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Update  on  AK  Dermoscopy  and  

Impact  on  Management  

Susana  Puig  Hospital  Clinic  Barcelona  

Spain  

Course  005f:  Advance  Dermoscopy  

Final Program

2018 AAD Annual MeetingSan Diego, CaliforniaFebruary 16-20, 2018SAN DIEGO CONVENTION CENTER

FinalProgram

Zalaudek  I,  Giacomel  J,  Argenziano  G,  et  al.  Dermoscopy  of  facial  nonpigmented  acKnic  keratosis.  Br  J  Dermatol.  2006  Nov;155(5):951-­‐6  

Strawberry  paUern    

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“rosette” like structures

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Cuellar F et al. Arch Dermatol 2009

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Non  pigmented  AK  Cross-­‐Polarized  dermoscopy  

Erythematous  pseudonetwork  Surface  scale  Follicular  opennings/  plugs  Linear  wavy  vessels      

RoseUe  sign  

dermoscopic grading of actinic keratosis

I° II° III°

red pseudo-network and fine scales background erythema and white hair follicles strawberry pattern

structureless white-yellow keratotic areas

Zalaudek et al. Clinics in Dermatology 2014 Courtesy  Iris  Zalaudek  

RED  and  WHITE  color  

Progression  model  of  AK  

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Lallas  A,  Apalla  Z,  Lefaki  I,  SoKriou  E,  Lazaridou  E,  Ioannides  D,  et  al.  Dermoscopy  of  discoid  lupus  erythematosus.  Br  J  Dermatol2013;168:284-­‐8.  

Dermatoscopy of flat pigmented facial lesions: diagnostic challenge between pigmented actinic keratosis and lentigo maligna.Akay BN, Kocyigit P, Heper AO, Erdem C.Br J Dermatol. 2010;163(6):1212-7

PAK =67, LM/LMM (20) and LPLK (2) Slate-grey dots (70%) Annular-granular pattern (39%) Rhomboidal structures (36%) Pseudonetwork (36%) Black globules (34%) Slate-grey globules (33%) Black dots (30%) Asymmetrical pigmented follicular openings (25%) Hyperpigmented rim of follicular openings (21%) Slate-grey areas (18%) Streaks (3%)

Asymmetric pigmented follicular openings

Dark rhomboidal structures

Homogeneous areas

Slate-grey dots and globules

Dermoscopic features indicating Lentigo maligna melanoma (LMM)

Mod

el o

f pro

gres

sion

Schiffner R et al. J Am Acad Dermatol 2000;42:25-32

Dermoscopic features indicating Lentigo maligna melanoma (LMM)

Cognetta AB et al. Dermatol Clin 2001;19:307-318

Circles, circle in a circle (Isobar) pattern

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GENERAL DERMATOLOGYBJD

British Journal of Dermatology

Dermoscopic clues to differentiate facial lentigo malignafrom pigmented actinic keratosisA. Lallas,1 P. Tschandl,2 A. Kyrgidis,1 W. Stolz,3 H. Rabinovitz,4 A. Cameron,5 J.Y. Gourhant,6 J. Giacomel,7

H. Kittler,2 J. Muir,8 G. Argenziano,9 R. Hofmann-Wellenhof10 and I. Zalaudek10

1Skin Cancer Unit, Arcispedale Santa Maria Nuova IRCCS, Viale Risorgimento 80, 42100 Reggio Emilia, Italy2Department of Dermatology, Medical University of Vienna, Vienna, Austria3Department of Dermatology, Allergology and Environmental Medicine II, Hospital Thalkirchner Straße, St€adtisches Klinikum Munich, Munich, Germany4Skin and Cancer Associates, Plantation, FL, U.S.A.5School of Medicine, University of Queensland, Brisbane, Qld, Australia6Dermatology Practice, Nemours, France7Skin Spectrum Medical Services, Como, WA, Australia8School of Medicine, The University of Queensland, Brisbane, Qld, Australia9Dermatology Unit, Second University of Naples, Naples, Italy10Department of Dermatology and Venerology, Non-Melanoma Skin Cancer Unit, Medical University of Graz, Graz, Austria

CorrespondenceAimilios Lallas.E-mail: emlallas@gmail.com

Accepted for publication26 November 2015

Funding sourcesThis study was supported in part by the ItalianMinistry of Health (RF-2010-2316524).

Conflicts of interestNone declared.

DOI 10.1111/bjd.14355

Summary

Background Dermoscopy is limited in differentiating accurately between pigmentedlentigo maligna (LM) and pigmented actinic keratosis (PAK). This might berelated to the fact that most studies have focused on pigmented criteria only,without considering additional recognizable features.Objectives To investigate the diagnostic accuracy of established dermoscopic criteriafor pigmented LM and PAK, but including in the evaluation features previouslyassociated with nonpigmented facial actinic keratosis.Methods Retrospectively enrolled cases of histopathologically diagnosed LM, PAKand solar lentigo/early seborrhoeic keratosis (SL/SK) were dermoscopically evalu-ated for the presence of predefined criteria. Univariate and multivariate regressionanalyses were performed and receiver operating characteristic curves were used.Results The study sample consisted of 70 LMs, 56 PAKs and 18 SL/SKs. In a mul-tivariate analysis, the most potent predictors of LM were grey rhomboids (sixfoldincreased probability of LM), nonevident follicles (fourfold) and intense pigmen-tation (twofold). In contrast, white circles, scales and red colour were signifi-cantly correlated with PAK, posing a 14-fold, eightfold and fourfold probabilityfor PAK, respectively. The absence of evident follicles also represented a frequentLM criterion, characterizing 71% of LMs.Conclusions White and evident follicles, scales and red colour represent significantdiagnostic clues for PAK. Conversely, intense pigmentation and grey rhomboidallines appear highly suggestive of LM.

What’s already known about this topic?

• Dermoscopy is insufficient to differentiate between lentigo maligna (LM) and pig-mented actinic keratosis (PAK).

What does this study add?

• White and evident follicles, scales and red colour represent significant diagnosticclues for PAK.

• Intense pigmentation and grey rhomboidal lines appear highly suggestive of LM.

• These novel findings might improve the early detection of LM, while reducingunnecessary biopsies for PAK.

© 2015 British Association of Dermatologists British Journal of Dermatology (2016) 174, pp1079–1085 1079

presented in Tables 4 and 5 and yielded a sensitivity of88!6%, a specificity of 74!3% and an AUC of 0!89 for LM;and a sensitivity of 78!6%, a specificity of 94!3% and an AUCof 0!94 for PAK.

Aiming to increase the clinical applicability of our findings,we created a scoring scheme for the diagnosis of LM(Table 6). We elected to use arbitrary multipliers, rather thanexact odds ratios, to maximize clinical applicability. In thescoring scheme we included all of the significant multivariateLM predictors and one additional criterion, namely grey cir-cles. The latter variable was included on the basis of previousevidence suggesting a high specificity for the diagnosis of LM,in combination with the fact that it represented the mostpotent predictor in our sample in the univariate analysis.Although not reaching significance in the multivariate model,a trend was evident (P = 0!07, 93% certainty). In the boot-strap analyses, the P-value of grey circles was marginal(P = 0!050), a finding that further concurs with its inclusionin the scoring scheme. The bootstrapping process for the totalscore of the six variables from the score chart did not reportany bias.

Discussion

Our study largely confirms previous data regarding the fre-quency and accuracy of clinical and dermoscopic pigmented

criteria in the diagnosis of PFLs, but also provides additionalnovel clues for differentiating LM from PAK.In line with previous studies, dermoscopy showed a signifi-

cantly higher accuracy in the diagnosis of PFL compared withthe unaided eye.3,12 The overall frequencies of criteria in LMand PAK in our study appear similar to those in previousreports, including both the ‘traditional’ pigmented criteria andthe most recently described vascular structures of LM.3–12,20 Incontrast, there is a discrepancy between our and previousstudies concerning the dermoscopic criteria of SL/SK, whichexhibited grey colour in about half of our cases, while grey

Table 4 Clinical and dermoscopic predictors of lentigo maligna.Univariate and multivariate regression analysis

VariableOddsratio

95% Confidenceinterval P-value

UnivariateNonevident follicles 6!33 3!06–12!98 < 0!001Grey circles 5!90 2!76–12!65 < 0!001Pigmentation intensity(heavy)

2!15 1!40–3!29 < 0!001

Grey rhomboidal lines 1!95 1!01–3!79 0!048White circles 0!05 0!01–0!16 < 0!001Scaly surface 0!13 0!06–0!18 < 0!001MultivariateGrey rhomboidal lines 6!18 1!89–20!24 0!003Nonevident follicles 3!82 1!26–11!63 0!018Grey circles 2!86 0!92–8!91 0!070Pigmentation intensity(heavy)

2!08 1!06–4!09 0!034

White circles 0!08 0!01–0!46 0!005Scaly surface 0!28 0!09–0!89 0!030

Odds ratios for lentigo maligna compared with other lesions inthe study (both pigmented actinic keratosis and seborrhoeic ker-atosis). All dermoscopic variables were included in the multivari-ate model. Relative risks approximated by odds ratios, 95%confidence intervals and P-values were calculated with condi-tional multivariate logistic regression conditional backward elim-ination. Relative risks were mutually adjusted for variables in themodel. Logit for all independent dichotomous variables = no.Alpha level set to P < 0!05. Cut-off value set to < 0!10.

