Absence of TERT promoter mutations in primary melanocytic tumours of the central nervous system

Telomerase reverse transcriptase (TERT) encodes the catalytic subunit of telomerase, which adds telomere repeats to chromosome ends, enabling repeated rounds of cell replication without cells becoming genetically instable and apoptotic or senescent [1–3]. Recent studies uncovered C > T mutations at Chr.5.1295228 and Chr.5.1295250, further referred to as C228 and C250, respectively, in the TERT promoter region in different human cancers, including hepatocellular carcinomas, thyroid cancer, bladder cancer, gliomas and skin melanomas [1–3]. These mutations appear to increase TERT transcriptional activity, by creating ETS transcription factor binding sites. Killela et al. proposed that TERT promoter mutations may be more common in cancers derived from terminally differentiated cells with low self-renewing capacity, whereas rapidly renewing tissues have alternative mechanisms to maintain telomerase lengthening [3]. The incidence of TERT mutations in skin melanoma and melanoma cell lines appears to be very high, up to 70% of the cases examined, and seems to exceed the cumulative frequency of BRAF and NRAS mutations. However, it is still to be determined whether TERT mutations can occur in other melanoma variants. While a recent study showed the absence of TERT promoter mutations in ocular melanomas, no information regarding its mutational status in primary melanocytic tumours of the central nervous system is available [4]. Primary melanocytic tumours occurring in the central nervous system (CNS) are rare neoplasms [5,6]. Histologically, they represent a spectrum of lesions ranging from welldifferentiated melanocytomas to malignant melanomas [5,6]. They are believed to derive from melanocytes normally present in the leptomeninges and share molecular and histological features with uveal melanomas. FFPE tissues corresponding to 25 primary and 3 relapsing primary melanocytic tumours of the CNS (8 melanocytomas, 18 melanocytomas of intermediate grade and 2 melanomas respectively) arising in 25 patients (13 male and 12 female; age range 20–77; mean age 53.7 years), 2 melanotic schwannomas and 23 brain metastases of systemic, non-CNS melanomas (13 male and 10 female; mean age: 58,08; age range 37–78) were retrieved from the Institute of Neuropathology, University of Bonn, Germany, from the DGNN German Brain Tumor Reference Center, Bonn, Germany, from the Institute of Neuropathology, University Duisburg-Essen, Germany, from the Department of Pathology, Catholic University, Rome, Italy, and from the Department of Neuropathology, Royal Prince Alfred Hospital, Sydney, Australia. All tumours were classified according the 2007 WHO classification of CNS tumours [5]. Moreover, 10 uveal melanoma FFPE tissues (5 male, 5 female; age range 29–84, years; mean age 55,4 years) were provided from the Institute of Pathology, University of Messina, Italy and from the Section of Anatomic Pathology, University of Catania, Italy. The study was performed in accordance with the guidelines of the ethical policies of the involved institutions. In particular, the specimens from the Royal Prince Alfred Hospital (RPAH) were collected under an RPAH Human Research Ethics Committee approval. After assignment of the required information to the samples, these have been anonymized. For mutation analysis of the TERT promoter hotspots (C228 and C250) we developed a pyrosequencing assay (Figure 1). The DNA from FFPE tumour tissue was extracted using the QIAamp DNA Mini Tissue Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s instructions. A 169 bp fragment of the 5′ region of TERT containing the C228 and C250 coding region was amplified using the primers TERT fwd 5′-CCTGCCCCTTCACCTTCCAG-3′ and TERT rev 5′-biotinAGGACGCAGCGCTGCCTGAA-3′ using 50 ng genomic DNA as template. For pyrosequencing the primer TERT Py-5′-ACCCCGCCCCGTCCCGACCCC-3′ was used with the nucleotide dispensation order GTCGTCCGCATGCCTC to sequence TT/CCCGGGTCCCCGGCCCAGCCCCT/CTCCG. Ten glioblastomas harbouring TERT promoter (C228 or C250) mutations have been used as positive controls (Figure 1); The GNAQ, GNA11 (hotspot codon 209) and NRAS (exons 2 and 3) have been analysed for the presence of mutations as previously described [7]. None of the primary melanocytic tumours of the CNS (0/25) showed TERT promoter mutations at position