Response and acquired resistance to everolimus in anaplastic thyroid cancer

Abstract

Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is effective in treating tumors harboring alterations in the mTOR pathway. Mechanisms of resistance to everolimus remain undefined. Resistance developed in a patient with metastatic anaplastic thyroid carcinoma after an extraordinary 18-month response. Whole-exome sequencing of pretreatment and drug-resistant tumors revealed a nonsense mutation in TSC2, a negative regulator of mTOR, suggesting a mechanism for exquisite sensitivity to everolimus. The resistant tumor also harbored a mutation in MTOR that confers resistance to allosteric mTOR inhibition. The mutation remains sensitive to mTOR kinase inhibitors.

Figure 1. Histologic Findings and Computed Tomographic (CT) Scans in a Patient with Metastatic Anaplastic Thyroid Carcinoma

Hematoxylin and eosin staining of a total-thyroidectomy specimen (Panel A, top) shows anaplastic thyroid carcinoma; MIB-1 staining (Panel A, bottom) reveals the high proliferative rate of the tumor. A histologic section (hematoxylin and eosin) of an enlarged mediastinal lymph node, obtained after 18 months of a response to everolimus monotherapy, shows recurrent thyroid carcinoma (Panel B). Axial CT scans of the chest show a right-sided hilar mass (arrow) before treatment with everolimus (Panel C), 6 months after treatment initiation (Panel D), and at the time of everolimus resistance, 18 months after treatment initiation (Panel E).

Figure 2. Mutations in TSC2 and MTOR in Anaplastic Thyroid Carcinoma Revealed by Whole-Exome Sequencing

Whole-exome sequencing of the tumor tissue before treatment and after the development of resistance to everolimus revealed a TSC2 nonsense mutation in both tumors (Panel A) and an F2108L mutation in MTOR in the resistant tumor that was undetectable in the pretreatment tumor (Panel B). Representative genome images from the Integrative Genomics Viewer (Broad Institute), along with the number of reads for the reference allele and the variant allele, are shown for each alteration. A comparison of the proportion of cancer cells harboring specific mutations in the pretreatment biopsy sample (Panel C, x axis) versus those in the post-resistance biopsy sample (y axis) is shown. Mutations in TSC2, TP53, and FLCN were present in all cancer cells from both biopsy samples (yellow triangle at upper right), whereas the MTOR mutation was detected in all cancer cells in the post-resistance biopsy sample but was not detected in the pretreatment biopsy sample (red triangle at upper left). Additional mutations were detected in all cancer cells in the pretreatment biopsy sample but in no cancer cells in the post-resistance biopsy sample (light blue triangle at lower right). Other mutations were predicted to occur in a subgroup of the cancer cells in the pretreatment biopsy but in none of the cancer cells in the post-resistance biopsy sample (dark blue) or in a subgroup of the cancer cells in the post-resistance biopsy sample but in none of the cancer cells in the pretreatment biopsy sample (dark red). Shaded areas denote Bayesian posterior probability distributions over cancer-cell fraction values for each mutation. Gray shading indicates mutations with cancer-cell fraction distributions having considerable uncertainty, with lighter shading indicating greater uncertainty.

Figure 3. Effects of Nonmutant mTOR and mTOR^F2108L

Ribbon rendition of mTOR crystal structure (PDB 1FAP) for nonmutant mTOR (Panel A) and mTOR^F2108L (Panel B) shows the FKBP (FK506 binding protein)–rapamycin binding (FRB) domain, FKBP12, and rapamycin. Enlarged views show that the substitution of leucine for phenylalanine sterically hinders rapamycin binding (Panels A and B, right side). Ribbon rendition of mTOR crystal structure (PDB 4JSX) (Panel C) highlights the kinase domain (KD) C-terminal lobe, KD N-terminal lobe, FRB domain, and FAT domain. As shown, residue F2108 is not expected to be involved with binding of torin 2. Growth-inhibition curves are shown for rapamycin (Panel D) and torin 1 (Panel E) in human embryonic kidney (HEK) 293T cells stably expressing nonmutant mTOR or MTOR^F2108L. Constructs expressing mTOR^F2108L or nonmutant mTOR were expressed in HEK 293T cells (Panel F). The levels of phosphorylated and total S6 kinase 1 (S6K1), the downstream target of mTOR, are shown for the HEK 293T cells after treatment with 0 nM, 0.5 nM, 2.5 nM, or 12.5 nM of rapamycin or torin 1, as indicated. GAPDH denotes glyceraldehyde 3-phosphate dehydrogenase.