Table 5 Clinical and dermoscopic predictors of pigmented actinickeratosis. Univariate and multivariate regression analysis

VariableOddsratio

95% Confidenceinterval forEXP (B) P-value

UnivariateWhite circles 37!80 12!06–118!47 < 0!001Scaly surface 14!12 6!23–31!80 < 0!001Evident follicles 12!45 5!34–29!06 < 0!001Red rhomboidal lines 6!07 2!67–13!98 < 0!001Grey circles 0!11 0!04–0!28 < 0!001Brown circles 0!16 0!03–0!70 0!015Pigmentation intensity(heavy)

0!55 0!36–0!84 0!006

MultivariateWhite circles 13!52 2!11–86!55 0!006Scaly surface 7!67 2!24–26!28 0!001Red colour 3!60 1!07–12!10 0!039Pigmentation intensity(heavy)

0!31 0!13–0!75 0!009

Odds ratios for pigmented actinic keratosis compared with otherlesions in the study (both lentigo maligna and seborrhoeic ker-atosis). All dermoscopic variables were included in the multivari-ate model. Relative risks approximated by odds ratios, 95%confidence intervals and P-values were calculated with condi-tional multivariate logistic regression conditional backward elim-ination. Relative risks were mutually adjusted for variables in themodel. Logit for all independent dichotomous variables = no.Alpha level set to P < 0!05. Cut-off value set to < 0!10.

Table 6 Scoring scheme for lentigo maligna based on dermoscopicand clinical predictors

Criterion Score

Grey rhomboidal lines +2Nonevident follicles +1Grey circles +1Pigmentation intensity (heavy) +1White circles "2Scaly surface "1Total score in our sample (range) "3 to +5

A total score ≥ 1 is suggestive of lentigo maligna with a speci-ficity of 55!4% and a sensitivity of 92!9%.

© 2015 British Association of Dermatologists British Journal of Dermatology (2016) 174, pp1079–1085

Dermoscopic differentiation of lentigo maligna, A. Lallas et al. 1083

The most frequent local features of LM were grey circles(39 of 70, 56%), grey rhomboidal structures (39 of 70, 56%)and brown rhomboidal structures (26 of 70, 37%) (Fig. 1).In PAK, evident follicles (47 of 56, 84%), white circles (36 of56, 64%) and grey rhomboidal structures (29 of 56, 52%)represented the most frequent dermoscopic criteria (Fig. 2).Brown circles were the most common feature of SL/SKs (nineof 18, 50%), followed by grey circles (seven of 18, 39%) andevident follicles (six of 18, 33%).The inter-rater reliability among evaluators for each dermo-

scopic criterion ranged from 0!61 to 0!81. The variables withthe best intraobserver agreement were ‘grey colour’ (0!81)and ‘red colour’ (0!77), while those scoring worst were

‘brown rhomboidal lines’ (0!61) and ‘brown circles’ (0!63).The inter-rater reliability of the new variable ‘evident follicles’was 0!67.Overall, the diagnostic accuracy of dermoscopy [which cor-

rectly classified 80!6% of cases, area under curve (AUC) 0!89]was superior to the clinical criteria (which correctly classified75!0% of cases, AUC 0!59).The results of the univariate analysis for the diagnosis of

LM are presented in Table 4. In a following step, logisticregression was used to model the influence of each variableon the final histopathological diagnosis, using the dichoto-mous variables to determine the diagnostic accuracy of clinicaland dermoscopic criteria for each diagnosis. These models are

(a) (b)

Fig 1. Melanomas in situ displaying greycircles (circles) and grey granules/dots(arrows). The follicular openings appear smalland equal in size and are difficult to visualize.

(a) (b)

Fig 2. (a) Background erythema, scales andbrown-to-grey rhomboidal lines (circles) canbe dermoscopically seen in this pigmentedactinic keratosis. Note that the pigmentation isstrictly limited to the interfollicular space anddoes not invade the follicular openings. (b)Surface scaling, background erythema andevident follicular openings showing whitecircles (circles) allow the diagnosis ofpigmented actinic keratosis even in thepresence of grey dots and rhomboidal linesaround the follicular openings. Although notincluded as a criterion in this study, thisimage also shows the characteristic ‘rosettes’or ‘four dots in a square’ (arrows).

© 2015 British Association of DermatologistsBritish Journal of Dermatology (2016) 174, pp1079–1085

1082 Dermoscopic differentiation of lentigo maligna, A. Lallas et al.

PAK LM/LMM

The  importance  of  follicular  changes  

follicular  keratoKc  plugs    

invasion  of  the  follicle    

Courtesy  Iris  Zalaudek  

Dermoscopic features in AK and pigmented AK

Dermoscopic features in LMM

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Dermoscopy

Confocal microscopy Histology

24  

ORIGINAL ARTICLE

Treatment monitoring of 0.5% 5-fluorouracil and 10%salicylic acid in clinical and subclinical actinic keratoseswith the combination of optical coherence tomography andreflectance confocal microscopyJ. Malvehy,1,2,3,* I. Alarcon,1,4 J. Montoya,1 R. Rodr!ıguez-Azeredo,4 S. Puig1,2,3

1Melanoma Unit, Dermatology Department, Hospital Cl!ınic, Barcelona, Spain2Institut d’Investigacions Biom"ediques August Pi I Sunyer (IDIBAPS), Barcelona, Spain3Centre on Biomedical Research on Rare Diseases (CIBERER), Instituto Carlos III, Barcelona, Spain4Almirall SA, Barcelona, Spain*Correspondence: J. Malvehy. E-mail: jmalvehy@gmail.com

AbstractBackground Reflectance confocal microscopy (RCM) and high-definition optical coherence tomography (HD-OCT)allow the observation in vivo of dynamic changes in response to non-surgical treatment of actinic keratosis and field ofcancerisation.Objective To non-invasively assess the pharmacodynamic changes induced by treatment with low dose 5-fluorouraciland 10% salicylic acid by means of RCM and high-definition OCT in field cancerization and actinic keratosis.Methods Twenty patients aged >50 years with diagnosis of actinic keratosis on the head and face and indication fortreatment with 0.5% 5-fluorouracil and 10% salicylic acid were enrolled. An area of 25 cm² including visible type I and IIAK and subclinical AK was treated once daily during 6 weeks and examination was performed with RCM and HD-OCTbefore treatment and 2 weeks after the end of treatmentResults High-definition optical coherence tomography results at baseline of mean thickness of the stratum corneumand epidermis were in AK 10.4 (SD = 4.99) and 43.3 (SD = 24.01) lm respectively and in subclinical AK 3.7 (SD = 2.15)and 30.05 (SD = 16.85) lm. At week 8 (2 weeks after the end of treatment) measurements of stratum corneum andepidermis were significantly reduced in AK and subclinical AK for stratum corneum and epidermis. In RCM at week 8scaling, detached corneocytes, atypical honeycomb, round nucleated cells in the spinosum granulosum layer, roundvessels (dermal papillae), inflammatory cells and total AK score were significantly reduced in AK and subclinical AK.Conclusions Evaluation of AK and subclinical AK by RCM and HD-OCT showed objective improvement after treat-ment with 5-fluorouracil and 10% salicylic acid. These methods allowed the study of dynamic changes in the tissue at asubclinical level.Received: 26 April 2015; Accepted: 4 September 2015

Conflicts of interestJosep Malvehy has served as a consultant for Almirall, S.A. and MAVIG GmbH. Ivette Alarcon has receivedhonoraria from Almirall during the course of the study. Rosario Rodr!ıguez Azeredo is an employee of Almirall S.A

Funding sourcesThe study was sponsored by Almirall, S.A

IntroductionActinic keratoses (AK) are the result of the mutation and abnor-

mal growth of keratinocytes due to the cumulative long-term

exposure of the skin to ultraviolet radiation.1,2 Some authors

consider AK a very early stage of cancer or carcinoma in situ,

since the majority of invasive squamous cell carcinoma (SCC)

arise from AK; thus treatment of these lesions is recommended

to limit the risk of progression into invasive SCC.3,4

The concept of field cancerization was first introduced by

Slaughter et al.5 in 1953 when studying the presence of histologi-

cally abnormal tissue surrounding oral squamous cell carci-

noma. On the basis of molecular findings, the following

definition of field cancerization has been proposed: ‘the presence

of one or more areas consisting of epithelial cells that have

genetic alterations’.6 The presence of a field with genetically

altered cells appears to be a continuous risk factor for cancer.7,8

© 2015 European Academy of Dermatology and VenereologyJEADV 2015

DOI: 10.1111/jdv.13445 JEADV

cost-effectiveness in the treatment of AKs.22–24 Recently,

Ulrich et al. reported a series of eight patients treated with

this combination in areas with refractory AK and field

cancerisation. In six patients, a complete response was

observed by in vivo RCM.25 The authors observed in two

patients persistent inflammatory response in AK and

subclinical AK at the end of the treatment.

