TERT promoter mutations in actinic keratosis before and after treatment.

Abstract

Actinic keratoses (AK), intraepithelial skin neoplasms, are regarded as field precursors of squamous cell carcinoma (SCC).1 Five to ten percent of AKs progress to invasive SCC, with increased rate of progression in patients with multiple lesions.2 Due to the potential risk of progression to invasive SCC, treatment at the earliest stage is recommended. AK lesions are mostly confined to the basal layer of the epidermis and are less aggressive, characterized by parakeratotic and orthokeratotic hyperkeratosis.3 In contrast to AK, Morbus Bowen (MB; SCC in situ) is an intra-epidermal carcinoma with atypia of keratinocytes at every epidermal layer, characterized by parakeratotic and bulbous rete ridges.3 Treatment of AK primarily involves lesion-directed destruction or field treatment with topical medications. The efficacy of photodynamic therapy (PDT) in the treatment of localized and disseminated lesions has been reported in several studies.1, 4 AKs are associated with chronic sun-exposure and ultraviolet (UV)-induced mutations in the p53 gene occur before the clinical appearance and p53 expression serve as marker of UV-B damage.5 Mutations in other genes in the lesions have remained uninvestigated. We investigated the telomerase reverse transcriptase (TERT) promoter mutations, which create binding sites for ETS transcription factors, in AK lesions from 27 patients undergoing treatment with daylight-mediated PDT1, 4, 6, 7 (Fig. 1). In addition, we sequenced DNA from 20 MB tumors and 11 SCC tumors for mutations in the TERT promoter from 29 different patients, including two patients with both MB and SCC. The study was approved by the Ethics Committee and all methods were performed in accordance with relevant guidelines and regulations. A written informed consent was signed by all study participants. The data that support the findings of our study are available on request from the corresponding author. The patients with symmetrical AK on face or scalp were subjected to daylight-mediated PDT. Two AK lesions ≥6 mm in diameter, one each from right and left side were biopsied from each patient before and after treatment. For one patient, we could biopsy only the left sided lesion before and after treatment. Thus, 53 lesions from 27 patients prior to treatment were investigated. The same lesions were biopsied 3 months after the treatment. The median patient age at diagnosis was 79 years (IQR: 75–80 years) with 12 men and 15 women1, 4 (Fig. 1). Biopsy specimens were processed, embedded in paraffin and stained for hematoxylin and eosin and p53 immunohistochemistry based on standard methods.1 The diagnoses and the clearance of AK lesions were histologically verified. The presence of AK and grade of dysplasia (Grade I—mild dysplasia, Grade II—intermediate dysplasia and Grade III—severe dysplasia) were assessed from routine hematoxylin and eosin-stained slides.1, 8 Specimens not fulfilling the diagnostic criteria for AK were regarded as healthy. The p53 reactivity was quantified by counting the percentage of positive nuclei in three consecutive high-power fields from the region of highest reactivity and expressed as the average of the three high power fields (<10% low expression and ≥10% high expression)1 (Fig. 1). For the mutational analysis, DNA was PCR amplified from the punch biopsies. The amplified products were Sanger sequenced with a dideoxy terminator kit and forward and reverse primers in separate reactions (BigDye Terminator v3.1 Cycle sequencing kit). Sequencing reaction products were analyzed on a capillary sequencer (ABIPrism 3130xl Genetic Analyzer platform) as described.9 The associations between TERT promoter mutations and other clinical parameters before and after treatment were determined by χ2 test; odds ratio (OR) and p values were calculated to determine the statistical significance. TERT promoter mutations were detected in 11 of 53 (20.8%) lesions before treatment. Of 26 AKs from the right side, four (15.4%) lesions carried TERT promoter mutations that included two lesions with the −146C>T mutation (of those, one lesion additionally carried a −126C>T mutation); one with the −138/−139CC>TT and one with the −124/−125CC>TT mutation. Of 27 lesions from the left side, seven (25.9%) lesions harbored TERT promoter mutations that included the −146C>T mutation in four lesions, the −124C>T in two lesions, and the −138/−139CC>TT mutation in one lesion. All detected mutations were heterozygous with mutant allele peak ranging between 10 and 50% compared to the wild-type allele. At 3 months after treatment, 27/53 (50.9%) lesions were histologically clear. TERT promoter mutation (−124C>T) was detected only in one lesion on the left side (Grade I) of one patient. No other lesion after treatment had a detectable mutation (Fig. 1). The mutations in 12 patients were present in either right or left-sided lesions but not simultaneously in both lesions. And 10 out of 12 lesions with the TERT promoter mutations had increased (>10%) p53 expression. Mutation frequency in lesions before the treatment was statistically significantly higher than after (OR 0.07, 95% confidence intervals [CI] 0.01–0.55, p < 0.01). We observed statistically significant decreased p53 expression (<10%; OR 0.27, 95% CI 0.11–0.69, p < 0.006) and decrease in the AK grading (OR 0.17, 95% CI 0.06–0.49, p < 0.001) in lesions after the treatment.1 The treatment with daylight-mediated PDT seems to decrease the frequency of TERT promoter mutations and p53 expression burden. Out of the 20 MB tumors, six (30%) lesions carried the TERT promoter mutations that included three tumors with the −146C>T mutation, two tumors with the −138/−139CC>TT and one with the 124/−125CC>TT mutation. Of the 11 SCC tumors, five (45.4%) tumors carried TERT promoter mutations that included three tumors with the −124C>T mutation and two with the −146C>T mutation. TERT promoter mutations in general occur in tumors arising from nonself-renewing tissues that lack telomerase. However, cells of basal epidermis and presenescent fibroblasts contain rate-limiting levels of TERT and telomerase.6 We for the first time, as an exception to that generalization, report TERT promoter mutations in AKs. We observed that the frequency of those mutations in MB and SCC were in accordance with previous reports and higher than that in AKs.10 The TERT promoter mutations in AKs contrast with the absence of such mutations in melanocytic nevi despite high frequencies in melanoma.6 Yours sincerely, Nalini Srinivas Noora Neittaanmäki Barbara Heidenreich Sivaramakrishna Rachakonda Toni T. Karppinen Mari Grönroos Taneli T. Tani Mari Salmivuori Erna Snellman Kari Hemminki Rajiv Kumar Our study was supported by a TRANSCAN (JTC2013) award though German Ministry of Education and Research (BMBF) under grant number 01KT15511. Authors declare no conflict of interest.