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Prognostic Utility of CpG Island Hypermethylated Phenotype in Early-Stage Invasive Primary Melanomas

  • Matias A. Bustos
    Affiliations
    Department of Translational Molecular Medicine, Saint John’s Cancer Institute, Providence Saint John’s Health Center, Santa Monica, California, USA
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  • Dave S.B. Hoon
    Correspondence
    Correspondence: Dave S. B. Hoon, Department of Translational Molecular Medicine, Saint John’s Cancer Institute, Providence Saint John’s Health Center, 2200 Santa Monica Boulevard, Santa Monica, California 90404, USA.
    Affiliations
    Department of Translational Molecular Medicine, Saint John’s Cancer Institute, Providence Saint John’s Health Center, Santa Monica, California, USA
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Published:January 11, 2022DOI:https://doi.org/10.1016/j.jid.2021.12.013
      In their article in the Journal of Investigative Dermatology,
      • Conway K.
      • Tsai Y.S.
      • Edmiston S.N.
      • Parker J.S.
      • Parrish E.A.
      • Hao H.
      • et al.
      Characterization of the CpG island hypermethylated phenotype (CIMP) subclass in primary melanomas [e-pub ahead of print].
      describes the CpG island methylator phenotype (CIMP) subclass occurring in 16% of invasive primary melanomas (Figure 1). Cutaneous melanoma is one of the deadliest forms of skin cancer in North America, Europe, and Australia. Early diagnosis and prognostic classification of primary melanoma are of critical importance to reduce morbidity and healthcare costs and improve survival (
      • Siegel R.L.
      • Miller K.D.
      • Fuchs H.E.
      • Jemal A.
      Cancer statistics, 2021 [published correction appears in CA Cancer J Clin 2021;71:359].
      ). The utilization of molecular omics in the study of primary melanomas is rapidly growing, and the molecular factors involved in the progression of primary melanoma are being delineated. Understanding these molecular alterations may improve the early identification of patients who will eventually develop aggressive metastatic disease. This would inform clinical decisions on diagnosis, prognosis, and treatment strategies. To date, the major emphasis of molecular omics analyses of primary melanomas has been related to specific sequence variations. However, only a single nucleotide variation, the BRAF V600E single nucleotide variation, occurs with an average frequency >50%, and other single nucleotide variation's are much less frequent in primary melanomas. There is a need for a more useful molecular omics assessment that has more generalized clinical utility for predicting poor outcomes of invasive primary melanomas.
      • CpG island methylator phenotype (CIMP) is an epigenetic prognostic factor for overall survival in primary melanomas.
      • Invasive primary melanomas with CIMP subclass have more ulceration and higher T stage.
      • CIMP in primary melanomas has low T-cell infiltrates and limited antitumor immunity.
      Figure thumbnail gr1
      Figure 1Schematic representation of methylation subtypes in invasive primary cutaneous melanomas. Using HM450K analysis in 89 patients with invasive primary melanoma, three subtypes have been proposed: LM, IM, and CIMP subtype. Each methylation subtype was associated with the histological subtypes: SSM, NM, LMM, ALM, and other/unclassified. Invasive primary melanoma with CIMP subtype is associated with poor clinical outcomes. AJCC, American Joint Committee on Cancer; ALM, acral lentiginous melanoma; CIMP, CpG island methylator phenotype; IM, intermediate methylation; LM, low methylation; LMM, lentigo maligna melanoma; NM, nodular melanoma; SSM, superficial spreading melanoma.