Although the study was not designed to assess toxicity

and tolerance to treatment with 0.5% 5-fluorouracil and

10% salicylic acid, interestingly no patient presented toxicity

that required interruption of the treatment. The toxicities

were all mild to moderate skin adverse effects reported in

previous studies and consisted of erythema, scaling and

itching.

Cros

sEn

face

Figure 3 HD-OCT of AKs exhibiting specific features. Image on the left side exhibiting a cross-sectional mode (cross) and en face image(en face) with alternating atrophy and hypertrophy (red double arrow), hyperkeratosis (blue arrow), parakeratosis (yellow arrow) and out-lined DEJ (orange arrow). Left side: en face image with atypical honeycomb pattern, adnexal involvement (loss of cocade sign withdyskeratosis in the infundibulum) (red arrow) and presence of dyskeratotic cells in the epidermis (yellow arrow).

Figure 4 RCM of AK at the level of the stratum corneum showingscale (score = 2), polygonal nucleated cells stratum corneum(score = 2), detached corneocytes (score = 2) (upper images). Inthe same lesion at week 8, straum corneum is homogeneous withabscence of scale (score = 0), polygonal nucleated cells stratumcorneum (score = 0), detached corneocytes (score = 0) (lowerimages).

Figure 5 RCM at the stratum spinosum level in subclinical AKexhibiting atypical honeycomb (score = 2) (left). In the same lesionat week 8, the honeycomb pattern is typical (score = 0) (right).

© 2015 European Academy of Dermatology and VenereologyJEADV 2015

Monitoring of field cancerization by RCM and HD-OCT 5

Acta Derm Venereol 94

INVESTIGATIVE REPORT

Acta Derm Venereol 2014 Preview

© 2014 The Authors. doi: 10.2340/00015555-1860Journal Compilation © 2014 Acta Dermato-Venereologica. ISSN 0001-5555

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Accepted Apr 1, 2014; Epub ahead of print Apr 3, 2014

Acta Derm Venereol

Dr. Josep Malvehy, Melanoma Unit, Dermatology De-partment, Hospital Clínic, C/Villarroel, 170, ES-08036 Barcelona, Spain. E-mail: jmalvehy@gmail.com

,Q�WKH�VNLQ��³¿HOG�FDQFHULVDWLRQ´��������GHVFULEHV�VXEFOLQL-FDO� OHVLRQV�ZLWK� ³QRUPDO� DSSHDULQJ� VNLQ´� VXUURXQGLQJ�FOLQLFDOO\�YLVLEOH�DFWLQLF�NHUDWRVLV��$.���SURYLGLQJ�WKH�ED-sis for clonal expansion of genetically altered neoplastic FHOOV���±����7KH�GLDJQRVLV�RI�$.�LV�JHQHUDOO\�EDVHG�RQ�clinical examination. However, biopsy and histological evaluation are performed in clinically indistinct cases, or LI�LQYDVLYH�VTXDPRXV�FHOO�FDUFLQRPD��6&&��LV�VXVSHFWHG��While visual inspection may fail to detect early subclini-cal changes, repeated invasive histological analysis of areas with extensive actinic damage often may not be feasible. $.V�DUH�FRPPRQO\�SUHVHQW�LQ�DUHDV�RI�FKURQLF�DFWLQLF�

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In thinking about the various diagnostic modalities currently available, it is clear that a diagnosis based on FOLQLFDO�JURXQGV�DORQH�PD\�QRW�EH�VXI¿FLHQWO\�UHOLDEOH��while the feasibility of obtaining biopsies of all affected sides and surrounding skin is limited. Hence, in recent years interest has risen in the development of novel non-invasive diagnostic techniques that not only allow SUHFLVH�GLDJQRVLV�RI�FOLQLFDO�$.V�EXW�DOVR�DOORZ�GHWHFWLRQ�DQG�GH¿QLWLRQ�RI�VXEFOLQLFDO�GLVHDVH��In vivo�UHÀHFWDQFH�FRQIRFDO�PLFURVFRS\��5&0��JHQHUDWHV�KRUL]RQWDO�VNLQ�sections with quasi-histological resolution. Its use in WKH�GHWHFWLRQ�RI�$.V�DQG�¿HOG�FDQFHULVDWLRQ��DV�ZHOO�DV�the evaluation of response to topical treatments has been FOHDUO\�HVWDEOLVKHG�������±�����

Several new therapeutic options have become avail-able over recent years, and there remains debate about WKH�EHVW�WUHDWPHQW�RSWLRQV�IRU�$.V�DQG�¿HOG�FDQFHULVD-WLRQ��7UHDWPHQW�RSWLRQV�IRU�$.V�LQFOXGH�D�ZLGH�UDQJH�of ablative modalities and topical treatments such as diclofenac sodium gel, imiquimod, ingenol mebutate JHO� RU� ��ÀXRURXUDFLO�ZLWK� VDOLF\OLF� DFLG��'LFORIHQDF�VRGLXP� ���JHO� LV� D� QRQ�VWHURLGDO� DQWL�LQÀDPPDWRU\�topical agent that demonstrates preferential inhibition of F\FOR�R[\JHQDVH����&2;�����DQ�HQ]\PH�LPSOLFDWHG�LQ�XOWUDYLROHW��89��LQGXFHG�VNLQ�FDUFLQRJHQHVLV����±�����7KH� WUHDWPHQW� RI�$.V�ZLWK� WRSLFDO� ���GLFORIHQDF� LQ������K\DOXURQLF�DFLG��6RODUD]H®, Almirall, S.A., Barce-ORQD��6SDLQ��KDV�EHHQ�H[WHQVLYHO\�GRFXPHQWHG�LQ�WKH�ODVW�GHFDGH� ������+RZHYHU��SUHFLVH� LQIRUPDWLRQ� UHJDUGLQJ�in vivo morphological changes induced by this drug in the skin is needed. In the present study we aimed to describe the changes in the skin of patients with clinical DQG�VXEFOLQLFDO�$.�GXULQJ�DQG�DIWHU�WRSLFDO�WUHDWPHQW�ZLWK����GLFORIHQDF�VRGLXP�ZLWK������K\DOXURQLF�DFLG�E\�5&0��DQG�GHVFULEH� WKH�FRUUHODWLRQ�EHWZHHQ�5&0�

Monitoring Treatment of Field Cancerisation with 3% Diclofenac 5QFKWO������*[CNWTQPKE�#EKF�D[�4GƀGEVCPEG�%QPHQECN�/KETQUEQR[��A Histologic CorrelationJosep MAlVEHy�±�, 5RGULJR�52/'È1�0$5Ë11,4, Pablo�,*/(6,$6�*$5&Ë$1, Alba�'Ë$=� and Susana PUIg�±�

1Melanoma Unit, Dermatology Department, Hospital Clínic, 2Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), 3Centre on Biomedical Research on Rare Diseases (CIBERER), Instituto Carlos III, 4Almirall S.A., Global Dermatology Business Unit, and 5Pathology Department, Hospital Clínic, Barcelona, Spain

2 J. Malvehy et al.

DQG� KLVWRORJ\��7KH� VWXG\�ZDV not designed to assess clinical response or to compare this drug with other alternatives but to demonstrate the changes in vivo induced by the drug.

