Photochemotherapy (PUVA) has been used for decades with great success and at a constantly increasing rate in the management of psoriasis 1. Combining PUVA with systemic retinoids (Re-PUVA) has been shown to be another effective treatment modality for psoriasis 2. Because of several advantages to both patients and physicians, PUVA remains an important therapeutic option not only for psoriasis but also for several other skin diseases 1.
As UV rays play an important role in the development of skin cancer, clinicians have been concerned about the possible carcinogenic effects of UV-based phototherapy 3. UVA (320–400 nm) penetrates the skin deeply, causing oxidative damage to nucleic acids, membrane lipids, and cell proteins through the production of reactive oxygen species (ROS) 4. These ROS interrupt normal cellular transduction pathways and cell–cell signaling, causing altered proliferation 5.
Guanine has the lowest oxidation potential among DNA bases, rendering it the most easily oxidizable nucleic acid base and hence the most abundant and well-characterized DNA lesion generated by ROS 6. 8-Oxoguanine (8-oxoG) is among the most mutagenic oxidative DNA modifications that induce replication errors and interfere with transcription 7. If DNA replication occurs before the repair of the 8-oxoG lesion, replicative DNA polymerases frequently misincorporate adenine opposite 8-oxoG 8.
In contrast, extensive preclinical, epidemiological, and early clinical evidence for the beneficial use of retinoids in cancer treatment and prevention is already available. Acitretin was reported to exhibit antitumour activity in actinic keratosis, verrucous carcinomas, and in squamous cell carcinoma (SCC), without significant side effects or toxicity 9.
Accordingly, this study was carried out to compare the possible carcinogenic and/or protective effects of PUVA versus Re-PUVA through measurement of serum levels of 8-oxoG before and after therapy.
Patients and methods
This study is a prospective, randomized, controlled study in which 20 patients with psoriasis were recruited from the Dermatology Outpatient Clinic of Kasr Al-Aini University Hospital between March and September 2015.
Inclusion and exclusion criteria
Patients of all ages and of both sexes were recruited; however, priority was given to men. For women to be included in the study, they should have been married and using at least two methods of contraception (intrauterine device should be one of them).
Patients receiving any systemic treatment for psoriasis in the last 3 months were excluded from the study. Further, patients known to have contraindications to either PUVA therapy (e.g. hepatic patients, cataract, photosensitivity, previous skin cancer) or retinoids (e.g. pregnancy, hyperlipidemia, hepatic patients) were also excluded.
The 20 patients were then randomly divided into two groups: group A (PUVA) and group B (Re-PUVA). Each of the 20 patients of both groups received 30 sessions of PUVA photochemotherapy. Patients of group B received additional oral retinoids 2 weeks before the start of session, which continued until the end of the PUVA sessions.
The study was approved by the Dermatology Research Ethics Committee, which is approved by the Research Ethics Committee of the Faculty of Medicine, Cairo University. Written informed consent was taken from each patient before participation in this study. Thorough history taking was taken from each patient, including name, age, weight (to determine the dose of 8-methoxypsoralen if receiving PUVA), occupation (sun exposed or not), and any special habits of medical importance. Skin examination was performed to determine the distribution and clinical variant of the disease, skin type, and percentage of body involvement using the rule of 9 9.
Before initiation of photochemotherapy, ophthalmologic examination using a slit lamp was performed to exclude lens opacity. Full liver profile was determined for all patients to exclude any possible hepatic affection, which, if present, would exclude the patient from the study. Although the married women included in the study were already using two methods of contraception, a pregnancy test was conducted as an additional precautionary step.
Psoriasis Area Severity Index (PASI) score was determined for all patients with psoriasis to assess the severity of psoriasis. It included assessment of erythema, infiltration, desquamation, and extent of the disease 10.
In both groups A and B, PUVA was delivered by UVA cabins (PUVA 7001 and PUVA 1000; Herbert Waldmann GmbH & Co. KG, Villingen-Schwenningen, Germany) with F85/100 W fluorescent lamps (40 and 26 lamps, respectively) that emitted UV light in the wavelength of 315–400 nm with a peak emission at 365 nm, with an integrated UV photometer. Psoralen was given 2 h before sessions at a dose of 0.5–0.7 mg/kg of 8-methoxypsoralen. The starting dose and subsequent increments were skin-type dependent. The dose was increased every other PUVA and Re-PUVA session until improvement in PASI occurred, and then the dose was maintained. Each patient received 30 sessions, at a rate of three sessions every alternate day each week.
In group B (Re-PUVA group), systemic retinoids were given in the form of 25–50 mg acitretin/day, and they were given 2 weeks before the start of the session and continued until the 30th session of PUVA.
