A pilot study for distinguishing basal cell carcinoma from normal human skin tissues using visible resonance Raman spectroscopy

Abstract

Aim: The aim of the study is to test visible resonance Raman (VRR) spectroscopy for rapid skin cancer diagnosis, and evaluate its effectiveness as a new optical biopsy method to distinguish basal cell carcinoma (BCC) from normal skin tissues.

Methods: The VRR spectroscopic technique was undertaken using 532 nm excitation. Normal and BCC human skin tissue samples were measured in seconds. The molecular fingerprints of various native biomolecules as biomarkers were analyzed. A principal component analysis - support vector machine (PCA-SVM) statistical analysis method based on the molecular fingerprints was developed for differentiating BCC from normal skin tissues.

Results: VRR provides a rapid method and enhanced Raman signals from biomolecules with resonant and near-resonant absorption bands as compared with using a near-infrared excitation light source. The VRR technique revealed chemical composition changes of native biomarkers such as tryptophan, carotenoids, lipids and proteins. The VRR spectra from BCC samples showed a strong enhancement in proteins including collagen type I combined with amide I and amino acids, and a decrease in carotenoids and lipids. The PCA-SVM statistical analysis based on the molecular fingerprints of the biomarkers yielded a 93.0% diagnostic sensitivity, 100% specificity, and 94.5% accuracy compared with histopathology reports.

Conclusion: VRR can enhance molecular vibrational modes of various native biomarkers to allow for very fast display of Raman modes in seconds. It may be used as a label-free molecular pathology method for diagnosis of skin cancer and other diseases and be used for combined treatment with Mohs surgery for BCC.

Keywords

Visible resonance Raman spectroscopy / human skin / basal cell carcinoma / principal component analysis / supports vector machine / molecular fingerprints / tryptophan / carotenoids

References

[1] Rogers HW,Harris AR,Feldman SR.Incidence estimate of nonmelanoma skin cancer in the united states..Arch Dermatol2010;146:283-7

[2] Brewster DH,Inglis JHC,Doherty VR.Recent trends in incidence of nonmelanoma skin cancers in the east of Scotland, 1992-2003..Brit J Dermatol2007;156:1295-300

[3] American Cancer Society. Cancer Facts and Figures 2017. Available from: https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2017.html. [Last accessed on 2 Jan 2019]

[4] The Skin Cancer Foundation. Available from: https://www.skincancer.org/skin-cancer-information/skin-cancer-facts. [Last accessed on 9 Jan 2019]

[5] Mogensen M.Diagnosis of nonmelanoma skin cancer/keratinocyte carcinoma: a review of diagnostic accuracy of nonmelanoma skin cancer diagnostic tests and technologies..Dermatol Surg2007;33:1158-74

[6] Raab SS.Quality in cancer diagnosis..CA Cancer J Clin2010;60:139-65

[7] Smith L.State of the art in non-invasive imaging of cutaneous melanoma..Skin Res Technol2011;17:257-69

[8] Kong K,Varma S,Leach IH.Diagnosis of tumors during tissue-conserving surgery with integrated autofluorescence and Raman scattering microscopy..Proc Natl Acad Sci U S A2013;110:15189-94 PMCID:PMC3780864

[9] Heuke S,Meyer T,Kluschke F.Detection and discrimination of non-melanoma skin cancer by multimodal imaging..Healthcare2013;1:64-83 PMCID:PMC4934506

[10] Alfano RR,Cordero J,Longo FW.Laser induced fluorescence spectroscopy from native cancerous and normal tissue..IEEE J Quantum Elect1984;20:1507-11

[11] Alfano RR,Pradhan A,Choy DSJ.Fluorescence spectra from cancerous and normal human breast and lung tissues..IEEE J Quantum Elect1987;QE-23:1806-11

[12] Liu CH,Sha WL,Akins DL.Human breast tissues studied by IR Fourier transform Raman spectroscopy..Lasers Life Sci1991;4:23-8

[13] Liu CH,Zhu HR,Deckelbaum LI.Near-IR Fourier transform Raman spectroscopy of normal and atherosclerotic human aorta..Laser in the Life Sciences1992;43:257-64