0$7(5,$/�$1'�0(7+2'67ZHQW\�YROXQWHHU�PDOH�SDWLHQWV��DJHG�!���\HDUV�ROG��)LW]SDWULFN¶V�VNLQ�SKRWRW\SH�,±,,,��ZLWK�D�PLQLPXP�RI���FOLQLFDOO\�YLVLEOH�$.�on the bald scalp, were included in this study. Written informed consent was obtained from each participant before evaluation. Evaluation consisted of clinical examination, dermoscopic LPDJLQJ� DQG�5&0�HYDOXDWLRQ� RI� D� FOLQLFDOO\� YLVLEOH�$.� DQG�³QRUPDO�DSSHDULQJ´�DGMDFHQW�VNLQ��,Q�HDFK�SDUWLFLSDQW��OHVLRQV�FOLQLFDOO\�GLDJQRVHG�DV�$.�ZHUH�VHOHFWHG�IRU�LPDJLQJ�DQG�ELRSV\�as well as adjacent, apparently unaffected skin. An area of 2 × 2 FP�RQ� WKH�VFDOS� LQFOXGLQJ�DW� OHDVW���$.�ZDV�VHOHFWHG��$W� WKH�¿UVW�YLVLW��D���PP�SXQFK�ELRSV\�ZDV�SHUIRUPHG�LQ�RQH�$.�DQG�another 3-mm punch biopsy was performed in normally appa-UHQW�VXUURXQGLQJ�VNLQ��VXEFOLQLFDO�$.���)LJ������$GGLWLRQDOO\��RQH�FOLQLFDO�$.�DQG�RQH�VXEFOLQLFDO�$.�ZHUH�DOVR�VHOHFWHG�DW�EDVHOLQH� �W��� WR� EH� ELRSVLHG� DW� WKH� HQG� RI� WKH� WUHDWPHQW� �W����7UDQVSDUHQF\�ERG\�FKDUWV�ZHUH�XVHG�WR�RXWOLQH�WKH�VHOHFWHG�VNLQ�areas at baseline and to permit co-localisation. Highlighted skin areas were documented on the chart by using permanent marker pens in a colour-coded fashion, correlating individual colours to VHOHFWHG�HYDOXDWLRQ�GDWHV��(YDOXDWLRQ�ZLWK�UHÀHFWDQFH�FRQIRFDO�PLFURVFRS\�ZDV�SHUIRUPHG�EHIRUH�WUHDWPHQW��WLPH�����DW�ZHHN����WLPH�����DW�ZHHN����WLPH�����DW�HQG�RI�WUHDWPHQW��WLPH����LQ���VXE�DUHDV����î���PP��LQVLGH�WKH�VHOHFWHG���î���FP�DUHD��6LWHV�RI�ELRSVLHV�DW�W��ZHUH�DYRLGHG�IRU�WKH�5&0�HYDOXDWLRQ�)ROORZLQJ� WKH� DSSURYHG� LQGLFDWLRQ� WUHDWPHQW�ZLWK� WRSLFDO�

GLFORIHQDF� VRGLXP� ��� LQ� ����� K\DOXURQLF� DFLG�ZDV� DSSOLHG�twice daily for 90 days over the affected skin area, covering both FOLQLFDOO\�DIIHFWHG�$.V�DQG�DSSDUHQWO\�XQDIIHFWHG�DGMDFHQW�VNLQ��

A commercially available in vivo reflectance confocal micros-FRSH��9LYDVFRSH�������/XFLG�7HFK��,QF���+HQULHWWD��1<��86$��0$9,*�*PE+��0XQLFK��*HUPDQ\��ZDV�XVHG�WR�LPDJH�$.V�DQG�VXUURXQGLQJ�³QRUPDO�DSSHDULQJ´� VNLQ��$�GHWDLOHG�GHVFULSWLRQ�of the technique and the device has been published previously ���±����� )RU� HDFK� VNLQ� VLWH� DQDO\VHG�� V\VWHPDWLF� KRUL]RQWDO�PDSSLQJ�ZDV�SHUIRUPHG�DQG��±��LPDJHV�ZHUH�FDSWXUHG�LQ�D[LDO�sections beginning with the stratum corneum, through the entire epidermis and into the upper reticular dermis using the “Viva-EORFN´�IXQFWLRQ�PRGDOLW\� �HDFK�YLYDEORFN�ZDV����î���� �����LPDJHV�FRPSRVLWH���)XUWKHUPRUH��D�YHUWLFDO�PDSSLQJ�XVLQJ�WKH�³9LYDVWDFN´�ZDV�SHUIRUPHG�LQ����P�VWHSV�WR�D�PD[LPXP�GHSWK�RI� �����P�EHJLQQLQJ�ZLWK� WKH� VWUDWXP� FRUQHXP� WKURXJK� WKH�HQWLUH�HSLGHUPLV�DQG�LQWR�WKH�VXSHUILFLDO�GHUPLV��5&0�LPDJHV�were individually subjected to evaluation.

'LDJQRVWLF�UHIOHFWDQFH�FRQIRFDO�PLFURVFRS\��5&0��FULWHULD�IRU�$.�DUH�VKRZQ�LQ�7DEOH�6,D1. 'LDJQRVWLF�FULWHULD�IRU�$.�LQFOXGHG�SDUDNHUDWRVLV��K\SHUNHUD-

tosis, individual detached keratinocytes, architectural disarray, keratinocyte atypia and pleomorphism as shown in 7DEOH�6,1. $Q�LQYHVWLJDWRU�REWDLQHG�LPDJHV�RI�DOO�WHVW�VLWHV��55���7KH�FRU-

UHVSRQGLQJ�5&0�LPDJHV�ZHUH�WKHQ�VXEMHFW�WR�EOLQGHG�HYDOXDWLRQ�by 2 independent experts in the field of confocal microscopy �-0�DQG�63���(YDOXDWRUV�ZHUH�EOLQGHG�WR�SDUWLFLSDQW¶V�QDPH��age and treatment duration.6NLQ�SXQFK�ELRSVLHV����PP�LQ�GLDPHWHU��ZHUH�REWDLQHG�XQGHU�

sterile conditions after local anaesthesia with lidocaine 1% from D�SUHYLRXVO\�VHOHFWHG�VNLQ�OHVLRQ�FOLQLFDOO\�GLDJQRVHG�DV�$.��and also from apparently unaffected adjacent skin at t1 and end RI�WUHDWPHQW��W����7KH�VNLQ�VDPSOHV�ZHUH�IL[HG�LQ�����IRUPDOLQ�and processed by routine histopathology. Staining with haema-toxylin and eosin was performed and slides were evaluated by a ERDUG�FHUWLILHG�SDWKRORJLVW��$'���,PPXQRKLVWRFKHPLFDO�VWDLQV�ZHUH�SHUIRUPHG�WR�HYDOXDWH�&2;���H[SUHVVLRQ��S���PXWDWLRQ��.L����3&1$��S���DQG�&'���EHIRUH�DQG�DW�WKH�HQG�RI�WUHDWPHQW��7KH�KLVWRSDWKRORJLFDO�DQG� LPPXQRKLVWRFKHPLFDO�VFRUHV�XVHG�are detailed in 7DEOH�6,E1.

Statistical analysis7KH�VWDWLVWLFDO�DQDO\VHV�IRU�FDWHJRULFDO�YDULDEOHV�ZHUH�SHUIRU-PHG�ZLWK� D� FKL�VTXDUHG� WHVW�� RU� )LVKHU¶V� WHVW� IRU� ��î��� WDEOHV�ZKHQ�UHTXLUHG�GHSHQGLQJ�RQ�WKH�VDPSOH�VL]H��)RU�FRQWLQXRXV�variables; the mean score for each parameter before treatment �W��� KLVWRORJ\� RU�5&0��ZDV� FRPSDUHG�ZLWK� WKH�PHDQ� VFRUH�GXULQJ��W���W���5&0��DQG�DW�WKH�HQG�RI�WKH�VWXG\��W���KLVWRORJ\�DQG�5&0��XVLQJ�6WXGHQW�t-test for paired samples for normally GLVWULEXWHG�GDWD��$129$�ZDV�XVHG�IRU�PXOWLSOH�FRPSDULVRQV�DW�GLIIHUHQW�WLPHV��W���W���W���W����

5(68/76

At the end of the study 14 patients were able to complete the treatment course and evaluation. Six patients dropped RXW�RI�WKH�VWXG\��7ZR�SDWLHQWV�ZLWKGUHZ�FRQVHQW�DQG�GLG�not follow-up with treatment or evaluation after inclusion �W����)RXU�SDWLHQWV�ZHUH�H[FOXGHG�IURP�WKH�VWXG\�GXULQJ�the treatment course because they were unwilling to continue with treatment or follow-up due to local adverse skin reaction to the topical application of 3% diclofenac VRGLXP�ZLWK������K\DOXURQLF�DFLG��7KHVH�ORFDO�DGYHUVH�

Fig. 1��$FWLQLF�NHUDWRVLV��D��DQG�VXEFOLQLFDO�DFWLQLF�NHUDWRVLV��E��E\�URXWLQH�KLVWRORJ\��+(���������Presence of parakeratosis, architectural disarray, keratinocyte atypia seen to a varying degree.

