Profiling cytochrome P450 CYP1 enzyme expression in primary melanoma and disseminated disease utilizing spectral imaging microscopy (SIM)

Abstract

8556 Background: The aim of this study was to profile cytochrome CYP1 family (CYP1A1/1A2, and CYP1B1) mono-oxygenase enzymes during the malignant progression of primary melanoma and metastatic disease. Methods: Tissue microarrays of primary (n = 75), and metastatic (n = 104) melanoma were constructed with the patient demographics: (1) primary melanoma; age 22 to 93 (median 59); sex M/F 36/44; Breslow thickness 0.4 to 15 mm (median 2.5 mm); ulceration 25/80, and (2) metastatic melanoma; age 26 to 92 (median 60 mm); sex M/F 54/49; ulceration 30/104; number of nodes 1 to 15 (median 2); extra-capsular spread 20/95. CYP1 protein was detected by IHC using validated selective poly- and monoclonal antibodies. Vector SG (grey) stain for CYP1 was used with nuclear fast red counterstain to aid spectral resolution from background melanin. Staining intensity was scored visually (negative 0, weak 1, moderate 2, strong 3) and using SIM at every pixel of a captured image of each melanoma core. Reference spectra of individual chromophores were used to spectrally ‘un-mix’ CYP1 staining before the mean normalised absorbance intensity was determined. Grading was by the 2002 AJCC classification system: primary stage I n = 27 (1A 8, 1B 19), and stage II n = 48 (2A 22, 2B 16, 2C 10), lymph node metastasis stage III n = 98 (3B 53, 3C 45), visceral metastasis stage IV n = 6. Normal skin (n = 27), benign naevi (n = 14), and dysplastic naevi (n = 21) were also included. Results: CYP1B1 was not in normal skin but was over-expressed in both primary and metastatic melanoma (visual: 71% & 65%, SIM: 91% & 83%). Primary melanoma (stage I & II) was significantly greater (p = 0.004) than metastasis (stage III & IV). CYP1B1 did not correlate with ulceration or Breslow thickness but did correlate with N stage lymph node metastasis (p = 0.005). CYP1B1 expression in dysplastic naevi indicated up-regulation at an early stage of melanoma progression. CYP1A1/1A2 was not expressed in normal skin nor primary/metastatic melanoma. Conclusions: CYP1B1 protein expression is maintained with advancing AJCC stage from primary through to visceral metastasis. Future work will seek to correlate protein expression with functionality with a view to exploiting CYP1B1 in the enzyme/prodrug therapy of malignant melanoma. No significant financial relationships to disclose.