurements. Spectra were collected in mid-infrared region using a FTIR spectrometer on ATR sampling mode. Principal Component-Linear Discriminant Analysis (PC-LDA) was applied on preprocessed second derivatives spectra. Biochemical changes were assessed using PCA-loadings and accuracy of classification was obtained from PC-LDA .
  
  
  
  Sub-bands of Amide I (1,624 and 1,650 cm(-1) ) and Amide II (1,517 cm(-1) ) indicated a protein overexpression in non-treated and post-PDT neoplastic tissue compared with healthy skin, as well as a decrease in collagen fibers (1,204, 1,236, 1,282, and 1,338 cm(-1) ) and glycogen (1,028, 1,082, and 1,151 cm(-1) ) content. Photosensitized neoplastic tissue revealed shifted peak position and decreased β-sheet secondary structure of proteins (1,624 cm(-1) ) amount in comparison to non-treated neoplastic lesions. PC-LDA score plots discriminated non-treated neoplastic skin spectra from post-PDT cutaneous lesions with accuracy of 92.8%, whereas non-treated neoplastic skin was discriminated from healthy tissue with 93.5% accuracy and post-PDT cutaneous lesions was discriminated from healthy tissue with 89.7% accuracy.
  
  
  
  PC-LDA was able to discriminate ATR-FTIR spectra of non-treated and post-PDT neoplastic lesions, as well as from healthy skin. Thus, the method can be used for early diagnosis of premalignant skin lesions, as well as to evaluate the response to photodynamic treatment. Lasers Surg. Med. 48:538-545, 2016. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, Universidade de Sao Paulo, Sao Paulo SP, 05508 000, Brazil.
authors.forename: Cassio A
authors.initials: CA
authors.lastname: Lima
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, Universidade de Sao Paulo, Sao Paulo SP, 05508 000, Brazil.
authors.forename: Viviane P
authors.initials: VP
authors.lastname: Goulart
authors.affiliation: Faculdade de Odontologia, Universidade de São Paulo, São Paulo SP, 05508 000, Brazil.
authors.forename: Luciana
authors.initials: L
authors.lastname: Correa
authors.affiliation: Instituto de Pesquisas Energéticas e Nucleares, IPEN - CNEN/SP, Universidade de Sao Paulo, Sao Paulo SP, 05508 000, Brazil.
authors.forename: Denise M
authors.initials: DM
authors.lastname: Zezell
mesh_terms: D000622:Aminolevulinic Acid / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D016002:Discriminant Analysis; D005260:Female; D051379:Mice; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology* / Q000627:therapeutic use; D011230:Precancerous Conditions / Q000175:diagnosis / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D025341:Principal Component Analysis; D012867:Skin / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D012878:Skin Neoplasms / Q000175:diagnosis / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D014408:Biomarkers, Tumor; D017319:Photosensitizing Agents; D000622:Aminolevulinic Acid
keywords: 5-aminolevulinic acid (ALA); Fourier transform infrared spectroscopy; attenuated total reflectance (ATR); biochemical characterization; cutaneous squamous cell carcinoma; neoplastic lesion; principal component analysis