1KWWS���ZZZ�PHGLFDOMRXUQDOV�VH�DFWD�FRQWHQW�"GRL ���������������������

Acta Derm Venereol 94

Acta Derm Venereol 94

INVESTIGATIVE REPORT

Acta Derm Venereol 2014 Preview

© 2014 The Authors. doi: 10.2340/00015555-1860Journal Compilation © 2014 Acta Dermato-Venereologica. ISSN 0001-5555

9LVXDO� LQVSHFWLRQ� PD\� IDLO� WR� DFFXUDWHO\� HYDOXDWH� ¿HOG�cancerisation (subclinical actinic keratoses [AKs]). We DLPHG�WR�GHVFULEH�¿HOG�FDQFHULVDWLRQ�E\�FRQIRFDO�UHÀHF-WDQFH�PLFURVFRS\�DQG�FKDQJHV�LQGXFHG�E\�WKH�DSSOLFDWLRQ�RI� ��� GLFORIHQDF� VRGLXP� JHO� LQ� ����� K\DOXURQLF� DFLG��)RXUWHHQ�PDOH�SDWLHQWV��!����\HDUV�ROG��ZLWK�$.V�RQ�WKH�EDOG�VFDOS�ZHUH�LQFOXGHG��&OLQLFDO�H[DPLQDWLRQ��FRQIRFDO�PLFURVFRS\� DQG� KLVWRORJLFDO� VWXG\� RI� FOLQLFDOO\� YLVLEOH�OHVLRQV� DQG� ³QRUPDO� DSSHDULQJ´� DGMDFHQW� VNLQ� EHIRUH�DQG�DIWHU�WUHDWPHQW�ZDV�FRPSOHWHG��5HÀHFWDQFH�FRQIRFDO�PLFURVFRS\�VKRZHG�D�GHFUHDVH�LQ�VFDOLQJ��p ��������DQG�DW\SLD�RI�WKH�KRQH\FRPE�SDWWHUQ��p ��������DW���ZHHNV�RI�WUHDWPHQW��&KDQJHV�LQ�SDUDNHUDWRVLV��LQÀDPPDWLRQ�DQG�GHUPDO�FROODJHQ�UHPRGHOOLQJ�ZHUH�DOVR�REVHUYHG��+LVWR-ORJ\� FRUUHODWHG�ZLWK� FRQIRFDO� IHDWXUHV� LQ�$.� DQG� VXE-FOLQLFDO�$.��5HÀHFWDQFH�FRQIRFDO�PLFURVFRS\�ZDV�XVHIXO�LQ�WKH�HYDOXDWLRQ�RI�¿HOG�FDQFHULVDWLRQ�DQG�PRQLWRULQJ�RI�WUHDWPHQW�UHVSRQVH��$�UDSLG�LPSURYHPHQW�LQ�HSLGHUPDO�DW\SLD�ZDV�REVHUYHG��.H\�ZRUGV��¿HOG�FDQFHULVDWLRQ��DF-WLQLF�NHUDWRVLV��UHÀHFWDQFH�FRQIRFDO�PLFURVFRS\��KLVWRORJ\��GLFORIHQDF��K\DOXURQLF�DFLG.

Accepted Apr 1, 2014; Epub ahead of print Apr 3, 2014

Acta Derm Venereol

Dr. Josep Malvehy, Melanoma Unit, Dermatology De-partment, Hospital Clínic, C/Villarroel, 170, ES-08036 Barcelona, Spain. E-mail: jmalvehy@gmail.com

,Q�WKH�VNLQ��³¿HOG�FDQFHULVDWLRQ´��������GHVFULEHV�VXEFOLQL-FDO� OHVLRQV�ZLWK� ³QRUPDO� DSSHDULQJ� VNLQ´� VXUURXQGLQJ�FOLQLFDOO\�YLVLEOH�DFWLQLF�NHUDWRVLV��$.���SURYLGLQJ�WKH�ED-sis for clonal expansion of genetically altered neoplastic FHOOV���±����7KH�GLDJQRVLV�RI�$.�LV�JHQHUDOO\�EDVHG�RQ�clinical examination. However, biopsy and histological evaluation are performed in clinically indistinct cases, or LI�LQYDVLYH�VTXDPRXV�FHOO�FDUFLQRPD��6&&��LV�VXVSHFWHG��While visual inspection may fail to detect early subclini-cal changes, repeated invasive histological analysis of areas with extensive actinic damage often may not be feasible. $.V�DUH�FRPPRQO\�SUHVHQW�LQ�DUHDV�RI�FKURQLF�DFWLQLF�

GDPDJH��+LVWRORJLFDOO\��$.V�KDYH� EHHQ� FODVVL¿HG� DF-

cording to keratinocyte atypia, nuclear pleomorphism, K\SHUNHUDWRVLV�� SDUDNHUDWRVLV�� D� GHUPDO� LQÀDPPDWRU\�LQ¿OWUDWH�DQG�FRQFRPLWDQW�VRODU�HODVWRVLV������$.�LV�RQH�stage of a biologic continuum in the progression from subclinical keratinocyte dysplasia to invasive SCC ��±�����+RZHYHU�� LQ�WKH�FOLQLFDO�VHWWLQJ��SDWLHQWV�RIWHQ�SUHVHQW�ZLWK�QXPHURXV�$.V�DW�YDULRXV�VWDJHV�RI�JUDGHG�differentiation and the extent of subclinical disease is GLI¿FXOW�WR�GHWHUPLQH��

In thinking about the various diagnostic modalities currently available, it is clear that a diagnosis based on FOLQLFDO�JURXQGV�DORQH�PD\�QRW�EH�VXI¿FLHQWO\�UHOLDEOH��while the feasibility of obtaining biopsies of all affected sides and surrounding skin is limited. Hence, in recent years interest has risen in the development of novel non-invasive diagnostic techniques that not only allow SUHFLVH�GLDJQRVLV�RI�FOLQLFDO�$.V�EXW�DOVR�DOORZ�GHWHFWLRQ�DQG�GH¿QLWLRQ�RI�VXEFOLQLFDO�GLVHDVH��In vivo�UHÀHFWDQFH�FRQIRFDO�PLFURVFRS\��5&0��JHQHUDWHV�KRUL]RQWDO�VNLQ�sections with quasi-histological resolution. Its use in WKH�GHWHFWLRQ�RI�$.V�DQG�¿HOG�FDQFHULVDWLRQ��DV�ZHOO�DV�the evaluation of response to topical treatments has been FOHDUO\�HVWDEOLVKHG�������±�����

Several new therapeutic options have become avail-able over recent years, and there remains debate about WKH�EHVW�WUHDWPHQW�RSWLRQV�IRU�$.V�DQG�¿HOG�FDQFHULVD-WLRQ��7UHDWPHQW�RSWLRQV�IRU�$.V�LQFOXGH�D�ZLGH�UDQJH�of ablative modalities and topical treatments such as diclofenac sodium gel, imiquimod, ingenol mebutate JHO� RU� ��ÀXRURXUDFLO�ZLWK� VDOLF\OLF� DFLG��'LFORIHQDF�VRGLXP� ���JHO� LV� D� QRQ�VWHURLGDO� DQWL�LQÀDPPDWRU\�topical agent that demonstrates preferential inhibition of F\FOR�R[\JHQDVH����&2;�����DQ�HQ]\PH�LPSOLFDWHG�LQ�XOWUDYLROHW��89��LQGXFHG�VNLQ�FDUFLQRJHQHVLV����±�����7KH� WUHDWPHQW� RI�$.V�ZLWK� WRSLFDO� ���GLFORIHQDF� LQ������K\DOXURQLF�DFLG��6RODUD]H®, Almirall, S.A., Barce-ORQD��6SDLQ��KDV�EHHQ�H[WHQVLYHO\�GRFXPHQWHG�LQ�WKH�ODVW�GHFDGH� ������+RZHYHU��SUHFLVH� LQIRUPDWLRQ� UHJDUGLQJ�in vivo morphological changes induced by this drug in the skin is needed. In the present study we aimed to describe the changes in the skin of patients with clinical DQG�VXEFOLQLFDO�$.�GXULQJ�DQG�DIWHU�WRSLFDO�WUHDWPHQW�ZLWK����GLFORIHQDF�VRGLXP�ZLWK������K\DOXURQLF�DFLG�E\�5&0��DQG�GHVFULEH� WKH�FRUUHODWLRQ�EHWZHHQ�5&0�

