Ataxia-Telangiectasia Mutated Loss of Heterozygosity in Melanoma

Abstract

ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. However, ATM loss of heterozygosity (LOH) has never been investigated in melanoma and, therefore, a causal association with melanoma development has not been established yet. The purpose of this study was to functionally characterize 13 germline ATM variants found in high-risk melanoma patients-and classified by in silico tools as pathogenic, uncertain significance, or benign-using multiple assays evaluating ATM/pATM expression and/or LOH in melanoma tissues and cell lines. We assessed ATM status by Immunohistochemistry (IHC), Western Blot, Whole-Exome Sequencing/Copy Number Variation analysis, and RNA sequencing, supported by Sanger sequencing and microsatellite analyses. For most variants, IHC results matched those obtained with in silico classification and LOH analysis. Two pathogenic variants (p.Ser1135_Lys1192del and p.Ser1993ArgfsTer23) showed LOH and complete loss of ATM activation in melanoma. Two variants of unknown significance (p.Asn358Ile and p.Asn796His) showed reduced expression and LOH, suggestive of a deleterious effect. This study, showing a classic two-hit scenario in a well-known tumor suppressor gene, supports the inclusion of melanoma in the ATM-related cancer spectrum.

Keywords: ATM; germline variant; loss of heterozygosity; melanoma; susceptibility.

Figure 1

The lollipop plot shows the distribution of the ATM variants included in this study. TAN = Tel1/ATM N-terminal motif domain; FAT = FRAP–ATM–TRRAP domain; PI3/PI4K = phosphatidylinositol 3-kinase/phosphatidylinositol 4-kinase-related kinase domain.

Figure 2

Representative images of ATM and pATM expression by immunohistochemistry. (A) Metastatic melanoma with pathogenic ATM variant (p.Ser1993ArgfsTer23), showing a lack of ATM and pATM nuclear expression in tumor cells (magnification 10X, inset 20X; scale bars 200 μm and 100 μm, respectively). (B) Metastatic melanoma with ATM variant of uncertain significance (p.Asn796His), showing partial conservation of nuclear expression for ATM and for pATM (magnification 10X, inset 20X; scale bars 200 μm and 100 μm, respectively). (C) Skin melanoma metastasis with benign ATM variant (p.Pro604Ser) showing nuclear ATM and pATM staining (magnification 10×, inset 20×; scale bars 200 μm and 100 μm, respectively).

Figure 3

Multiple assays showing loss of heterozygosity of the ATM p.Ser1993ArgfsTer23 PV in Mel_3. (A) WES showing prevalence of the mutant allele (c.5979_5983del), with an AF of 74%. (B) RNA sequencing showing a mutant allele (c.5979_5983del) with a frequency of 94%. (C) The LogR plot (left) derived from WES analysis shows the presence of CNV: a region on chromosome 11q that encompasses the ATM gene (chr11: 100650080-115623321) shows copy-number loss; in the ploidy plot (right), the same region of the tetraploid sample contains three copies of a single allele (red bar), whereas the other allele is deleted (green bar). (D) Sanger sequencing showing the heterozygous germline allele (top), partial loss of the WT allele (middle) in the FFPE sample, and the complete loss of the WT allele in the patient’s derived melanoma cell line (bottom). (E) Microsatellite analysis showing LOH in one of the FFPE samples and in the patient’s derived melanoma cell line: D11S2179 LOH in tumor is determined by comparing the peak-height ratio of blood sample (top) with those of tumor (bottom left) and cell line (bottom right) DNA. (F) Western blotting. ATM protein expression was tested on A549, a NSCLC cell line expressing functional ATM, and on two primary cell lines derived from metastatic cutaneous melanoma biopsies, Mel_3 and Mel_15. A549 and Mel_15 cells express ATM and upmodulate the expression of its phosphorylated form upon irradiation at 4 Gray. Conversely, the Mel_3 cell line does not express either ATM or p-ATM. Cropped images are obtained from the same blot subsequently probed with the different antibodies. Molecular weight markers are shown. Abbreviations: AF = Allele Frequency. FFPE = formalin fixed paraffin embedded.