A volume of 3 ml of blood was taken from each of the 20 patients before initiation of phototherapy or retinoid therapy and after the last PUVA session (session 30). The samples were used to measure 8-oxoG levels before and immediately after the last PUVA and Re-PUVA sessions.
A measure of 3 ml of blood was collected and serum was separated and stored at a temperature of −80°C until use. The DNA of 8-oxoG was measured by enzyme-linked immunosorbent assay before and after photo(chemo)therapy.
The serum samples were used without dilution. 8-oxoG is an oxidized derivative of deoxyguanosine and one of the major products of DNA oxidation.
A standard blank, or sample of 50 μl was added per well. Immediately 50 μl of detection A working solution was added to each well. The plate was gently tapped to ensure thorough mixing and incubated for 1 h at 37°C. Each well was aspirated and washed. The process was repeated three times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, any remaining wash buffer was removed by aspirating or decanting. The plate was inverted and blotted against clean paper towels. A detection reagent B working solution of 100 μl was added to each well and incubated for 45 min at 37°C. The aspiration/wash process was repeated five times as done in step 3. A substrate solution of 90 μl was added to each well, and incubated for 15–30 min at 37°C with protection from light. A stop solution of 50 μl was added to each well. The optical density of each well was determined immediately using a microplate reader at 450 nm.
Calculation of results
A standard curve was created by reducing the data using computer software capable of generating a four-parameter logistic curve fit. The concentrations of 8-oxo-dG in serum (ng/ml) were determined from the standard curve.
Data were statistically described in terms of mean±SD, median and range, or frequencies (number of cases) and percentages when appropriate. Comparison of numerical variables between the study groups was done using the Mann–Whitney U-test for nonparametric samples. Within-group comparison of pretreatment and post-treatment numerical variables was performed using the Wilcoxon signed-rank test for paired (matched) samples. For comparing categorical data, the χ2-test was performed. The exact test was used when the expected frequency was less than 5. P values less than 0.05 were considered statistically significant. All statistical calculations were performed using statistical package for the social science (SPSS Inc., Chicago, Illinois, USA) release 15 for Microsoft Windows (2006).
This study included 20 patients with psoriasis vulgaris who were randomly divided, according to the type of therapy, into two groups – group A (PUVA) and group B (Re-PUVA) – using the sealed envelopes method.
Patients from both groups completed their scheduled sessions with no dropouts. The clinical data of the patients are presented in Table 1.
A significant drop in PASI score was detected in both groups receiving PUVA and Re-PUVA (Table 2). Although the drop in PASI score was greater in the Re-PUVA patients, the difference was found to be statistically nonsignificant (P=0.064) (Table 2). The percentage of reduction in PASI score was assessed and a higher percentage was detected in the Re-PUVA group; however, the difference was found to be statistically nonsignificant (P=0.082) (Table 2).
Part of the clinical evaluation in the current study was that of the onset of clinical response to therapy, which turned out to be significantly earlier in the Re-PUVA group (P=0.037) (Table 2). Predictably, the total cumulative dose of UVA at the end of sessions was found to be significantly lower in the Re-PUVA group (P=0.002) (Table 2).
Rise in the serum level of 8-oxoG level was noticed in psoriasis patients following their PUVA therapy; however, the increase was not significant (P=0.114) (Table 3). In contrast, a drop in the serum level of 8-oxoG was noticed following RE-PUVA therapy. On comparing the mean levels of 8-oxoG and the percentage of change following therapy in both groups, the difference was found to be statistically significant (0.023 and <0.005, respectively) (Table 3).
Both PUVA and Re-PUVA improved psoriasis, with lower post-treatment PASI score and higher percentage reduction in PASI in the Re-PUVA group; however, these differences were not significant. This result was consistent with that of Tanew et al.2, who observed complete or significant clinical improvement in 96 versus 80% of patients suffering from severe plaque-type psoriasis treated with Re-PUVA and PUVA, respectively. This could be attributed to the additional and synergistic effect of acitretin.
In addition, the onset of clinical response was significantly earlier (6.4±2.547 vs. 10.2±5.350) and accordingly the total cumulative UVA dose was significantly lower (66.81±25.8 vs. 109.13±14.31) with Re-PUVA than with PUVA therapy. In the current study, the dose was increased every other PUVA and Re-PUVA session until improvement in PASI occurred, and then the dose was maintained. The Re-PUVA group took shorter time and, consequently, lower total cumulative UVA dose to achieve improvement in PASI and thus in maintenance of the UVA dose. This comes in agreement with the results of Tanew et al.2, who showed that patients treated with Re-PUVA (n=23) needed approximately six fewer PUVA exposures to achieve clearance compared with patients treated with PUVA (n=25). A significantly lower total UVA dose was also found in the Re-PUVA group (58.7±17.9 vs. 101.5±15.8). Again these results were consistent with those of Magis et al.11, who demonstrated that Re-PUVA not only accelerates clinical remission by an average of 18 days but also decreases the cumulative dose of UVA by 30–50% as compared with PUVA alone. This could also be attributed to the additional and synergistic effect of acitretin given 2 weeks before beginning PUVA therapy in their study.
