Somatic Mutations in the ATRX Gene Reported for the First Time in Pheochromocytomas/ Paragangliomas

Lauren Fishbein MD, PhD, MTR

Somatic mutations in the ATRX gene are observed in about 13% of pheochromocytomas/paragangliomas, the first such report of somatic ATRX mutations in these tumors. Somatic genetic changes identified from whole exome sequencing of tumor samples from patients with sporadic or inherited PCC/PGL were presented by Lauren Fishbein MD, PhD, MTR, at the 2014 Neuroendocrine Tumor Symposium.

Pheochromocytomas (PCCs) form in the center of the adrenal gland, causing it to make too much adrenaline. Although PCCs are typically benign, they can result in conditions such as elevated blood pressure, severe headaches, and heart palpitations, according to the NCI. Paragangliomas (PGLs) are also usually benign and found near the adrenal glands and some blood vessels and nerves. Those which develop in the adrenal glands are PCCs.

Novel therapeutic targets in PCC/PGL are lacking because tumorigenesis is not well understood, yet a number of PCC/PGL susceptibility genes involved in multiple pathways in the cell are known. “Other than the inherited mutations, not too much else is known about the genetics,” she said.

These tumors can be divided by their expression analysis and their global methylation status. For expression analysis, they are divided into cluster 1 tumors, which are the succinate dehydrogenase inherited (SDHx) susceptibility mutation and von Hippel—Lindau (VHL). Cluster 2 represents the RET/NF1 TMEM127/MAX–associated tumors. Sporadic tumors are divided into these two groups.

More recently, global methylation analysis of PGL/PCC identified three clusters: tumors of the M1 cluster (SDHx and SDHB-related tumors as displaying a hypermethylator phenotype), whereas the M2 (VHL) and the M3 clusters did not display strong hypermethylation.

Older studies have found gene-specific somatic mutations are usually <5%, but more recently, the HRAS gene has been found to have a somatic mutation rate in up to 10% of sporadic PCC/PGL. Genes involved in the inherited syndrome have higher somatic mutation rates, particularly NF1 in 24% to 41%. Almost no somatic mutations are found in SDHx genes.

Fishbein’s group investigated somatic mutations through whole exome sequencing of 21matched tumor/germline pairs from patients with either sporadic or inherited PCC/PGL. “The idea was to compare clinically benign versus clinically aggressive PCC/PGL in order to identify markers of tumorigenesis as well as malignant potential,” she said.

In the discovery set, somatic variants in ATRX were identified in two of seven SDHB-associated tumors, which were considered the clinically aggressive tumors (metastatic disease). Through the use of a filtering algorithm, regardless of mutation group, the tumors were found to have a relatively low number of somatic mutations. The mean number of variants per tumor was 17 (range 4-26), “which is very comparable to that seen in neuroblastoma [12-18 per tumor] as well as the nonfunctional, well-differentiated pancreatic neuroendocrine tumors [pNETs; 16 per tumor],” explained Fishbein, instructor in medicine, Division of Endocrinology, Diabetes, and Metabolism at the University of Pennsylvania in Philadelphia.

“The one standout gene was ATRX,” she said. “I found two SDHB-associated tumors that had somatic ATRX mutation.” One mutation was a stop gain, and the other was a missense mutation in the conserved helicase domain at an amino acid residue that has been reported in neuroblastoma.

By immunohistochemistry, the ATRX protein was absent in tumors with ATRX variant. ATRX is part of the Swi/Snf family of chromatin remodelers. It heterodimerizes with the protein DAXX, which recruits histone H3.3 for heterochromatin formation. Therefore, the complex plays a role in maintenance of genomic integrity, said Fishbein.

Somatic mutations are observed in several neuroendocrine tumors; DAXX or ATRX mutations are the second most common mutations (behind MEN1) in the well-differentiated nonfunctional pNETs, and a significant proportion of tumors in neuroblastoma have point mutations or deletions of ATRX. In metastatic pNETs, ATRX/DAXX mutations are associated with increased survival, whereas an absence is associated with worse prognosis.

In a separate validation set of 103 samples, 12.6% of PCC/PGLs were found to have potentially damaging mutations in ATRX. Twenty five percent were frameshift mutations, and 75% were missense. “Out of the validation set where we knew the clinical information, four of the six tumors had SDHx mutations, one VHL, and one NF1, in terms of their inherited mutation status, and all but the NF1 tumor had clinically aggressive behavior,” she said.

PCC/PGLs with somatic ATRX variants were associated with alternative lengthening of telomeres and clinically aggressive behavior. Six of 22 samples (27%) demonstrated alternative lengthening of telomeres on telomere fluorescence in situ hybridization analysis, an alternative marker for telomere lengthening, and four of the six had an inherited SDHB mutations as well as somatic ATRX mutations.

“It seems that ATRX mutations or alternative lengthening of telomeres are more highly associated with clinically aggressive PCC/PGL,” she concluded.

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