A Randomized, Vehicle-Controlled Phase 3 Study of Aminolevulinic Acid Photodynamic Therapy for the Treatment of Actinic Keratoses on the Upper Extremities

Home , Aminolevulinic acid, Tretinoin

Shang I. Brian Jiang, MD,* Steven Kempers, MD,† Phoebe Rich, MD,‡ Stuart Marcus, MD, PhD,x Anna Houlihan, MA,║ Daniel Piacquadio, MD,¶ and David Pariser, MD**

BACKGROUND Blue-light aminolevulinic acid photodynamic therapy (ALA-PDT) after broad-area application and 3-hour incubation is efficacious for actinic keratosis (AK) lesion clearance on upper extremities, with use of occlusive dressing significantly increasing efficacy.

OBJECTIVE To prove the safety and efﬁcacy of ALA-PDT versus vehicle (VEH-PDT) in the spot treatment of multiple AKs on upper extremities.

METHODS Aminolevulinic acid or VEH was spot applied only to lesions on one upper extremity 3 hours before blue-light exposure. Treated extremity was covered with occlusive dressing during incubation. Identical treatment was repeated at Week 8 if AK lesions were present in the treated area.

RESULTS Thirty-one percent (42/135) of subjects treated with ALA-PDT had complete clearance at Week 12, compared with 13% (17/134) of the subjects treated with VEH-PDT (p = .0001). The mean AK lesion clearance rate for ALA-treated subjects at Weeks 8 and 12 was 53% and 69%, respectively, compared with 26% and 30% for the VEH-treated group (p < .0001, linear mixed model). Safety proﬁle observed in this study is consistent with previous studies/reports in the literature, and the therapy was well tolerated overall.

CONCLUSION Aminolevulinic acid-PDT spot treatment using a 3-hour occluded incubation was superior to VEH-PDT for AK lesion clearance of the upper extremity.

Supported by DUSA Pharmaceuticals, Inc. The authors have indicated no signiﬁcant interest with commercial supporters. S.I. Brian Jiang, S. Kempers, P. Rich, and D. Pariser were investigators on the actinic keratosis study presented in this article; S. Marcus and A. Houlihan were employed by DUSA Pharmaceuticals, Inc.; D. Piacquadio is employed by Therapeutics, Inc., the company providing CRO services.

ctinic keratoses (AKs) are dysplastic epidermal potential to progress to squamous cell carcinoma Alesions that occur in fair-skinned individuals who (SCC). The results of a published study suggest that have been chronically exposed to sunlight. AKs are AKs may give rise to nonmelanoma skin cancers at a usually found on sun-exposed areas such as the face, greater rate than previously acknowledged.1 Criscione bald scalp, forearms, and backs of hands. Individual and colleagues found, based on following 7,784 AKs are identiﬁed as scaly, rough lesions that may individual AKs on the face and ears of 169 disappear and reappear over months or years. AKs are participants, that the risk of progression of AK to considered to be precancerous lesions with the primary SCC was 0.6% at 1 year and 2.57% at 4

*Mohs and Dermatologic Surgery, University of California, San Diego, San Diego, California; †Department of Dermatology, Minnesota Clinical Study Center, University of Minnesota, Fridley, Minnesota; ‡Department of Dermatology, Oregon Dermatology and Research Center, Oregon Health and Science University, Portland, Oregon; xDUSA Pharmaceuticals, Inc., Wilmington, Massachusetts; ║Clinical Research, DUSA Pharmaceuticals, Inc., Wilmington, Massachusetts; ¶Therapeutics, Inc., San Diego, California; **Department of Dermatology, Eastern Virginia Medical School, Virginia Clinical Research, Inc., Norfolk, Virginia

