Epigenetic Dysregulation in Cutaneous Malignant Melanoma: Advancing Approaches to Diagnosis, Prognosis, and Therapeutic Targeting

................................................................................................................................... 1 LIST OF ABBREVIATIONS ......................................................................................................... 4 BACKGROUND AND INTRODUCTION ................................................................................... 5 The ‘black cancer’: A historian’s lens .................................................................................... 5 The ‘modern black plague’: Mechanistic insight from the 21 century ................................. 7 Genomic sequencing technologies and personalized medicine: Advancing discoveries in melanoma pathobiology .......................................................................................................... 9 Epigenetic alterations in melanoma: A new frontier in cancer pathobiology ....................... 10 DNA demethylation and 5-hydroxymethylcytosine: Epigenetic fidelity unraveled ............. 11 ‘Loss’ of 5-hydroxymethylcytosine: From bench to bedside ............................................... 12 Melanoma in focus: A troublesome diagnosis ...................................................................... 14 Melanoma pseudomaturation and the pre-existing nevus: The challenge of histologically heterogeneous, malignant melanocytic lesions ..................................................................... 15 Nodal nevus: A diagnostic pitfall of sentinel lymph node evaluation .................................. 17 OBJECTIVES ............................................................................................................................... 19 MATERIALS AND METHODS .................................................................................................. 20 Ethics statement .................................................................................................................... 20 Acquisition of histopathologic samples for immunohistochemical study ............................ 20 Immunohistochemistry protocol, clinical data acquisition, and quantitative analysis of primary cutaneous melanomas ............................................................................................. 21 Immunohistochemistry protocol, clinical data acquisition, and analysis of sentinel lymph node biopsy cases .................................................................................................................. 22 Acquisition of patient melanoma tissue samples for next-generation sequencing and corresponding clinical data ................................................................................................... 23 DNA isolation and next-generation sequencing ................................................................... 23 Quantitative and additional analytics of somatic mutation data ........................................... 24 RESULTS ..................................................................................................................................... 26 I. Melanoma pseudomaturation or pre-existing nevus associated with primary cutaneous melanomas ................................................................................................................................ 26 Histopathologic and clinical outcome data ........................................................................... 26 5-hydroxymethylcytosine immunoreactivity in MPEN versus MPM .................................. 28 II. Metastatic melanoma or nodal nevus in sentinel lymph node biopsies ............................... 36 Clinical and histopathologic data of primary cutaneous melanomas corresponding leading to sentinel lymph node biopsy .................................................................................................. 36 Detailed histopathologic and microanatomic data of sentinel lymph node biopsies ............ 36 5-hydroxymethylcytosine immunoreactivity within sentinel lymph node biopsies ............. 39 III. Targeted next-generation sequencing of patient melanoma samples ................................. 44 General somatic mutation distribution and characteristics ................................................... 44 High frequency of mutations identified in key epigenetic regulators ................................... 44 High frequency of UVB-signature mutations among key epigenetic regulators .................. 51 DISCUSSION ............................................................................................................................... 55 5-hydroxymethylcytosine immunoreactivity highlights pseudomaturing subpopulation of melanoma cells while distinguishing pre-existing nevus from bona fide melanoma ........... 55