[14] Brancaleon L,Tu JH,Fallon JD.In vivo fluorescence spectroscopy of nonmelanoma skin cancer..Photochem Photobiol2001;73:178-83

[15] Panjehpour M,Phan MN,Overholt S.Laser-induced fluorescence spectroscopy for in vivo diagnosis of non-melanoma skin cancers..Lasers Surg Med2002;31:367-73

[16] Longo C,Ragazzi M,Gardini S.Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue..Br J Dermatol2014;171:561-70

[17] Rao BK,Wassef C.In vivo confocal microscopy in clinical practice: comparison of bedside diagnostic accuracy of a trained physician and distant diagnosis of an expert reader..J Am Acad Dermatol2013;69:e295-300

[18] Giambrone D,Masud A,Rao B.The diagnostic accuracy of in vivo confocal microscopy in clinical practice..J Am Acid Dermal2015;73:317-9

[19] Flores ES,Kose K,Rossi A.Intraoperative imaging during Mohs surgery with reflectance confocal microscopy: initial clinical experience..J Biomed Opt2015;20:61103 PMCID:PMC4405085

[20] Mosterd K,Nieman FH,Essers BA.Surgical excision versus Mohs’ micrographic surgery for primary and recurrent basal-cell carcinoma of the face: a prospective randomised controlled trial with 5-years’ follow-up..Lancet Oncol2008;9:1149-56

[21] Choe CS,Darvin ME.Analysis of human and porcine skin in vivo/ex vivo for penetration of selected oils by confocal Raman microscopy..Skin Pharmacol Physiol2015;28:318-30

[22] Kourkoumelis N,Moulia V,Gaitanis G.Advances in the in vivo Raman spectroscopy of malignant skintumors using portable instrumentation..Int J Mol Sci2015;16:14554-70 PMCID:PMC4519858

[23] Gniadecka M,NieIseW OF,Hercogova J.Distinctive molecular abnormalities in benign and malignant skin lesions: studies by Raman spectroscopy..Photochem Photobiol1997;66:418-23

[24] Notingher I,Romanska H,Polak JM.In situ characterization of living cells by Raman spectroscopy..Spectroscopy2002;16:43-51

[25] Sigurdsson S,Hansen LK,Gniadecka M.Detection of skin cancer by classification of Raman spectra..IEEE Trans Biomed Eng2004;51:1784-93

[26] Nijssen A,Heule F,Hayes DP.Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy..J Invest Dermatol2002;119:64-9

[27] Nunes LD,Silviera L.FT-Raman spectroscopy study for skin cancer diagnosis..Spectrosc Int J2003;17:602

[28] Choi J,Chung H,Park J.Direct observation of spectral differences between normal and basal cell carcinoma (BCC) tissues using confocal Raman microscopy..Biopolymers2005;77:264-72

[29] Nijssen A,Santos LF,Bakker Schut TC.Discriminating basal cell carcinoma from perilesinal skin using high wave-number Raman spectroscopy..J Biomed Opt2007;12:034004

[30] Egawa M.Comparison of the depth profiles of water and water-binding substances in the stratum corneum determined in vivo by Raman spectroscopy between the cheek and volar forearm skin: effects of age, seasonal changes and artificial forced hydration..Br J Dermatol2008;158:251-60

[31] Kumar S,Mukherjee R,Somasundaramc K.Raman and infrared microspectroscopy: towards quantitative evaluation for clinical research by ratiometric analysis..Chem Soc Rev2016;45:1879-900

[32] Opitz CA,Sahm F,Tritschler I.An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor..Nature2011;478:197-203

[33] Waller JM.Age and skin structure and function, a quantitative approach (I):blood flow, pH, thickness, and ultrasound echogenicity..Skin Res Technol2005;11:221-35

[34] Ly E,Durlach A,Manfait M.Polarized Raman microspectroscopy canreveal structural changes of peritumoral dermis in basal cell carcinoma..Applied Spectroscopy2008;62:1088-94

[35] Caspers PJ,Carter EA,Puppels GJ.In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles..J Invest Dermatol2001;116:434-42

[36] Caspers PJ,Wolthuis R,Puppels GJ.In vitro and in vivo Raman spectroscopy of human skin..Biospectroscopy1998;4:S31-S9