© 2019 by the American Society for Dermatologic Surgery, Inc. Published by Wolters Kluwer Health, Inc. All rights reserved. ISSN: 1076-0512 · Dermatol Surg 2019;00:1–8 · DOI: 10.1097/DSS.0000000000001760

years; in addition, approximately 65% of all primary 4 weeks after the second treatment were 58.4% 6 SCCs diagnosed during the trial arose in lesions that 22.2% and 24.8% 6 20.6% for the ALA and VEH had earlier been clinically diagnosed as AKs. The sides, respectively. The mean lesion count reduction cellular damage and atypia observed at the histologic for the ALA-treated side was significantly greater than level of AKs is similar to that of surrounding the mean reduction for the VEH-treated side nonlesional skin,2,3 suggesting that skin surrounding (p = .0004). AKs may also be at increased risk of skin cancer. Schmieder and colleagues12 examined the effect of Photodynamic therapy (PDT) using a 20% topical occlusion on ALA-PDT treatment to AKs of the upper solution of aminolevulinic acid-HCl (ALA, Levulan extremities. Seventy subjects were treated with ALA- Kerastick) and activation by 10 J/cm2 blue light (Blue PDT or VEH-PDT using broad-area application and a Light Photodynamic Therapy Illuminator, BLU-U) 3-hour incubation period followed by 10 J/cm2 of blue delivered at 10 mW/cm2 is FDA approved for the spot light; one extremity of each subject was covered with treatment of minimally to moderately thick AKs of the occlusive dressing during the incubation period. The face or scalp after a 14- to 18-hour incubation period median AK lesion clearance rate at Week 12 was (drug-light interval), in the United States and Canada, 88.7% for extremities treated with occluded ALA and the spot treatment of minimally to moderately (ALA + OCC), 70.0% for extremities treated with thick AKs of the upper extremities after a 3-hour nonoccluded ALA, 16.7% for extremities treated with incubation period in the United States. occluded VEH (VEH + OCC), and 5.6% for extremities treated with nonoccluded VEH (p < .0001). When ALA is applied to dysplastic lesions, it is ALA + OCC resulted in a significantly higher lesion absorbed by cells and preferentially metabolized to clearance rate compared with the nonoccluded protoporphyrin IX (PpIX) relative to normally divid- extremity at Week 8 (p = .0006) and Week 12 ing keratinocytes. When PpIX is activated by light of (p = .0029). Thirty-four percent (12/35) of extremities an appropriate wavelength and energy in the presence treated with ALA + OCC had complete clearance of of oxygen, it results in a cytotoxic process. There lesions at Week 12 compared with 0% (0/35) of is minimal damage to surrounding untreated skin, and extremities treated with VEH + OCC (p = .0002). cutaneous photosensitivity is transient (usually clear- ing within 24–48 hours) due to metabolic conversion The current study is designed to prove the safety and of the remaining PpIX to heme.4 efficacy of blue-light ALA-PDT in the treatment of multiple AKs on the upper extremities. ALA-PDT, when used on-label for the 14- to 18-hour incubation, has been shown to be safe and highly Methods effective in the treatment of minimally to moderately 5,6 thick AKs of the face and scalp. There are a few Subjects published studies using ALA and red light7–10 and ALA and blue light11–13 to treat AKs on the arms and hands. Eligible participants were male or nonpregnant, non- lactating females aged 18 years or older, with 4 to 15 Using a bilateral, vehicle-controlled study design, Grade 1 or 2 AKs on one upper extremity (dorsal Taub and Garretson11 treated 15 subjects with at least hand/forearm). Key exclusion criteria included: Grade 3 4 AKs on the dorsal hands/forearms with 2 blue-light and/or atypical AKs within the treatment area; lesions ALA-PDT treatments spaced 8 weeks apart. Amino- suspicious or proven for skin cancer within the treat- levulinic acid or placebo vehicle (VEH) was initially ment area; a history of cutaneous photosensitization; applied to AK lesions, followed by a second applica- any condition with associated immunosuppression; tion to the entire treatment area, occluded with plastic keratolytics including >5% urea, alpha hydroxy acids wrap, and allowed to incubate for 2 hours before blue- (AHAs) (e.g., glycolic acid and >5% lactic acid), >2% light treatment. The mean lesion count reductions salicylic acid within 2 days of initiation of treatment;

