Noninvasive fluorescence monitoring of protoporphyrin IX production in actinic keratosesfollowing short-contact application of 5-aminolevulinate

Christine B. Warren, MD, MS1; Brian W. Pogue, PhD&, Philip L. Bailin, MD*, and Edward V. Maytin, MD, PhD*#.

Abstract

Nonmelanoma skin cancers (NMSC) account for more than one

third of all adult cancers in the U.S. Photodynamic therapy (PDT)

is an attractive alternative to current surgical treatment options,

due to its improved cosmetic outcome. Aminolevulinic acid (ALA)-

PDT is currently FDA approved for treatment of actinic keratoses

(precancerous lesions) but not for NMSC. ALA is applied for 1 to

24 hours to lesions before exposing them to visible light. A

biological rationale for PDT using short-contact times (under 2

hours) remains an open question.

Objective: To study the kinetics of protoporphyrin IX (PpIX)

accumulation in actinic keratoses, a type of premalignant skin

lesion, under routine clinical conditions.

Hypothesis: The rate of PpIX accumulation in actinic keratoses

may differ widely among individuals.

1Department of Medicine, Georgetown University Hospital, Washington, DC; &Thayer School of Engineering, Dartmouth College, Hanover, NH;*Dermatology and Plastic Surgery Institute, Cleveland Clinic; #Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA

Introduction

Results

Summary Conclusions

Introduction: Topical 5-aminolevulinic acid (ALA) is widely used in photodynamic therapy of actinic keratoses (AK), a type of premalignant skin lesion. However, the optimal time between ALA application and exposure to light has not been carefully investigated.

Objectives: To study the kinetics of protoporphyrin IX (PpIX) accumulation in AK.

Patients/Methods: Using a noninvasive dosimeter, PpIX fluorescence was measured at 20 min intervals for 2 hr following ALA application, for 63 AK in twenty patients, . Data were analyzed for maximal fluorescent signal obtained, kinetic slope, relationships to erythema, and anatomic (regional) variation.

Results: PpIX accumulation was linear over time, becoming statistically higher than background in 48% of all lesions by 20 min, 92% of lesions by 1 hr, and 100% of lesions by 2 hr. PpIX accumulation was roughly correlated with changes in lesional erythema post- PDT. Also, PpIX in photodamaged, nonlesional skin was higher than in normal skin.

Conclusions: Significant amounts of PpIX are produced in nearly all AK lesions by 2 hr. The linear kinetics of accumulation suggest that shorter ALA application times may be efficacious in many patients. Fluorescence dosimetry of PpIX may be useful to delineate areas of precancerous changes in the skin.

Using a noninvasive dosimeter, PpIX fluorescence was measured at 20

min intervals for 2 hr following ALA application, for 63 AK in twenty

patients (mean 65.6 years;45% women). Measurements were also

obtained from 126 sites adjacent to AK lesions. ALA application was

followed by blue light therapy for 1000 sec to activate PpIX. Data were

analyzed for maximal fluorescent signal obtained, kinetic slope,

relationships to erythema, and anatomic (regional) variation.

• Fluorescence dosimeter measurements are useful in

determining the rate of PpIX production in AK

lesions.

• Maximal levels of PpIX attained in AK after 2 hr of

ALA on the skin, show wide variation (up to 10 fold),

with less variation among lesions in a given patient.

• PpIX accumulation is linear.

• PpIX signal reached 200% baseline in 46% of

lesions by 1 hr, although 23% failed to do so,

consistent with incomplete treatment response.

• Positive correlation between PpIX levels and overall

erythema status that was statistically significant.

• PpIX measurements after ALA application may help

detect precancerous lesions that are not apparent

clinically (field cancerization effect).

Fig 2.(a) Raw data for an AK lesion of Subject #113: PpIX signal as function of time after ALA. (b) Summary of all intralesional readings taken from facial AK (n=45), expressed relative to the max fluorescence, after background subtraction. PpIX was expressed as % of the max reading at 2 hr. (c) Box and whisker plot shows variability of max PpIX accumulation in 45 facial AK at 2 hr after ALA application. The PpIX signal expressed on the y-axis is the ratio of fluorescence readings taken

post-application and pre-application.

Fig. 3. Correlation between erythema and the maximal increase in PpIX fluorescence, at 2 hr after ALA application. The Erythema Combined Score on the x-axis was calculated for 60 AK lesions. Asterisk, significant difference from erythema "1" and from erythema "0" values; analysis of these data using ANOVA confirmed significant differences between the groups.

Fig.1.(a) Fluorescence dosimeter probe, (b) Close-up of probe tip, viewed end-

on. Central optical fiber carries excitation light (405 nm) to the skin, and the

peripheral optical fibers carry the fluorescent PpIX emissions back to the detector.

(c) Patient's forehead prior to illumination with blue light. Relative

locations of the clinical lesion (AK), the immediate perilesional zone (Peri), and a

distal region (Distant) that were selected for fluorescence measurements. (d)

Same forehead area following 2 hr of topical 5-ALA and a 1000-sec exposure to

blue light.

MethodsNoninvasive monitoring shows linear rates of PpIX accumulation in AK lesions and a wide variation in maximal amount of PpIX formed in each AK

Positive correlation between PpIX levels and overall erythema status

Fig. 4. Examples of regional patterns of PpIX-specific fluorescence. Dosimeter readings were taken at the intralesional, perilesional, and distant locations defined in Fig. 1c, at 2 hr after ALA application. Readings were plotted for all AK lesions, and examples were chosen here to illustrate the patterns (a, b, c) typically encountered. Gray zone, range of dosimeter signals in normal skin. PB, photobleached.

0.9949

Relationship between PpIX levels, photodamage, and anatomical distance from AK lesions

Georgetown University