[37] Zhou Y,Sun Y,Boydston-White S.Human brain cancer studied by resonance Raman spectroscopy..J Biomed Opt2012;17:116021 PMCID:PMC3499405

[38] Liu CH,Sun Y,Zhou LX.Resonance Raman and Raman spectroscopy for breast cancer detection..Technol Cancer Res Treat2013;12:371-82

[39] Zhou Y,Li JY,Chen K.Tumor margin detection using optical biopsy techniques..SPIE Proc2014;8940:894014

[40] Zhou Y,Zhou LX,Liu YL.Resonant Raman spectra of grades of human brain glioma tumors reveal the content of tryptophan by the 1588 cm-1 mode..Proc SPIE2015;9318:931810

[41] Liu CH,Wang WB,Shi LY.Optical pathology study of human abdominal aorta tissues using confocal micro resonance Raman spectroscopy..Proc SPIE2016;9703:97031S

[42] Liu CH,Weisberg A,Sordillo LA.Vulnerable atherosclerotic plaque detection by resonance Raman spectroscopy..J Biomed Opt2016;21:127006 PMCID:PMC5174785

[43] Liu CH,Boydston-White S,Zhang C.Resonance Raman of BCC and normal skin..Proc SPIE2017;10060:100601B

[44] Massart DL,Deming SM,Kaufman L.Chemometrics: a Textbook.1988;AmsterdamElsevier

[45] Cortes C.Support-vector networks..Machine Learning1995;20:273-97

[46] Wu B,Mukherjee S.Quantitative diagnosis of bladder cancer by morphometric analysis of HE images..Proc SPIE2015;9303:930317

[47] Jain M,Wu B,Mukherjee S.Exploring multiphoton microscopy as a novel tool to differentiate chromophobe renal cell carcinoma from oncocytoma in fixed tissue sections..Arch Pathol Lab Med2017;

[48] Wu B,Jain M.A new method using multiphoton imaging and morphometric analysis for differentiating chromophobe renal cell carcinoma and oncocytoma kidney tumors..Proc SPIE2016;9712:97121O

[49] Mazet V,Brie D,Humbert B.Background removal from spectra by designing and minimising a non-quadratic cost function..Chemometr Intell Lab Syst2005;76:121-33

[50] Jolliffe IT.Principal Component Analysis.1986;New YorkSpringer

[51] Turk M.Eigenfaces for recognition..J Cogn Neurosci1991;3:71-86

[52] Wu B,Cai W,Gayen SK.Diffuse optical Imaging using decomposition methods..Int J Opt2012;2012:185435

[53] Golub GH.Singular value decomposition and least squares solutions..Numer Math1970;14:403-20

[54] Metz CE.Basic principles of ROC analysis..Semin Nucl Med1978;8:283-98

[55] Zweig MH.Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine..Clin Chem1993;39:561-77

[56] Platt JC.Probabilistic outputs for support vector machines and comparisons to regularized likelihood methods. Advances in Large Margin Classifiers.1999;Cambridge, MAMIT Press61-74

[57] Hanley JA.The meaning and use of the area under a receiver operating characteristic (ROC) Curve..Radiology1982;143:29-36

[58] Leeflang MMG,Reitsma JB.Bias in sensitivity and specificity caused by data-driven selection of optimal cutoff values: mechanisms, magnitude, and solutions..Clin Chem2008;54:729-37

[59] Hata TR,Ermakov IV,Khachik F.Non-invasive Raman spectroscopic detection of carotenoids in human skin..J Invest Dermatol2000;115:441-8

[60] Skin Cancer Treatment (PDQ®)-Patient Version. Available from: https://www.cancer.gov/types/skin/patient/skin-treatment-pdq. [Last accessed on 4 Jan 2019]

[61] Sandby-Møller J,Wulf HC.Epidermal thickness at different body sites: relationship to age, gender, pigmentation, blood content, skin type and smoking habits..Acta Derm Venereol2003;83:410

[62] Ermakov IV,Ermakova M.Resonance Raman detection of carotenoid antioxidants in living human tissue..J Biomed Opt2005;10:064028 PMCID:PMC3086339