Monitoring Treatment of Field Cancerisation with 3% Diclofenac 5QFKWO������*[CNWTQPKE�#EKF�D[�4GƀGEVCPEG�%QPHQECN�/KETQUEQR[��A Histologic CorrelationJosep MAlVEHy�±�, 5RGULJR�52/'È1�0$5Ë11,4, Pablo�,*/(6,$6�*$5&Ë$1, Alba�'Ë$=� and Susana PUIg�±�

1Melanoma Unit, Dermatology Department, Hospital Clínic, 2Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), 3Centre on Biomedical Research on Rare Diseases (CIBERER), Instituto Carlos III, 4Almirall S.A., Global Dermatology Business Unit, and 5Pathology Department, Hospital Clínic, Barcelona, Spain

3Field cancerisation treatment assessed by confocal microscopy with histologic correlation

events consisted mainly of moderate to severe erythema, mild to moderate scaling, dryness and pruritus. $�WRWDO�RI��������5&0�LPDJHV�ZHUH�REWDLQHG��7KH�

presence of scaling, the area of atypical honeycomb and the degree of atypical honeycomb decreased after ��ZHHNV�RI�WUHDWPHQW��p ��������p ��������p ���������7KHVH� FKDQJHV� SHUVLVWHG� DIWHU� WKH� HQG� RI� WUHDWPHQW��p �������IRU�DOO�����)LJ������$IWHU���ZHHNV�RI�WUHDWPHQW��the presence of polygonal nucleated cells, detached LQGLYLGXDO� FRUQHRF\WHV� DQG� LQÀDPPDWLRQ� LQFUHDVHG��p ��������p ��������p ���������7DEOH�6,,1���)LJV���DQG�����7KHVH�LQFUHDVHV�LQ�SRO\JRQDO�QXFOHDWHG�XSSHU�FHOOV�and detached corneocytes were normalised at the end RI�WUHDWPHQW��p ��������p ���������7DEOH�6,,1���2Q�WKH�FRQWUDU\�� DQ� HOHYDWHG� OHYHO� RI� LQÀDPPDWLRQ�SHUVLVWHG�GXULQJ�WKH�HQWLUH�SHULRG�RI�VWXG\��p ��������VKRZLQJ�WKH�SUHVHQFH�RI�VPDOO�EULJKW�LQÀDPPDWRU\�FHOOV�DQG�VRPH�dendritic cells in the spinous cell layer as well as in WKH�XSSHU�GHUPLV��)LJ������&KDQJHV�LQ�GHUPDO�FROODJHQ�WKLFNHQLQJ�DQG�DUUDQJHPHQW�RI�¿EUHV�LQ�WKH�XSSHU�GHUPLV�ZHUH�REVHUYHG�DW�WKH�HQG�RI�WUHDWPHQW��)LJ������+XGGOHV�RI�FROODJHQ�DQG�FXUOHG�¿EUHV��WKDW�KDYH�EHHQ�GHVFULEHG�LQ�HODVWRVLV��ZHUH�UHSODFHG�E\�DOLJQHG�WKLQQHU�¿EUHV�

Histology before and after the treatment course sho-ZHG�DQ�LPSRUWDQW�UHGXFWLRQ�LQ�HSLGHUPDO�DW\SLD�LQ�$.��p ��������DQG�VXEFOLQLFDO�$.��p ��������EHWZHHQ�W��DQG�t4. Immunohistochemical analysis demonstrated a reduc-

tion in cellular proliferation measured by the expression RI�.L���LQ�$.��p �������DQG�VXEFOLQLFDO�$.��p ��������DW�WKH�HQG�RI�WUHDWPHQW��,Q�VXEFOLQLFDO�$.V��WKHUH�ZDV�D�marked reduction in the expression of the vascular marker &'����p ��������DIWHU�WUHDWPHQW��(YDOXDWLRQ�RI�&2;���H[SUHVVLRQ�ZDV�DYDLODEOH�LQ�MXVW���SDLUHG�$.�DQG���SDLUHG�VXEFOLQLFDO�$.�VDPSOHV��W�±W���GHFUHDVLQJ�VLJQL¿FDQWO\�

Fig. 2�� 5HÀHFWDQFH� FRQIRFDO� PLFURVFRS\� �5&0��LPDJH��YLYDEORFN�����PP2��ZLWK�DW\SLFDO�KRQH\FRPE�in the stratum spinosum of a subclinical actinic NHUDWRVLV� DW� WLPH���EHIRUH� WUHDWPHQW� �D���3UHVHQFH�RI�DW\SLFDO�NHUDWLQRF\WHV�ZLWK�LUUHJXODU�VL]H��VKDSH�DQG�UHIUDFWLYLW\��white arrows���2Q�WKH�ULJKW�LPDJH��E��5&0�RI�WKH�VDPH�OHVLRQ�DIWHU�WKH�WUHDWPHQW�DW�WLPH����1RUPDOLVDWLRQ�RI�KRQH\FRPE�SDWWHUQ�LQ�WKH�same lesion is clearly seen.

Fig. 3.�5HÀHFWDQFH� FRQIRFDO�PLFURVFRS\� LPDJH� DW� WKH� VWUDWXP�FRUQHXP������î������P��DW�ZHHN����'\VNHUDWRVLV�ZLWK�SUHVHQFH�RI�QXFOHDWHG�FHOOV��black arrows��DQG�GLVFRKHVLYH�NHUDWLQRF\WHV��white arrows��

)LJ�����3UHVHQFH�RI�LQÀDPPDWRU\�FHOOV��white arrows��DW�time 2 of treatment in subclinical actinic keratosis seen XQGHU� UHÀHFWDQFH� FRQIRFDO� PLFURVFRS\� LQ� WKH� VWUDWXP�VSLQRVXP� RI� WKH� HSLGHUPLV� �����î�����PP��ZLWK� URXQG�QXFOHDWHG� FHOOV� FRUUHVSRQGLQJ� WR� O\PSKRF\WHV� �D�� DQG�GHQGULWLF�FHOOV�FRUUHVSRQGLQJ�WR�/DQJHUKDQV¶�FHOOV��E��

Acta Derm Venereol 94

14/2/18  

5  

CASE REPORT

Dermoscopy and Reflectance Confocal Microscopyfor Monitoring the Treatment of Actinic Keratosiswith Ingenol Mebutate Gel: Report of Two Cases

Caterina Longo . Stefania Borsari . Elisa Benati . Elvira Moscarella .

Roberto Alfano . Giuseppe Argenziano

To view enhanced content go to www.dermtherapy-open.comReceived: December 16, 2015 / Published online: February 4, 2016! The Author(s) 2016. This article is published with open access at Springerlink.com

ABSTRACT

Introduction: A relatively novel application for

dermoscopy and reflectance confocal

microscopy (RCM) is their use in the

monitoring of topical treatment response for

non-melanoma skin cancer. Actinic keratosis

(AK) is the early phase of a multistep biologic

continuum leading to invasive squamous cell

carcinoma. A number of topical therapies are

now available for the treatment of AK but their

disadvantages include long treatment duration

and prolonged local reactions. Ingenol

mebutate is a newer therapy for AK which is

only applied for 2 or 3 days.

Case Report: Dermoscopy and RCM findings in

two patients with AK treated with ingenol

mebutate confirm that it induces rapid lesion

necrosis and specific neutrophil-mediated,

antibody-dependent cellular cytotoxicity.

Necrosis occurs via mitochondrial membrane

disruption, with subsequent eradication of

residual tumor cells via transient inflammation.

Local skin reactions to ingenol mebutate should

be considered part of the drug’s mechanism of

action rather than an adverse effect.

Conclusion: Ingenol mebutate is a valuable

therapy for the treatment of AK. This case

report adds further evidence to the usefulness

of dermoscopy and RCM in the assessment and

monitoring of treatment outcome.

Keywords: Actinic keratosis; Dermoscopy;

Field cancerization; Ingenol mebutate;

Reflectance confocal microscopy

INTRODUCTION

Treatments for the so-called field cancerization

in actinic keratosis (AK) include imiquimod,

fluorouracil, diclofenac and photodynamic

therapy [1, 2]. Drawbacks to the self-applied

Electronic supplementary material The onlineversion of this article (doi:10.1007/s13555-016-0094-9)contains supplementary material, which is available toauthorized users.

