Prognostic Significance of Circulating RET M918T Mutated Tumor DNA in Patients With Advanced Medullary Thyroid Carcinoma.

Context: Interpretation of calcitonin measurement to predict the prognosis of medullary thyroid carcinoma (MTC) requires multiple measurements over an extended time period, making it an imperfect biomarker for evaluating prognosis or disease behavior. Single circulating cell-free DNA (cfDNA) values have been shown to be a valuable prognostic marker for several solid tumors. Objective: We tested the hypothesis that cfDNA containing the RET M918T mutation could be detected in the blood of patients with advanced MTC whose tumor harbored an M918T mutation and would be able to predict overall survival more reliably than calcitonin. Design: The level of cfDNA containing RET M918T mutation was measured in the plasma of patients with MTC via droplet digital polymerase chain reaction. Patients: Patients had a confirmed sporadic MTC diagnosis, a serum calcitonin measurement >100 pg/mL, and tumor tissue biopsy results providing RET M918T mutation status. There were 75 patients included in this study, 50 of whom harbored an RET M918T mutation by tissue biopsy. Results: RET M918T cfDNA was detected in 16 of 50 patients (32%) with a positive tissue biopsy. The detection of RET M918T cfDNA strongly correlated with worse overall survival and more accurately predicted a worse outcome than calcitonin doubling time. Conclusions: Liquid biopsy is able to detect RET M918T mutations in patient plasma with high specificity but low sensitivity. In patients with established somatic RET M918T mutations, the allelic fraction of circulating tumor DNA is prognostic for overall survival and may play a role in monitoring response to treatment.

Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

RET fusion as a novel driver of medullary thyroid carcinoma.

INTRODUCTION: Oncogenic RET tyrosine kinase gene fusions and activating mutations have recently been identified in lung cancers, prompting initiation of targeted therapy trials in this disease. Although RET point mutation has been identified as a driver of tumorigenesis in medullary thyroid carcinoma (MTC), no fusions have been described to date. OBJECTIVE: We evaluated the role of RET fusion as an oncogenic driver in MTC. METHODS: We describe a patient who died from aggressive sporadic MTC < 10 months after diagnosis. Her tumor was evaluated by means of next-generation sequencing, including an intronic capture strategy. RESULTS: A reciprocal translocation involving RET intron 12 was identified. The fusion was validated using a targeted break apart fluorescence in situ hybridization probe, and RNA sequencing confirmed the existence of an in-frame fusion transcript joining MYH13 exon 35 with RET exon 12. Ectopic expression of fusion product in a murine Ba/F3 cell reporter model established strong oncogenicity. Three tyrosine kinase inhibitors currently used to treat MTC in clinical practice blocked tumorigenic cell growth. CONCLUSION: This finding represents the report of a novel RET fusion, the first of its kind described in MTC. The finding of this potential novel oncogenic mechanism has clear implications for sporadic MTC, which in the majority of cases has no driver mutation identified. The presence of a RET fusion also provides a plausible target for RET tyrosine kinase inhibitor therapies.

Somatic mosaicism in the Wiskott-Aldrich syndrome: molecular and functional characterization of genotypic revertants.

The reasons underlying the occurrence of multiple revertant genotypes in Wiskott-Aldrich syndrome (WAS) patients remain unclear. We have identified more than 30 revertant genotypes in a C995T WAS patient having 10-15% revertant, WAS protein (WASp)-expressing circulating lymphocytes. Of 497 allospecific T-cell clones generated from the peripheral blood, 47.1% carried a revertant sequence. All revertant T-cell clones exhibited restoration of WASp expression. However, anti-CD3-induced proliferative responses varied greatly amongst revertants. Several revertant T-cell clones expressed an internally deleted WASp mutant lacking much of the proline-rich region. This potentially accounts for the reduced anti-CD3 proliferative responses of these T-cell clones. We found no evidence for an increased DNA mutation rate in this patient. We conclude that the diversity of revertant genotypes in our patient does not result from an extraordinary mutation rate and that the amino acid sequence space explored by WASp in revertant T-cells is significantly smaller than might have been predicted from the diversity of revertant genotypes.