[63] Ermakov IV,Redelfs AH,Stookey J.Skin carotenoids as biomarker for vegetable and fruit intake: validation of the reflection-spectroscopy based “Veggie Meter”..FASEB J2016;30:409

[64] Ermakov IV,Gellermann W.Noninvasive selective detection of lycopene and beta-carotene in human skin using Raman spectroscopy..J Biomed Opt2004;9:332-8

[65] Mayne ST,Scarmo S,Ermakov IV.Resonance Raman spectroscopic evaluation of skin carotenoids as a biomarker of carotenoid status for human studies..Arch Biochem Biophys2013;539:1-18 PMCID:PMC3818359

[66] Ren H,Mukamel S.Two-dimensional stimulated ultraviolet resonance Raman spectra of tyrosine and tryptophan; a simulation study..J Raman Spectrosc2013;44:544-59 PMCID:PMC3622277

[67] Darvin ME,Lademann J.The role of carotenoids in human skin..Molecules2011;16:10491

[68] Haag SF,Darvin ME,Lademann J.Determination of the antioxidative capacity of the skin in vivo using resonance Raman and electron paramagnetic resonance spectroscopy..Exp Dermatol2011;20:483-7

[69] Lademann J,Sterry W.Carotenoids in human skin..Exp Dermatol2011;20:377-82

[70] Wrona M,Rozanowska M,Truscott TG.Cooperation of antioxidants in protection against photosensitized oxidation..Free Radic Biol Med2003;35:1319-29

[71] Palozza P.beta-Carotene and alpha-tocopherol are synergistic antioxidants..Arch Biochem Biophys1992;297:184-7

[72] Darvin ME,Lademann J.Resonance Raman spectroscopy as an effective tool for the determination of antioxidative stability of cosmetic formulations..Journal of Biophotonics2010;3:82-8

[73] Puppels GJ,Sijtsema NM,Maraboeuf F.Development and Application of Raman microspectroscopic and Raman imaging techniques for cell biological studies..Journal of Molecular Structure1995;347:477-84

[74] Prendergast GC.Why tumours eat tryptophan..Nature2011;478:192-4

[75] Moroni F.Tryptophan metabolism and brain function: focus on kynurenine and other indole metabolites..European Journal of Pharmacology1999;375:87-100

[76] Fatokun AA,Ball HJ.Indoleamine 2,3-dioxygenase 2 (IDO2) and the kynurenine pathway: characteristics and potential roles in health and disease..Amino Acids2013;45:1319-29

[77] Sordillo PP,Helson L.The kynurenine pathway: a primary resistance mechanism in patients with glioblastoma..Anticancer Research2017;37:2159-71

[78] Shafaat HS.Resonance Raman analysis of the tryptophan cation radical..J Phys Chem Lett2014;5:3009-14

[79] Shafaat HS,Tauber MJ.Resonance Raman characterization of a stable tryptophan radical in an azurin mutant..J Phys Chem B2009;113:382-8

[80] Bleifuss G,Pötsch S,Bittl R.Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz..Biochemistry2001;40:15362-8

[81] Pandey R,Kang JW.Discerning the differential molecular pathology of proliferative middle ear lesions using Raman spectroscopy..Scientific Reports2015;5:13305 PMCID:PMC4542608

[82] Bodanese B,Albertini R,Pacheco MTT.Differentiating normal and basal cell carcinoma human skin tissues in vitro using dispersive Raman spectroscopy: a comparison between principal components analysis and simplified biochemical models..Photomed Laser Surg.2010;28:Suppl 1S119-S27

[83] Jermyn M,Mercier J,Pichette J.Intraoperative brain cancer detection with Raman spectroscopy in humans..Sci Transl Med2015;7:274ra19

[84] Bergholt MS,Ho KY,Yeoh KG.Fiberoptic confocal raman spectroscopy for real-time in vivo diagnosis of dysplasia in Barrett’s esophagus..Gastroenterology2014;146:27-32

[85] Lu L,Secor J.Resonance Raman scattering of β-carotene solution excited by visible laser beams into second singlet state..J Photochem Photobiol B2018;179:18-22