As regards serum 8-oxoG levels following therapy, it was elevated, but not significantly, following PUVA therapy. In contrast, Re-PUVA therapy was followed by both significant reduction in 8-oxoG level and a significantly higher percentage change in 8-oxoG level compared with the PUVA group.
In 1998, Cooke et al.12 examined 8-oxoG levels in both serum and urine in patients undergoing vitamin C supplementation, and both showed similar results; yet, serum levels showed significant changes earlier. A few years later, the same authors 13 recruited 14 volunteers and exposed them to a single suberythemal dose of UVA (15 J/cm2). Significant elevation of urinary 8-oxoG levels was detected at day 4, which returned to baseline at day 14.
UV induces both direct DNA damage, in the form of cyclobutane pyrimidine dimers, 6, 4-photoproducts and strand breaks, as well as indirect damage due to the production of damaging ROS. ROS, released by infiltrating inflammatory cells, including neutrophils and macrophages, and by activated epidermal keratinocytes, cause the formation of DNA adducts such as 8-oxoG 14.
Failure of repair mechanisms to properly deal with such a damage load has several detrimental consequences. The first is false pairing of 8-oxoG with adenine, resulting in increased frequency of replication errors 15. The second adverse effect of genomic 8-oxoG is erroneous bypass of the lesion by transcribing RNA polymerase II complexes, resulting in RNA mutagenesis and consequent production of aberrant proteins 16. Finally, 8-oxoG causes a powerful decrease in the transcriptional output of the damaged gene so that even a single lesion is sufficient to produce a significant effect 17.
In the current study, serum 8-oxoG was significantly reduced following Re-PUVA therapy when compared with its level following PUVA therapy, evidenced also by the highly significant percentage of change in its level (drop in that case) following Re-PUVA. The antitumor effect of acitretin may be mediated through the reduction of 8-oxoG formation, and the possible carcinogenic effect of PUVA may be canceled or even reduced with the addition of retinoids.
Systemic retinoids are known to have antiproliferative properties, to regulate the differentiation and growth of keratinocytes, to interfere with the process of tumor initiation, reduce regulation of proto-oncogenes, increase the expression of p53 and proapoptotic caspases, and to sensitize keratinocytes to apoptosis 18.
Retinoic acid-induced apoptosis is also associated with downregulation of Bcl-2 and survivin 19. Moreover, retinoic acid may be involved in extrinsic apoptotic signaling triggered by the death receptor Fas. Upon binding of Fas ligand, Fas aggregates to form a death-inducing signaling complex that contains an adapter protein and procaspase 8 20. The formation of the death-inducing signaling complex triggers the cleavage of caspase 8, initiating a caspase cascade 21.
Extensive preclinical, epidemiological, and early clinical evidence for the beneficial use of retinoids in cancer treatment and prevention is already available. Lebwohl et al.22 described a psoriatic patient who developed multiple SCCs after long-term PUVA therapy, and oral acitretin was administered to prevent the recurrence of skin cancer after surgical removal. Acitretin has also been shown to be effective in controlling and reducing actinic keratosis 23, and in preventing the development of SCC in organ transplant recipients 24,25.
Epidemiological data from around the world have demonstrated that exposure of skin to UV radiation is the main ecological reason for the development of both melanoma and nonmelanoma types of skin cancers. UV irradiation stimulates clonal expansion of aberrant skin cells, resulting in skin carcinogenesis through the involvement of multiple cellular signaling pathways 26. Several tumor suppressor genes and oncogenes have been reported to be activated by UV (p53, PTCH1, BRM, and Ras) and to be implicated in photocarcinogenesis 27.
There could be several explanations for the increased incidence of skin cancer in PUVA-treated patients. First, PUVA is mutagenic and carcinogenic and may itself induce skin cancer 28. Second, because PUVA treatment is immunosuppressive, it may permit the growth of skin cancers induced by other carcinogenic agents 29.
Re-PUVA was able to achieve the same clinical response as PUVA in psoriasis patients, but with an earlier onset of clinical response, lower UVA cumulative dose, less DNA damage in the serum, and hence a lower carcinogenic potential.
Conflicts of interest
There are no conflicts of interest.
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