C. Longo ! S. Borsari ! E. Benati ! E. MoscarellaSkin Cancer Unit, Arcispedale Santa Maria NuovaIRCCS, Reggio Emilia, Italy

R. AlfanoDepartment of Anesthesiology, Surgery andEmergency, Second University of Naples, Naples,Italy

G. Argenziano (&)Dermatology Unit, Second University of Naples,Naples, Italye-mail: g.argenziano@gmail.com

Dermatol Ther (Heidelb) (2016) 6:81–87DOI 10.1007/s13555-016-0094-9

significantly better than the surrounding

non-treated area (Fig. 6). Both dermoscopy and

RCM confirmed the clinical response by

showing normal skin and regular

keratinocytes, respectively.

Compliance with Ethics Guidelines

All procedures followed were in accordance

with the ethical standards of the responsible

committee on human experimentation

Fig. 1 a A 74-year-old man with several AKs on his leftcheek. b, e After 7 days’ treatment with ingenol mebutate, amoderate-to-severe reaction is visible clinically anddermoscopically, with the occurrence of confluent vesicles

and pustules. c, f After 15 days, complete healing of thetreated area is seen, with absence of residual AKs.d Dermoscopically, AKs are typified by a reddish pseudonetwork and whitish scales. AK Actinic keratosis

Fig. 2 a At baseline, RCM (0.5 9 0.5 mm) reveals thetypical findings seen in AKs, such as the presence ofparakeratosis and irregular keratinocytes. b After 7 days,vesicles formation with several inflammatory cells are seen,

followed, after 30 days, by a complete recovery of theepidermis that shows regularly spaced keratinocytes with awell-defined contour (c). RCM Reflectance confocalmicroscopy, AK Actinic keratosis

Dermatol Ther (Heidelb) (2016) 6:81–87 83

significantly better than the surrounding

non-treated area (Fig. 6). Both dermoscopy and

RCM confirmed the clinical response by

showing normal skin and regular

keratinocytes, respectively.

Compliance with Ethics Guidelines

All procedures followed were in accordance

with the ethical standards of the responsible

committee on human experimentation

Fig. 1 a A 74-year-old man with several AKs on his leftcheek. b, e After 7 days’ treatment with ingenol mebutate, amoderate-to-severe reaction is visible clinically anddermoscopically, with the occurrence of confluent vesicles

and pustules. c, f After 15 days, complete healing of thetreated area is seen, with absence of residual AKs.d Dermoscopically, AKs are typified by a reddish pseudonetwork and whitish scales. AK Actinic keratosis

Fig. 2 a At baseline, RCM (0.5 9 0.5 mm) reveals thetypical findings seen in AKs, such as the presence ofparakeratosis and irregular keratinocytes. b After 7 days,vesicles formation with several inflammatory cells are seen,

followed, after 30 days, by a complete recovery of theepidermis that shows regularly spaced keratinocytes with awell-defined contour (c). RCM Reflectance confocalmicroscopy, AK Actinic keratosis

Dermatol Ther (Heidelb) (2016) 6:81–87 83

REVIEW ARTICLE

A new vision of actinic keratosis beyond visible clinicallesionsJ. Malvehy*

Melanoma Skin Unit, University Hospital Clinic of Barcelona, Barcelona, Spain*Correspondence: J. Malvehy. E-mail: jmalvehy@gmail.com

AbstractIn actinic keratosis (AK), clinical and subclinical lesions coexist across large areas of sun-exposed skin resulting in fieldcancerization. The lesions are part of a disease continuum which can progress into invasive squamous cell carcinoma(SCC). Conventional biopsy sampling together with histopathological analysis of the excised tissue is still the gold stan-dard for differentially diagnosing AK from invasive SCC and identifying the characteristic pathophysiological features ofthese lesions. Given that biopsy sampling is invasive and not suited to the investigation of disease across large fields ofskin, several imaging technologies have been applied to non-invasively investigate AK. Widely available imaging technol-ogies such as cross-polarized light, fluorescence and dermoscopy can assist the dermatologist in diagnosing AK and inidentifying different types of AK lesions. Modern imaging technologies such as reflectance confocal microscopy (RCM)and high-definition optical coherence tomography (HD-OCT) provide high-resolution images of the skin. These tech-niques can be used to image the histological changes that characterize AK and so can be used to diagnose the diseaseand its severity. They can also identify the presence of subclinical lesions and non-invasively monitor the effects of AKtreatments on both subclinical and clinical lesions over time. Both RCM and HD-OCT have revealed a new vision of AKby visualizing in detail the cellular and histological changes that characterize both clinical and subclinical lesions, andconfirming that the disease affects the entire sun-exposed field. As a consequence of these findings, the target for thetreatment of AK now needs to be the detection and clearance of all clinical and subclinical lesions across the entire sun-exposed field.Received: 4 September 2014; Accepted: 6 October 2014

Conflict of interestConsultant for the following companies: Meda Pharma, Leo Pharma, Almirall, ISDIN, Roche Posay, Pierre Farma, Mavig,3Gen, Bristol Meyers Squibb, Amgen, Roche Pharma

Funding sourceThis supplement was funded by Meda Pharma GmbH & Co. KG.

IntroductionIn actinic keratosis (AK), the entire area of sun-exposed skin is

affected by disease resulting in field cancerization. The concept

of field cancerization was first introduced by Slaughter et al.1 in

1953 to describe histologically abnormal tissue surrounding pri-

mary oral squamous cell carcinoma (SCC) and to explain the

development of multiple primary tumours and locally recurrent

cancer within the field. This concept was extended by Braakhuis

et al.2 who in 2003 proposed that clusters of genetically altered

stem cell clonal units develop into individual and then contigu-

ous fields of pre-neoplastic cells. In AK, the visible clinical lesions

are the initial manifestation of a multi-step carcinogenesis pro-

cess or disease continuum that can progress from initial subclini-

cal keratinocyte dysplasia into invasive SCC. Field cancerization

develops since ultraviolet light causes neoplastic changes across

the entire sun-exposed field of skin. Within the cancerous field,

all stages of AK may coexist including individual UV-damaged

keratinocytes, subclinical (invisible, non-palpable) lesions, early

clinical lesions, late clinical lesions, and in some patients, inva-

sive SCC.3 According to Braakhuis et al.2 the consequence of this

pathophysiology is that the ‘treatment of epithelial cancers

should not only be focused on the tumour but also on the field

from which it developed’. In terms of AK, this means that all

lesions across an entire sun-exposed field need to be targeted and

eliminated to provide long-term disease remission and to pre-

vent disease recurrence.

The purpose of this article is to discuss how modern imaging

technologies, in particular reflectance confocal microscopy

© 2014 European Academy of Dermatology and VenereologyJEADV 2015, 29 (Suppl. 1), 3–8

DOI: 10.1111/jdv.12833 JEADV

(RCM) and high-definition optical coherence tomography

(HD-OCT), have been used to generate a new vision of AK.

These imaging techniques visualize both clinical and subclinical

lesions and so allow the full extent of a patient’s disease burden

to be evaluated. They also allow a patient’s response to field-

directed treatment and the ability of the treatment to clear clini-

cal and subclinical lesions to be assessed. High-resolution imag-

ing is currently mainly used in a research setting, though may be

increasingly used in a clinical setting in the future.

Histopathology of skin biopsiesAn initial vision and understanding of the pathophysiology of

AK were largely based on histopathological analysis of skin biop-

sies. Such investigations revealed characteristic histopathological

changes in the skin of AK patients which can be used to diagnose

the disease and to assess its severity.4,5 These changes include

parakeratosis and hyperkeratosis in the stratum corneum; abnor-

mal architecture, irregular acanthosis and cellular pleomor-

phisms in the stratum granulosum and spinosum; crowding of

keratinocytes and cellular pleomorphism in the stratum basale;

and solar elastosis and increased vascularity in the dermis.6 In

addition, the dermo-epidermal junction may appear irregular as

a result of small round buds which extend into the upper papil-

lary dermis from the basal cell layer.4 Histopathology of skin

biopsies from AK patients has also shown that AK lesions and

SCC are indistinguishable at a cellular level. Both AK lesions and

SCC contain atypical keratinocytes with loss of polarity, nuclear

polymorphism and disordered maturation.4 Investigations have

also shown that these two types of lesions contain similar genetic

mutations, e.g. in p53 and bcl-2 genes.7,8 As a consequence, AK is

now considered to be in situ SCC.3 Skin biopsies together with

histopathological analysis are recommended if invasive SCC is

suspected so that the patient may receive appropriate treatment.9

Non-invasive technologies are required to further understand

the pathophysiology of AK given the limitations of biopsy analy-

sis of skin tissue. In particular, the results from biopsies are only

applicable to the area from which the biopsy was taken and they

cannot be used to visualize pathophysiological changes across an

entire affected field of skin. In addition, repeat biopsies cannot

be taken from the same area to evaluate changes over time, e.g.

in response to an AK treatment. Furthermore, AK lesions can

sometimes occur in areas where biopsies are not feasible, and

biopsies involve patient discomfort, time and expense.