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cryotherapy within the previous 2 weeks; topical reti- Outcomes noids within the previous 4 weeks; microdermabrasion, Efficacy end points were complete (100%) clearance ablative laser, ALA-PDT, chemical peels, 5-fluorouracil, rate (CCR) at Weeks 8 and 12, AK lesion clearance diclofenac, imiquimod within the previous 8 weeks; and rate (AKCR) at Weeks 8 and 12 compared with systemic retinoids within the previous 6 months. baseline, and subject satisfaction and acceptability of treatment. Subjects were considered to have complete Procedure clearance if there were no AK lesions present in the Subjects were randomized to receive either ALA or treated area. Tolerability assessments, including ery- VEH as a spot application to AK lesions on one upper thema, edema, scaling and dryness, extremity. The treatment area was defined as the oozing/vesiculation/crusting, and changes in pigmen- dorsal extensor surface of the hand and forearm tation were assessed at each visit using a five-point between the elbow and the base of the fingers (the scale (0 = none to 4 = severe); and stinging/burning was fingers were not included in the treatment area). Before assessed using a four-point scale (0 = none to 3 = application of study solution, the skin was cleansed severe). The evaluating investigator for efficacy was with a mild cleanser, followed by an alcohol wipe. blinded to the treatment assignment. To maintain Two applications of solution were applied to AK blinding, personnel who were not involved in efficacy lesions on the treatment area, and the treatment area assessments administered PDT and assessed for tol- was then covered with an occlusive, polyethylene film, erability to treatment. Subjects were instructed not to held in place with elastic netting during the 3-hour discuss their treatment with the blinded evaluator, and incubation period. A cotton glove and long sleeves blinded/unblinded evaluators were instructed to avoid (from a shirt or other garment) were worn to block discussion of subjects. ambient light. Blue light (BLU-U) delivered at a power Statistical Analysis density of 10 mW/cm2 at the skin surface was administered to the treatment area for 16 minutes, 40 All data tabulations were performed using the SAS seconds for a total fluence of 10 J/cm2. Subjects were System for Windows (version 9.4; SAS Institute Inc., positioned with their upper extremity resting on a Cary, NC). The primary subject set for analysis of the Mayo table or other suitable stand, with the palm efficacy data is the intent-to-treat (ITT) population. facing downward within the BLU-U. Using a height The primary efficacy end point is the CCR at Week 12. adjustable table and chair, the heights were set to The key secondary efficacy end points are AKCR at ensure that the extensor surface of the forearm was Weeks 8 and 12, and the CCR at Week 8. Subjects who between 2$ and 4$ from the BLU-U surface. Follow- do not provide data are considered to be non- up was done at 24 hours and 2, 4, 8, and 12 weeks after responders. Using a two-sided test at the alpha = 0.05 initial treatment. A second treatment was given at level of significance, the CCRs in the 2 treatment Week 8 if the treatment area contained any AKs. groups were compared using the Cochran–Mantel– Subjects were instructed to prevent exposure of the Haenszel test stratified by analysis center. The treated skin to direct sunlight or bright indoor light for Breslow–Day test with a significance level of 0.1 was at least 40 hours after each treatment. used to test for treatment by analysis center interaction. A hierarchical (“gatekeeping”) testing pro- The study was conducted in accordance with the eth- cedure14 was used to control the overall level of ical principles of the 1975 Declaration of Helsinki and significance for the analyses of the primary and key in compliance with Good Clinical Practice Guidelines. secondary end points. The analysis of AKCR was The protocol was approved by an institutional review performed using a linear mixed model with fixed board for each study center. Written informed consent effects for treatment group (2 levels), time point (3 was obtained from all subjects. The study was regis- levels, Weeks 4, 8, and 12), and the treatment group by tered on ClinicalTrials.gov (Identifier: time point interaction. The unstructured covariance NCT02137785). model was used. The treatment group comparisons at

