Biomarkers of basal cell carcinoma resistance to methyl-aminolevulinate photodynamic therapy

Abstract

Background: Methyl-aminolevulinate photodynamic therapy (MAL-PDT) is an excellent option for the treatment of basal cell carcinoma (BCC). However, up to 25% of cases are resistant to this treatment modality.

Objective: The aim of this study was to identify potential biomarkers of BCC response to MAL-PDT.

Material and methods: Clinical, histological, and immunohistochemical (p53, Ki-67, CD-31, COX2, β-catenin, EGFR, and survivin) variables were analyzed in a retrospective study of consecutive BCC patients treated with MAL-PDT at the San Jorge Hospital, Huesca, Spain between January 2006 and December 2015. To deepen on these markers, the effects on p53 and cyclin D1 expression, in vitro response to MAL-PDT of 2 murine BCC cell lines (ASZ and BSZ), was also evaluated.

Results: The retrospective study examined the response to MAL-PDT of 390 BCCs from 182 patients. The overall clinical response rate was 82.8%, with a mean follow-up time of 35.96 months (SD = 23.46). Immunohistochemistry revealed positive p53 in 84.6% of responders but only 15.4% of nonresponsive tumors (p = 0.011). Tumors with increased peripheral palisading of basal cell islands to immunostaining β-catenin responded poorly to PDT (p = 0.01). In line with our findings in patients, in vitro studies revealed a better response to PDT in the p53-positive ASZ cell line than the p53-negative BSZ cell line (p<0.01). Multivariate analysis revealed that the following variables were significantly associated with response to PDT: age, nBCC, presence of peritumoral inflammatory infiltrate, and p53 immunopositivity. Patients with positive p53 immunostaining were 68.54 times more likely to achieve cure than p53-negative patients (CI95% 2.94-159.8).

Conclusion: Our finding suggest that certain clinicopathological and immunohistochemical variables, particularly p53 expression, may serve as indicators of BCC response to MAL-PDT, and thus facilitate the selection of patients who are most likely to benefit from this therapy.

Fig 1

(a) Peritumoral inflammation surrounding basal cell carcinoma in a MAL-PDT-sensitive BCC (10×). (b) p53 immunostaining in a MAL-PDT responsive BCC: 97% of cells exhibit positive p53 immunostaining (5×). (c) Intense β-catenin immunostaining in a MAL-PDT-sensitive BCC (10×) and (d) enhanced peripheral palisading in a MAL-PDT-resistant BCC (40x). Bar charts depict levels of perilesional inflammatory infiltrate, p53 immunoexpression, and the intensity and distribution of β-catenin immunostaining in BCCs treated with MAL-PDT (responsive and nonresponsive). Scale bar: 200 μm (A and C), 500 μm (B) and 100 μm (D).

Fig 2. Expression of p53, β-catenin and ciclin D1 in BCC mice lines.

(A) Expression pattern of p53, β-catenin and ciclin D1 on ASZ and BSZ cells by immunofluorescence. Scale bar: 20um. (B) Protein quantification by Western Blot of (a) p53 and (b) β-catenin. Load control: β-actin. (C) Relative fluorescence of cyclin d1 expression by immunofluorescence. *p< 0.05; ***p< 0.001.

Fig 3

(a) Cell viability of ASZ and BSZ cells as determined by MTT assay in drug control (0.3 mM MAL), light control (2.25 J/cm²), and PDT (0.3 mM MAL + 0.5 J/cm² or 0.3 mM MAL + 2.25 J/cm²) conditions. **p <0.01. (b) Photographs of ASZ and BSZ cells following PDT. Scale bar, 200 μm.