      Understanding CIMP subtype in early-stage melanoma lesions

      • Conway K.
      • Tsai Y.S.
      • Edmiston S.N.
      • Parker J.S.
      • Parrish E.A.
      • Hao H.
      • et al.
      Characterization of the CpG island hypermethylated phenotype (CIMP) subclass in primary melanomas [e-pub ahead of print].
      performed genome-wide methylation analysis using the HM450K BeadChip Illumina array (Illumina, Carlsbad, CA) and classified 89 invasive primary melanomas into three methylation subclasses integrated with clinical data: low methylation, intermediate methylation, and CIMP. Briefly, CIMP is a pattern of extensive DNA hypermethylation on cytosine in CpG islands of the human genome (
      • Greenberg E.S.
      • Chong K.K.
      • Huynh K.T.
      • Tanaka R.
      • Hoon D.S.
      Epigenetic biomarkers in skin cancer.
      ). The CIMP epigenetic phenomenon has been reported in the past as a universal pancancer epigenetic event that becomes more prominent in advanced stages of various cancers (
      • Miller B.F.
      • Sánchez-Vega F.
      • Elnitski L.
      The emergence of pan-cancer CIMP and its elusive interpretation.
      ).
      • Conway K.
      • Tsai Y.S.
      • Edmiston S.N.
      • Parker J.S.
      • Parrish E.A.
      • Hao H.
      • et al.
      Characterization of the CpG island hypermethylated phenotype (CIMP) subclass in primary melanomas [e-pub ahead of print].
      showed that patients with the CIMP subclass of invasive primary melanomas had a significantly worse melanoma-specific survival when considering known prognostic factors (hazard ratio = 11.84; confidence interval = 4.65‒30.20). A prominent feature of the CIMP subclass of primary melanomas is a poor tumor-infiltrating lymphocyte response associated with limited antitumor immunity. This study shows the clinical utility of CIMP in a subtype of early-stage invasive primary melanomas.
      CIMP involves the enhancement of methylation in variable CpG islands of gene promoter regions as well as other genomic regions. The hypermethylation of CpG (island) sites is not uniform. This epigenetic phenomenon may be due to a higher activity of DNA methyltransferase 1, which is involved in specific methylation of CpG sites during mitosis of cells (
      • Zhang X.
      • Bustos M.A.
      • Shoji Y.
      • Ramos R.I.
      • Iida Y.
      • Gentry R.
      • et al.
      Acetylated DNMT1 downregulation and related regulatory factors influence metastatic melanoma patients survival.
      ). Other epigenetic factors may also promote CIMP phenotypes.
      It is important to determine whether methylation of specific CpG sites in CIMP is responsible for regulation of selective gene expression. Using The Cancer Genome Atlas (TCGA) skin database, the authors found an association between CIMP and mRNA expression related to specific genes. However, a direct paired one-to-one correlation of specimens is required for validation of these findings. Although gene promoter regions were predominantly assessed for CpG island methylation, noncoding regions that include methylated-in-tumor loci as well as other noncoding loci (
      • Greenberg E.S.
      • Chong K.K.
      • Huynh K.T.
      • Tanaka R.
      • Hoon D.S.
      Epigenetic biomarkers in skin cancer.
      ;
      • Tanemura A.
      • Terando A.M.
      • Sim M.S.
      • van Hoesel A.Q.
      • de Maat M.F.
      • Morton D.L.
      • et al.
      CpG island methylator phenotype predicts progression of malignant melanoma.
      ) can be indicators of CIMP. The HM450K BeadChip arrays cannot accurately identify the CpG island methylation status of a large number of noncoding regions. The newer HM850K BeadChip Illumina array has more in-depth coverage of the whole genome that includes more CpG island sites at gene promoter and noncoding regions. A more comprehensive epigenomic platform that involves new approaches with in-depth whole-genome methylation sequencing with further validation using targeted quantitative PCR assays will allow for better profiling of CIMP events in tumors. These cutting-edge technologies, in combination with a better understanding of the CIMP subtype and improved bioinformatic analytical tools, will translate into the clinic enabling assessment and classification of more aggressive melanoma primary tumors at earlier stages.
      CIMP events are enhanced during advanced tumor progression in most solid tumor cancers, including malignant cutaneous melanoma. However, the identification of CIMP in a subset of invasive primary cutaneous melanomas suggests that it develops early and that it may be dependent on the persistence of tumor cell clones that have undergone progressive epigenetic changes. The analysis performed using methylation arrays only provides a bulk evaluation of the melanoma cells in the tumor sample. Further studies will be needed to assess clonal development and the heterogeneity of CIMP events in primary melanomas. CIMP involves the methylation silencing of many genes that influence differentiation, regulate transcription, and suppress tumors. In the latter group, RASSF1 is often downregulated and is related to CIMP in cutaneous melanomas and other cancers.
      • Conway K.
      • Tsai Y.S.
      • Edmiston S.N.
      • Parker J.S.
      • Parrish E.A.
      • Hao H.
      • et al.
      Characterization of the CpG island hypermethylated phenotype (CIMP) subclass in primary melanomas [e-pub ahead of print].
      showed increased methylation of RASSF1 in invasive primary melanomas with the CIMP subtype. Epigenetic silencing of this tumor suppressor gene has been significantly associated with melanoma progression and poor prognosis (
      • Spugnardi M.
      • Tommasi S.
      • Dammann R.
      • Pfeifer G.P.
      • Hoon D.S.
      Epigenetic inactivation of RAS association domain family protein 1 (RASSF1A) in malignant cutaneous melanoma.
      ), suggesting the RASSF1 gene as a key indicator of the onset of CIMP events.

