Immunohistochemistry for ATRX Can Miss ATRX Mutations

Neuroblastoma is the most common extracranial solid tumor of childhood with a median age of presentation of 17 months. A common theme in high-risk neuroblastoma is maintenance of telomeres, one mechanism for which involves alternate lengthening of telomeres (ALT) associated with ATRX gene mutations. Mutations are believed to result in loss of ATRX protein, and therefore immunohistochemistry is used to detect mutations. We screened 133 cases of neuroblastoma by ATRX immunohistochemistry, and found 9 cases with partial to total absence of ATRX. Sequencing for ATRX mutations detected a mutation in 1 of 9 cases, suggesting immunostaining was not reliable for detecting mutations. To correlate immunostaining with ALT, fluorescence in situ hybridization (FISH) for ALT was performed in 6 of these cases and 5 (from 4 patients) showed ALT, implying impaired ATRX protein function, despite the failure to identify a mutation. Two other cases with large deletions in the ATRX gene showed diffusely positive staining for ATRX protein but showed ALT by FISH. Four of the 6 patients with ALT-positive tumors were over 5 years old. Therefore, 29 additional patients 5 years old and above with ATRX-positive tumors were screened for ALT by FISH and 6 additional cases with ALT were detected, bringing the total to 29% (10/34) of children 5 years old and above, 70% of which showed positive ATRX immunohistochemistry. Patients with ATRX mutations in neuroblastoma tend to have a more chronic and progressive course of disease. Screening neuroblastoma tumors at diagnosis for ATRX mutations may help identify patients who might benefit from personalized therapy directed against ALT. However, relaying on negative immunohistochemistry for ATRX protein to identify ALT in neuroblastoma may miss a significant proportion of patients. The addition of FISH for ALT as part of the diagnostic workup, especially for older children (5 y old and above), would help ensure that patients are correctly identified for anti-ALT therapy.