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Week 12 and at Week 8 were tested using the results of compared with 13% (17/134) of the VEH-treated this model. All available data were used, with no subjects (p = .0001). imputation for missing data. At the final visit, subjects rated their overall satisfaction with improvement of the appearance of the area Results treated on a 4-point scale (0 = none/worse, 3 = excel- Demographics and Baseline lent). For overall satisfaction with response to treat- Disease Characteristics ment, 88% (118/134) of subjects treated with ALA- PDT were very or moderately satisfied with response Two hundred sixty-nine subjects (188 men, 81 to treatment, compared to 42% (55/131) of subjects women, mean age 68 years [range 45–90 years], with treated with VEH-PDT. Fitzpatrick skin Type I [12%], II [52%], III [26%], IV [10%]) were enrolled into the study at 14 sites and are Tolerability/Safety Results included in the efficacy analysis (ITT population). Subjects tolerated treatments well; all subjects received Ninety-seven percent (262/269) of the subjects com- full light dose with no subject requesting early termi- pleted the study. nation of light treatment. Stinging/burning during Efficacy Results light treatment was reported for 93% of ALA-treated subjects, compared with 17% of subjects treated with Table 1 summarizes the absolute AK count at baseline VEH (Table 3). For ALA-treated subjects, and the mean AKCR at Weeks 8 and 12; mean AKCR stinging/burning during light treatment was rated as is presented graphically in Figure 1. The ALA Week 12 moderate or severe for 64% of subjects (Table 4). response rates include 70% (95/135) of subjects who received a second treatment. The mean AKCR at Week The incidence of erythema increased over baseline 12 was 69.1 (637.4)% for ALA and 29.9 (651.5)% levels in both treatment groups immediately after light for VEH (p < .0001). treatment and seemed to be more frequent in the ALA- treated subjects than in the VEH-treated subjects Table 2 shows the number of subjects with AK com- (Table 3; 91% vs. 58%). The number of subjects with plete clearance (no lesions present) at Weeks 8 and 12. erythema returned to baseline levels for the VEH Thirty-one percent (42/135) of subjects treated with group at Week 4 and the ALA group at Week 8, ALA had complete clearance of lesions at Week 12, although the percentage of ALA-treated subjects with

TABLE 1. Actinic Keratosis Lesion Clearance Rate at Weeks 8 and 12

ALA-PDT (N = 135) VEH-PDT (N = 134) Baseline AK count Mean (SD) 8.5 (3.6) 8.6 (3.4) Range 4–15 4–15 AKCR Week 8, N 133 129 Mean (SD) 53.4* (45.0) 26.3 (44.3) Median† (SD) 60.0 (45.0) 16.7 (44.3) Week 12, N 132 130 Mean (SD) 69.1* (37.4) 29.9 (51.5) Median† (SD) 80.9 (37.4) 21.8 (51.5)

*p < .0001 compared with VEH; based on a linear mixed model with ﬁxed effects for treatment group, time point, and treatment group by time point interaction. †Median given for comparison with the study by Schmieder and colleagues12. ALA-PDT, aminolevulinic acid photodynamic therapy; VEH-PDT, vehicle photodynamic therapy.

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oozing/vesiculation/crusting at that time point. By Week 4, incidence of oozing/vesiculation/crusting in the ALA group approached near-baseline levels.

There were no serious adverse events related to the drug reported during the study. One adverse event (application site pain) was judged as related to treatment in VEH-treated subjects.

Two ALA-PDT–treated subjects and one VEH-PDT– treated subject, all with previous history of SCC, developed SCCs on their treated extremities during the post-treatment period of this study.

