Person:

Lee, Jonathan J.

Melanomas in the vertical growth phase (VGP) not infrequently demonstrate cellular heterogeneity. One commonly encountered subpopulation displays small cell/nevoid morphology. Although its significance remains unknown, such subpopulations may pose diagnostic issues when faced with differentiating such changes from associated nevus or mistaking such regions for nevic maturation (pseudomaturation). That ‘loss’ of the epigenetic biomarker, 5-hydroxymethylcytosine (5-hmC), is a hallmark for melanoma and correlates with virulence prompted us to explore the diagnostic utility and biological implications of 5-hmC immunohistochemistry (IHC) in melanomas with small cell/nevoid subpopulations. Fifty-two cases were included in this study, including melanomas with small cell/nevoid subpopulations (MSCN) or melanomas with pre-existing nevus (MPEN). Semiquantitative and computer-validated immunohistochemical analyses revealed invariable, uniform loss of 5-hmC in the conventional melanoma component. By contrast, the nevic components in MPEN cases demonstrated strong nuclear immunopositivity. In MSCN cases, there was partial to complete loss of 5-hmC restricted to these nevoid areas. Based on recent data supporting tight correlation between 5-hmC loss and malignancy, our findings indicate a potential ‘intermediate’ biological nature for small cell/nevoid subpopulations. Because 5-hmC assisted in differentiating such regions from associated nevus, the use of 5-hmC as an adjunct to microstaging in difficult cases showing VGP heterogeneity should be further explored.

BACKGROUND: Evidence is rapidly accumulating that epigenetic mechanisms, including DNA methylation and demethylation, histone modifications, and non-coding RNAs, may play a central role in the pathobiology of melanoma. Previous studies have demonstrated the utility of the novel epigenetic mark, 5-hydroxymethylcytosine, to distinguish between benign cutaneous nevi and primary cutaneous melanoma. Further applications of this epigenetic biomarker to enhance diagnostic accuracy and prognostic precision in the evaluation of a unique set of diagnostically-challenging primary cutaneous melanomas and sentinel lymph node biopsies will be explored. In addition, clinically-applicable next-generation sequencing platforms enable us to identify novel gene mutations present in patient melanoma samples. Accordingly, the prevalence and nature of mutations in genes encoding epigenetic regulators (as well as non-epigenetic regulators) in patient melanoma samples will also be explored in this study.

METHODS: Fifty-two histologic sections of primary cutaneous melanoma (NP=52) were obtained from the pathology archives of two academic institutions. These cases were intentionally selected based on the presence of pseudomaturation (nMPM=24) or associated pre-existing nevus (nMPEN=28). Immunohistochemistry for 5-hydroxymethylcytosine was performed on all primary melanoma cases (NP=52). In addition, a collection of twenty eight histologic sections of sentinel lymph node biopsies (NS=28) containing either metastatic melanoma (nMM=18) or nodal nevus (nNN=10) was also retrieved from the pathology archive of one academic institution. Dual-labeling direct immunofluorescence and immunohistochemistry for MART-1/5-hydroxymethylcytosine was performed on all sentinel lymph node biopsy cases. Finally, targeted next generation sequencing was performed on thirty-eight patient melanoma specimens (NM=38) to detect exonic mutations in 275 cancer genes, 41 of which encode known epigenetic regulators.

RESULTS: Collectively, regions containing pseudomaturing cells within primary melanomas with pseudomaturation demonstrated intermediate immunopositivity for 5-hydroxymethylcytosine, in stark contrast to the overlying melanoma, which showed complete, diffuse loss. The staining intensity in pseudomaturing regions was quantifiably distinct and intermediate to that of pre-existing nevi (strong, homogeneous positivity) and bona fide melanoma (complete, diffuse loss), providing further support to the hypothesis that pseudomaturing melanoma cells may reflect a more indolent subpopulation. 5-hydroxymethylcytosine immunoreactivity was strongly retained in 10 of 10 (100%) cases of nodal nevus but ‘lost’ in 18 of 18 (100%) of cases of metastatic melanoma, thus representing a useful adjunctive strategy to definitively diagnose histologically subtle micrometastases. Targeted next generation sequencing demonstrated that 20.2% of all somatic mutations (107 of 530) affected an epigenetic regulator, with 35 of 38 samples (92.1%) harboring at least one mutation in an epigenetic gene. Genes with the highest percentage of UVB-signature mutations encoded epigenetic regulators. In addition, MECOM, a novel, central epigenetic regulator, as well as TET2/IDH1, critical enzymatic and metabolic regulators of DNA 5-hydroxymethylation, were found to be more frequently mutated than previously described.

DISCUSSION/CONCLUSION: The present study provides direct genomic evidence that epigenetic regulators may be involved in the pathobiology of melanoma and that novel, personalized therapeutic targets may be revealed with next generation sequencing. In addition, our immunohistochemical investigations demonstrate that the epigenetic biomarker, 5-hydroxymethylcytosine, can enhance diagnostic and microstaging accuracy in the histopathologic evaluation of pseudomaturing primary cutaneous melanoma and refine prognostic evaluations by enabling the distinction of metastatic melanoma from its diagnostic mimic, nodal nevus, in sentinel lymph node biopsies.

Non-bullous congenital ichthyosiform erythroderma (NBCIE) is a hereditary disorder of keratinization caused by pathogenic variants in genes encoding enzymes important to lipid processing and terminal keratinocyte differentiation. Impaired function of these enzymes can cause pathologic epidermal scaling, significantly reduced skin barrier function. In this study, we have performed a focused, genetic analysis of a probrand affected by NBCIE and extended this to his consanguineous parents. Targeted capture and next-generation sequencing was performed on NBCIE associated genes in the proband and his unaffected consanguineous parents. We identified a homozygous nonsense variant c.814C>T (p.Arg272*) in ALOXE3 (NM_001165960.1) in the proband and discovered that his parents are both heterozygous carriers of the variant. The clinical manifestations of the proband’s skin were consistent with NBCIE, and detailed histopathological assessment revealed epidermal bulla formation and Majocchi’s granuloma. Infection with Trichophyton rubrum was confirmed by culture. The patient responded to oral terbinafine antifungal treatment. Decreased skin barrier function, such as that caused by hereditary disorders of keratinization, can increase the risk of severe cutaneous fungal infections and the formation of Majocchi’s granuloma and associated alopecia. Patients with NBCIE should be alerted to the possible predisposition for developing dermatophytoses and warrant close clinical follow-up.