      Lentigo maligna melanoma subtype has the highest incidence in CIMP

      This study found that 50% of invasive primary melanomas with the CIMP subtype had the lentigo maligna melanoma (LMM) histological subtype (Figure 1). LMM subtype is one of the four most frequent primary melanoma subtypes, most often diagnosed in patients aged >60 years. After surgical resection, LMM can recur late and require careful monitoring and long-term follow-up (
      • Collgros H.
      • Rodriguez-Lomba E.
      • Regio Pereira A.
      • Lo S.N.
      • Scolyer R.A.
      • Guitera P.
      Lentiginous melanoma (lentigo maligna and lentigo maligna melanoma) in Australia: clinicopathological characteristics, management and recurrence rates after 10-year follow-up at a tertiary centre.
      ). The more frequent CIMP phenotype in patients with LMM may be due to the spontaneous methylation of CpG islands in the human genome that occurs more frequently in normal tissues from older adults such as skin that is chronically exposed to the environment. LMM has been associated with skin damage due to sunlight through chronic UVR exposure (
      • Collgros H.
      • Rodriguez-Lomba E.
      • Regio Pereira A.
      • Lo S.N.
      • Scolyer R.A.
      • Guitera P.
      Lentiginous melanoma (lentigo maligna and lentigo maligna melanoma) in Australia: clinicopathological characteristics, management and recurrence rates after 10-year follow-up at a tertiary centre.
      ), and UV may be a CIMP driver. Understanding the early events promoting the CIMP subtype such as UVR dosage and exposure as well as epigenetic signatures associated with DNA repair deficiency after UV exposure may promote new approaches to prevention and intervention strategies for patients with primary melanoma. Future studies comparing invasive primary melanomas from chronic sun-exposed anatomical sites with those from nonsun-exposed sites may allow for determination of the contribution of UV-induced damage in the epigenomic events leading to CIMP. In addition, characterization of alterations on specific epigenetic regulatory genes such as those involved in the regulation of DNA demethylation in the early stages of primary melanomas may be responsible for the CIMP subtype.

