Lynch syndrome-associated ultra-hypermutated pediatric glioblastoma mimicking a constitutional mismatch repair deficiency syndrome.

Pediatric glioblastoma multiforme (GBM) has a poor prognosis as a result of recurrence after treatment of surgery and radiochemotherapy. A small subset of pediatric GBMs presenting with an ultra-high tumor mutational burden (TMB) may be sensitive to immune checkpoint inhibition. Here we report a 16-yr-old male with an ultra-hypermutated GBM. After incomplete surgical resection, molecular analysis of the tumor identified unusually high numbers of mutations and intratumor heterogeneity by a hotspot next-generation sequencing (NGS) panel. Further comprehensive molecular profiling identified a TMB of 343 mutations/Mb. An ultra-hypermutation genotype in pediatric GBMs is suggestive of a constitutive mismatch repair deficiency syndrome (CMMRD), which often acquires additional somatic driver mutations in replicating DNA polymerase genes. Tumor sequencing identified two MSH6 nonsense variants, a hotspot POLE mutation and a mutational signature supportive of a germline MMR deficiency with a somatic POLE mutation. However, constitutional testing identified only one nonsense MSH6 variant consistent with a Lynch syndrome diagnosis. This case represents the first confirmed Lynch syndrome case mimicking CMMRD by manifesting as an ultra-hypermutated pediatric GBM, following somatic mutations in MSH6 and POLE These findings permitted the patient's enrollment in an anti-PD-1 clinical trial for children with ultra-hypermutated GBM. Immunotherapy response has resulted in the patient's stable condition for over more than 1 year postdiagnosis.

Performance evaluation of two methods using commercially available reagents for PCR-based detection of FMR1 mutation.

The current workflow for clinical Fragile X testing is time consuming and labor intensive. Recently developed PCR-based methods simplify workflow, amplify full mutation alleles, and improve sensitivity for detecting low-level mosaicism. We evaluated the performance characteristics and workflow of two methods using commercially available reagents for determining FMR1 mutation status. We also tested each method's ability to detect mosaicism (range, 100% to 1% for males; 50% to 1% for females). One method used reagents from Asuragen (AmplideX FMR1 PCR, research use only). The second method used analyte specific reagents from Abbott Molecular, including FMR1 Primer 1 (for repeat sizing) and FMR1 Primer 2 (for screening of expanded alleles). Each reaction was evaluated for accuracy, precision, correlation with previous results, and workflow. Both methods performed equally well in accuracy and precision studies using NIST standards and previously characterized Coriell samples. Both methods showed 100% concordance with results from a previous consensus study and for previously analyzed patient samples. The Asuragen reagents were able to detect full mutation mosaicism down to 5% and premutation mosaicism to 1%. The Abbott Molecular Primer 2 reagents were able to detect both full mutation and premutation mosaicism down to 25%. Both PCR-based methods for the determination of FMR1 mutation status performed well, with expected results in their final diagnoses, and differed significantly only in their workflow.