Discussion

The results of this Phase 3, multicenter, vehicle- controlled, randomized, evaluator-blinded study using spot application conﬁrm the results of previous smaller studies, which tended to use broad-area ALA Figure 1. Mean AK lesion clearance rate by visit. ALA-PDT, application11–13 showing that blue-light ALA-PDT can aminolevulinic acid photodynamic therapy; VEH-PDT, be used to successfully treat AKs of the upper vehicle photodynamic therapy. extremities. Up to 2 ALA-PDT treatments to lesions on moderate or severe erythema declined from 51% on the full forearm using occlusion were able to clear 69% Day 2 to 3, to 7% at Week 2 (Table 4). Edema was of baseline AK lesions, compared with 30% for the observed after each light treatment and at Day 2 to 3 in VEH group (Table 1 and Figure 1). 30%-40% of ALA subjects and essentially resolved 2 weeks after each treatment. ALA-treated subjects The complete clearance rate of 31% (Table 2) and the exhibited an increase in incidence of scaling and dry- median AK lesion clearance rate of 81% (Table 1 and ness to 76% of subjects at Week 2 compared with Day Figure 2) observed at Week 12 in this study are similar 1 incidence of 39%, which resolved to near-baseline to those reported in the study by Schmieder and col- levels by Week 4 (Table 3). The majority of scaling and leagues (CCR 34%, median AKCR 89%) on the upper dryness at Week 2 was reported to be minimal or mild extremities.12 These 2 studies used identical treatment (Table 4). The percentage of subjects with methods, with the exception of ALA being applied to oozing/vesiculation/crusting gradually increased in individual lesions in the current study, whereas broad the ALA group after treatment, peaking at Week 2 area application to the entire extremity was used by with 32% (43/135) of subjects noted to have Schmieder and colleagues.

TABLE 2. Subject Complete Clearance Rates at Weeks 8 and 12

ALA-PDT (N = 135) VEH-PDT (N = 134) Subjects with complete clearance (100% lesion removal) Week 8, N (%) 35 (25.9)* 12 (9.0) Week 12, N (%) 42 (31.1)* 17 (12.7)

p-value based on the Cochran–Mantel–Haenszel test stratiﬁed by analysis center. *p = .0001 compared with VEH. ALA-PDT, aminolevulinic acid photodynamic therapy; VEH-PDT, vehicle photodynamic therapy.

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The safety proﬁle of treatment of AKs of the degree of tolerability for the treatment and satisfac- extremities with ALA-PDT is generally similar to that tion with its outcome. Although efﬁcacy in this study which has already been reported for treatment of the was similar to the previous Phase 2 study,12 some of approved indication areas of the face and scalp, the side effects were reported to be less frequent which is performed in a nonoccluded method,4 and and/or less severe than ALA + OCC in the earlier that of the Phase 2 extremity study by Schmieder and study. The stinging/burning sensation felt during colleagues12. Subjects in this study showed a high blue-light PDT activation was reported by a lower

TABLE 3. Percentage of Subjects With Acute Safety Symptoms at Speciﬁc Follow-up Visits by Treatment Group

ALA-PDT No. (%) VEH-PDT No. (%) Erythema Baseline 62/135 (45.9) 61/134 (45.5) Post-PDT 123/135 (91.0) 77/134 (57.5) Day 2–3 133/135 (98.5) 70/134 (52.2) Week 2 107/135 (79.3) 73/134 (54.5) Week 4 81/135 (60.0) 60/132 (45.5) Week 8 53/132 (40.2) 48/129 (37.2) Week 12 52/132 (39.4) 45/130 (34.6) Edema Baseline 2/135 (1.5) 0/134 (0.0) Post PDT 43/135 (31.9) 7/134 (5.2) Day 2–3 46/135 (34.1) 1/134 (0.7) Week 2 4/135 (3.0) 0/134 (0.0) Week 4 1/135 (0.7) 0/132 (0.0) Week 8 1/132 (0.8) 1/129 (0.8) Week 12 0/132 (0.0) 0/130 (0.0) Stinging/Burning Baseline 1/135 (0.7) 1/134 (0.7) During PDT 125/135 (92.6) 23/134 (17.2) Post-PDT 105/135 (77.8) 4/134 (3.0) Day 2–3 28/135 (20.7) 2/134 (1.5) Week 2 4/135 (3.0) 0/134 (0.0) Week 4 3/135 (2.2) 0/132 (0.0) Week 8 2/132 (1.5) 0/129 (0.0) Week 12 2/132 (1.5) 0/130 (0.0) Scaling and dryness Baseline 52/135 (38.5) 56/134 (41.8) Day 2–3 51/135 (37.8) 55/134 (41.0) Week 2 102/135 (75.6) 53/134 (39.6) Week 4 62/135 (45.9) 49/132 (37.1) Week 8 49/132 (37.1) 43/129 (33.3) Week 12 47/132 (35.6) 44/130 (33.8) Oozing, vesiculation, crust Baseline 7/135 (5.2) 5/134 (3.7) Day 2–3 19/135 (14.1) 7/134 (5.1) Week 2 43/135 (31.9) 5/134 (2.3) Week 4 14/135 (10.4) 3/132 (2.3) Week 8 7/132 (5.3) 5/129 (3.9) Week 12 9/132 (6.8) 5/130 (3.8)