      TET2 may have implications in promoting CIMP

      The Ten-eleven translocation (TET) enzymes are known for their important roles in DNA demethylation. Three proteins comprise the TET family: TET1, TET2, and TET3.
      • Conway K.
      • Tsai Y.S.
      • Edmiston S.N.
      • Parker J.S.
      • Parrish E.A.
      • Hao H.
      • et al.
      Characterization of the CpG island hypermethylated phenotype (CIMP) subclass in primary melanomas [e-pub ahead of print].
      determined that the CIMP methylation melanoma subclass is likely to have hypermethylation at the TET2 gene promoter. TET2 is a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. This is a critical step in cytosine demethylation such as in methylated CpG islands in the whole genome. Thus, the decreased level of TET2 transcription mediated by methylation promotes a feedback loop enhancing CIMP. Analysis of primary and metastatic CIMP melanomas in the TCGA skin dataset had higher TET2 hypermethylation with concordant lower mRNA expression. Previous reports have shown that epigenetic silencing of TET2 occurs in hypermethylated primary melanomas (
      • Koroknai V.
      • Szász I.
      • Hernandez-Vargas H.
      • Fernandez-Jimenez N.
      • Cuenin C.
      • Herceg Z.
      • et al.
      DNA hypermethylation is associated with invasive phenotype of malignant melanoma.
      ). However, the detailed mechanism prompting TET2 hypermethylation remains poorly characterized. A recent study by
      • Noreen F.
      • Küng T.
      • Tornillo L.
      • Parker H.
      • Silva M.
      • Weis S.
      • et al.
      DNA methylation instability by BRAF-mediated TET silencing and lifestyle-exposure divides colon cancer pathways.
      showed that BRAF V600E allelic variant promotes TET2 and TET1 hypermethylation in patients with colon cancer. This dependency of TET2 and TET1 expression on the BRAF V600E allelic variant is of interest because melanoma has about 50% BRAF V600E mutation frequency. The role of the BRAF V600E allelic variant in regulating TET1‒3 levels requires further studies in primary melanomas. Overall, the role of TET2 as well as those of TET1 and TET3 epigenomic status and the factors controlling TET2 levels need further investigation as related to the development and/or maintenance of CIMP in primary invasive melanomas.

      Clinical implication of CIMP in patients with early melanoma

      The study of
      • Conway K.
      • Tsai Y.S.
      • Edmiston S.N.
      • Parker J.S.
      • Parrish E.A.
      • Hao H.
      • et al.
      Characterization of the CpG island hypermethylated phenotype (CIMP) subclass in primary melanomas [e-pub ahead of print].
      showed that invasive primary melanomas with CIMP subclass have the worst melanoma-specific survival, which is the only significant epigenetic event known for early-stage invasive primary melanomas prognosis to date. Interestingly, CIMP of invasive primary melanomas is more likely in patients aged >65 years (P = 0.03) and in the LMM subtype (P = 0.02) than in IMs. The assessment of CIMP in LMM may be a valuable prognostic factor and improve follow-up management strategies for these high-risk lesions. Assessment of the epigenomic changes at early stages of invasive primary melanomas may be more beneficial and represent better prognostic tools than assessing genomic aberrations such as individual gene mutations. Clinical demographics suggest that primary melanomas from sites that have high sun exposure might be assessed for CIMP more aggressively. It would be interesting to determine whether there is an enhancement of cyclobutane pyrimidine thymine dimers (the most abundant DNA lesions produced after exposure to chronic UVR) in primary melanomas and whether these DNA lesions correlate with CIMP. Finally, further studies will be needed to validate the findings of the reported studies in a larger cohort of clinically annotated thin and intermediate invasive primary melanomas of multiple T stages and particularly of the LMM subtype.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      DSBH receives funding from Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.

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      Linked Article

      • Characterization of the CpG Island Hypermethylated Phenotype Subclass in Primary Melanomas
        Journal of Investigative DermatologyVol. 142Issue 7
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          Cutaneous melanoma can be lethal even if detected at an early stage. Epigenetic profiling may facilitate the identification of aggressive primary melanomas with unfavorable outcomes. We performed clustering of whole-genome methylation data to identify subclasses that were then assessed for survival, clinical features, methylation patterns, and biological pathways. Among 89 cutaneous primary invasive melanomas, we identified three methylation subclasses exhibiting low methylation, intermediate methylation, or hypermethylation of CpG islands, known as the CpG island methylator phenotype (CIMP).
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