New imaging techniques may also assist in the accurate non-

invasive diagnosis of AK and SCC. Diagnosing lesions on the

basis of clinical inspection alone can lead to a high rate of misdi-

agnosis. For example, the results from one study showed that

36% of lesions diagnosed clinically as AKs were actually invasive

SCC when classified with histology.10 A second study identified

a lower rate of clinical misdiagnosis: 9% of lesions identified

clinically as AK were shown by analysis of biopsies to have other

classifications such as SCC or basal cell carcinoma.11 Clinical

inspection of an AK patient also does not identify subclinical

lesions.

Cross-polarized light and fluorescenceCross-polarized light and fluorescence are relatively simple

imaging techniques which can be used to detect subclinical AK

lesions which are not normally visible.12 Cross-polarized light

can be used to enhance the imaging of vasculature, pigmentation

and structures below the skin surface and involves the use of spe-

cific filters on the source of illumination.12 Fluorescence involves

the topical application of methyl-aminolevulinic acid, which

induces porphyrin formation in the skin if clinical or subclinical

disease is present.12 Of these techniques, cross-polarized light is

easy to use, whereas fluorescence diagnosis is more time-con-

Figure 1 Vision of actinic keratosis withstandard light compared with multimodallight and fluorescence. In the image withstandard light (left), most of the patient’sactinic keratoses are not visible. With greenultraviolet light (right), actinic keratoses canbe seen as white scales.

© 2014 European Academy of Dermatology and VenereologyJEADV 2015, 29 (Suppl. 1), 3–8

4 Malvehy

suming but more sensitive for detecting subclinical lesions.12

The images generated by these techniques reveal that AK affects

the entire area of sun-exposed skin with many more lesions than

are visible to the naked eye (Fig. 1). In addition, the clinical AK

lesions themselves may be much larger than can be seen with

normal light.

DermoscopyDermoscopy can be used as a real-time, non-invasive imaging

technique to assist in the diagnosis of AK with a high level of

concordance between diagnoses made using this technique and

with histopathology.13 Dermoscopy allows the uncovering of

features such as vascular patterns which cannot be seen with the

naked eye.14 Four criteria for the diagnosis of facial AK with der-

moscopy have been established.14 These include erythema with a

marked pink-to-red pseudonetwork surrounding hair follicles;

white-to-yellow surface scale; fine, linear-wavy vessels surround-

ing hair follicles; and hair follicle openings filled with yellowish

keratotic plugs and/or surrounded by a white halo. These fea-

tures lead to a typical ‘strawberry pattern’ in 95% of patients

(Fig. 2). The features most consistently found with dermoscopy

of AK lesions are the erythematous pseudonetwork and follicular

openings.13

A recent study showed that the dermoscopic patterns associ-

ated with AK, intraepidermal carcinoma and invasive SCC are

significantly different and this may help in the early differential

diagnosis of in situ or invasive lesions so that they may be appro-

priately treated.15 A red pseudonetwork was significantly associ-

ated with AK, whereas dotted/glomerular vessels, diffuse yellow

opaque scales and microerosions were significantly more preva-

lent in intraepidermal carcinoma. Typical dermoscopic features

of invasive SCC were hairpin vessels, linear irregular vessels,

targetoid hair follicles, white structureless areas, a central mass

of keratin and ulceration.15

Reflectance confocal microscopyReflectance confocal microscopy uses infrared laser light to non-

invasively examine skin in vivo in real time without the use of

fluorescence, dyes or stains. The contrast observed in the RCM

image correlates to naturally occurring variations in the refrac-

tive index of organelles and micro-structures in the skin. RCM

Figure 2 Typical ‘strawberry pattern’ identified by dermoscopy ofskin from a patient with early actinic keratosis. Dermoscopyreveals a characteristic red pseudonetwork outlined by erythemainterrupted by hair follicles (magnification of 20x).

Figure 3 Comparison of images of reflectance confocal microscopy (Vivascope 1500) of subclinical actinic keratosis (left) and normalskin (right) of the same site in a patient before and after treatment. The image shows a field of 1.5 mm of the spinous layer of the epider-mis. On the left side, the changes associated with subclinical actinic keratosis are atypical honeycomb with irregularity in the size andshape of corneocytes. In contrast the confocal image on the right shows a typical honeycomb.

© 2014 European Academy of Dermatology and VenereologyJEADV 2015, 29 (Suppl. 1), 3–8

New imaging techniques for AK 5

images allow the visualization of cellular and subcellular struc-

tures in the skin, similar to the detail seen in histology sections,

but without the need for an invasive biopsy procedure.16 RCM

has been used in the diagnosis of several dermatological condi-

tions including melanoma and basal cell carcinoma.16

Reflectance confocal microscopy enables the histological

changes that characterize AK to be imaged and so can be used in

the diagnosis of this disease.6,17 RCM also enables the initial

changes in epidermal morphology and cellular atypia to be visual-

ized before the disease becomes clinically apparent and is there-

fore useful for the evaluation of AK field cancerization and the

detection of subclinical AK (Fig. 3).18 The main RCM features of

clinical AK lesions are: disruption and parakeratosis in the stra-

tum corneum; pleomorphism and architectural disruption in the

stratum granulosum and stratum spinosum with loss of the nor-

mal honeycomb pattern; and solar elastosis and increased vascu-

larity or blood vessel dilatation in the superficial dermis.6,16–18 Of

these features, cellular pleomorphism and architectural disarray

are considered the best predictors of AK.6 The principal RCM fea-

tures of subclinical AK lesions are pleomorphism and architec-

tural disruption in the stratum spinosum (Fig. 3), but not in the

stratum granulosum, and solar elastosis.18

Reflectance confocal microscopy can also be used in the dif-

ferential diagnosis of AK and SCC, which is important since the

two types of lesions require different therapeutic approaches. A

study which compared RCM images of AK lesions and SCC

demonstrated that SCC shows an increased frequency of abnor-

mal RCM features compared with AK.19 The key RCM features

of SCC are the presence of an atypical honeycomb pattern and

round nucleated cells corresponding to atypical keratinocytes in

the stratum spinosum and stratum granulosum, and round

blood vessels crossing the dermal papilla. These blood vessels are

scarcer in AK since these lesions are smaller and less developed

neoplasms.19

Reflectance confocal microscopy allows the effects of AK

treatments such as imiquimod or diclofenac sodium to be moni-

tored by non-invasively examining subclinical and clinical

lesions in vivo over time.18,20–22 An RCM investigation into the

effects of imiquimod on actinic field cancerization demonstrated

that this field-directed treatment unmasks previously invisible

subclinical lesions and is able to effectively treat both clinical

and subclinical lesions. Furthermore, RCM can detect any resid-

ual subclinical disease remaining after initial treatment with im-

iquimod and so can identify patients who may benefit from

additional cycles of therapy.18 After 2 weeks of imiquimod treat-

ment, RCM imaging of clinical and subclinical lesions revealed

the presence of inflammatory cells in the dermis and epidermis,

which correspond to the immunomodulatory response induced

by this treatment. Four weeks after treatment with imiquimod

was completed, RCM imaging demonstrated that clinical and

subclinical lesions had been cleared, e.g. by revealing that the

typical honeycomb pattern at the level of stratum granulosum

and spinosum had been re-established.18 Similarly, RCM imag-

ing revealed that 2 weeks of treatment with diclofenac sodium

resulted in a significant decrease in scaling and in the atypical

honeycomb pattern in AK patients.20 RCM has also been used to

non-invasively diagnose actinic cheilitis (AK on the lip) and to

visualize the response to treatment with diclofenac sodium by

demonstrating the absence of cellular atypia and rearrangement

of the epidermal architecture.21

High-definition optical coherence tomographyOptical coherence tomography (OCT) is a non-invasive imaging

technique that has been used to diagnose and monitor a variety

Figure 4 High-definition optical coherencetomography slice imaging of normal skin(top panel) and skin from a patient withactinic keratosis (lower panel) (Skintell,AGFA) (field of 1 mm in diameter). In theimage of normal skin, the epidermis isregular with normal thickness and thedermo-epidermal junction is clear and has atypical shape. In contrast, in the image ofthe patient with actinic keratosis, thethickness of the epidermis is irregular. Thestratum corneum is thickened with anincrease in contrast due to keratin and thedermo-epidermal junction is flat. Epid,epidermis; DEJ, dermo-epidermal junction.

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6 Malvehy

Dermoscopic features in AK and pigmented AK

Dermoscopic features in LMM

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