ALA-PDT, aminolevulinic acid photodynamic therapy; VEH-PDT, vehicle photodynamic therapy.

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TABLE 4. Erythema, Stinging/Burning, and Scaling and Dryness by Severity and Treatment Group at Acute Time Points

ALA-PDT No. (%) VEH-PDT No. (%) Erythema Day 2–3 133/135 (98.5) 70/134 (52.2) None/minimal/mild 66/135 (48.9) 130/134 (97.0) Moderate/severe 69/135 (51.1) 4/134 (3.0) Week 2 107/135 (79.3) 73/134 (54.5) None/minimal/mild 125/135 (92.6) 133/134 (99.3) Moderate/severe 10/135 (7.4) 1/134 (0.7) Stinging/burning During PDT 125/135 (92.6) 23/134 (17.2) None/minimal 49/135 (36.3) 134/134 (100.0) Moderate/severe 86/135 (63.7) 0/134 (0.0) After PDT 105/135 (77.8) 4/134 (3.0) None/minimal 100/135 (74.1) 134/134 (100.0) Moderate/severe 35/135 (25.9) 0/134 (0.0) Scaling and dryness Week 2 102/135 (75.6) 53/134 (39.6) None/minimal/mild 109/135 (80.7) 130/134 (97.0) Moderate/severe 26/135 (19.3) 4/134 (3.0)

ALA-PDT, aminolevulinic acid photodynamic therapy; VEH-PDT, vehicle photodynamic therapy.

percentage of subjects in the current study, with 64% of subjects reporting moderate to severe stinging/burning, compared with 86% in the previous study. Erythema also was noted to be slightly less frequent, less severe, and resolved quicker in the current study.

Two patients developed SCCs on their ALA-treated extremities during the post-treatment period of this study. These events are consistent with results previously observed in the study by Schmieder and col- leagues12 and a previous Phase 4 study on treatment of AKs of the face and scalp.6,15 In this study, all patients who developed SCCs also had a history of multiple SCC excisions. A possible explanation for this ﬁnding is that these lesions, which were initially designated AKs on clinical examination, were actually early SCCs (this was found to be the case for 5% of the lesions identiﬁed as AKs at baseline in the Phase 4 study15) with a deep component that persisted through PDT as occult lesions and then became visible during the follow-up period. Figure 2. Median AK lesion clearance rate by visit (for In conclusion, results from this study indicate that comparison with the study by Schmieder and colleagues). ALA-PDT, aminolevulinic acid photodynamic therapy; blue-light ALA-PDT after a 3-hour incubation, using VEH-PDT, vehicle photodynamic therapy.

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occlusive wrap, seems tolerable and efﬁcacious for AK 7. Szeimies RM, Karrer S, Sauerwald A, Landthaler M. Photodynamic therapy with topical application of 5-aminolevulinic acid in the lesion clearance of the upper extremities. treatment of actinic keratoses: an initial clinical study. Dermatology 1996;192:246–51. Acknowledgments The authors thank the patients 8. Jeffes EW, McCullough JL, Weinstein GD, Fergin PE, et al. Photodynamic therapy of actinic keratosis with topical 5- and investigators who participated in this study. The aminolevulinic acid: a pilot dose-ranging study. Arch Dermatol 1997; authors also thank Mary Beth Ferdon for her 133:727–32. contribution to statistical analysis and Michael 9. Kurwa HA, Yong-Gee SA, Seed PT, Markey AC, et al. A randomized paired comparison of photodynamic therapy and topical 5-ﬂuorouracil Guttadauro for leading project management in the treatment of actinic keratoses. J Am Acad Dermatol 1999;41(3 Pt activities for this study. Administrative support for 1):414–8. manuscript submission was provided by Denise 10. Sotiriou E, Apalla Z, Maliamani F, Zaparas N, et al. Intraindividual Freeman of Therapeutics, Inc. and was funded by left-right comparison of topical 5-aminolevulinic acid and photodynamic therapy vs 5% imiquimod cream for actinic keratoses on Sun Pharmaceutical Industries, Inc. the upper extremities. J Eur Acad Derm Venereol 2009;23:1061–5.

11. Taub AF, Garretson CB. A randomized, blinded, bilateral intraindividual, vehicle-controlled trial of the use of photodynamic References therapy with 5-aminolevulinic acid and blue light for the treatment of actinic keratoses of the upper extremities. J Drugs Dermatol 2011;10: 1. Criscione VD, Weinstock MA, Naylor MF, Luque C, et al. Actinic 1049–56. keratoses: natural history and risk of malignant transformation in the veterans affairs topical tretinoin chemoprevention trial. Cancer 2009; 12. Schmieder G, Huang E, Jarratt M. A multicenter, randomized, vehicle- 116:2523–30. controlled phase 2 study of blue light photodynamic therapy with aminolevulinic acid HCl 20% topical solution for the treatment of 2. Jonason AS, Kunala S, Price GJ, Restifo RJ, et al. Frequent clones of actinic keratoses on the upper extremities: the effect of occlusion during p53-mutated keratinocytes in normal human skin. Proc Natl Acad Sci the drug incubation period. J Drugs Dermatol 2012;11:1483–9. USA 1996;93:14205–9. 13. Galitzer B. Effect of retinoid pretreatment on outcomes of patients 3. Stahl PL, Stranneheim H, Asplund A, Berglund L, et al. Sun-induced treated by photodynamic therapy for actinic keratosis of the hand and nonsynonymous p53 mutations are extensively accumulated and tolerated forearm. J Drugs Dermatol 2011;10:1124–32. in normal appearing human skin. J Invest Dermatol 2011;131:504–8. 14. Westfall PH, Krishen A. Optimally weighted, ﬁxed sequence and 4. ALA Kerastick (Aminolevulinic Acid HCl) for Topical Solution, 20% gatekeeper multiple testing procedures. J Stat Plann Inference 2001;99: [package insert]. Wilmington, MA: DUSA Pharmaceuticals; 2010. 25–40.

5. Piacquadio DJ, Chen DM, Farber HF, Fowler JF, et al. Photodynamic 15. Ehrig T, Cockerell C, Piacquadio D, Dromgoole S. Actinic keratoses therapy with aminolevulinic acid topical solution and visible blue light and the incidence of occult squamous cell carcinoma: a clinico- in the treatment of multiple actinic keratoses of the face and scalp: histopathologic correlation. Dermatol Surg 2006;32:1261–5. investigator-blinded, phase 3, multicenter trials. Arch Dermatol 2004; 140:41–6.

6. Tschen EH, Wong DS, Pariser DM, Dunlap F, et al. Photodynamic Address correspondence and reprint requests to: Daniel therapy using aminolaevulinic acid for patients with nonhyperkeratotic Piacquadio, MD, Therapeutics, Inc., 9025 Balboa Avenue, actinic keratoses of the face and scalp: phase IV multicentre clinical trial Suite 100, San Diego, CA 92123, or e-mail: with 12-month follow up. Br J Dermatol 2006;155:1262–9. [email protected]

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