Genome Sequencing Identifies a Basis for Everolimus Sensitivity

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Science  12 Oct 2012:
Vol. 338, Issue 6104, pp. 221
DOI: 10.1126/science.1226344


Cancer drugs often induce dramatic responses in a small minority of patients. We used whole-genome sequencing to investigate the genetic basis of a durable remission of metastatic bladder cancer in a patient treated with everolimus, a drug that inhibits the mTOR (mammalian target of rapamycin) signaling pathway. Among the somatic mutations was a loss-of-function mutation in TSC1 (tuberous sclerosis complex 1), a regulator of mTOR pathway activation. Targeted sequencing revealed TSC1 mutations in about 8% of 109 additional bladder cancers examined, and TSC1 mutation correlated with everolimus sensitivity. These results demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs.

A long-standing problem in oncology is the variability of treatment response observed in early stage clinical trials. Drugs that fail to induce disease regression in most patients or prolong median progression-free survival (PFS) are deemed inactive and often abandoned, even when the drug exhibits profound activity in a small number of patients. We hypothesized that sequencing the tumor genomes of such “outlier” patients might identify unique somatic alterations that are the basis of their drug response, information that could in turn inform future clinical development.

We studied the tumor genome of a patient with metastatic bladder cancer who achieved a durable (>2 years) and ongoing complete response to everolimus, a drug targeting the mTORC1 (mammalian target of rapamycin) complex (Fig. 1A). The patient was enrolled in a phase II trial ( NCT00805 129) that failed to achieve its PFS end point. Whole-genome sequencing of DNA derived from the primary tumor and blood (1) identified 17,136 somatic missense mutations and small insertions and deletions (mutation rate of 6.21 per million bases). Of these, 140 were nonsynonymous mutations within protein-coding or noncoding RNA regions of the genome. Structurally, this tumor genome was intact, lacking significant copy number alterations or functional translocations (Fig. 1B). Among confirmed coding mutations were (i) a two-base-pair deletion in the TSC1 (tuberous sclerosis complex 1) gene, resulting in a frameshift truncation (c.1907_1908del, p.Glu636fs), and (ii) a nonsense mutation in the NF2 (neurofibromatosis type 2) gene, creating a premature stop codon (c.863C>G, p.Ser288*). These loss-of-function mutations were noteworthy (table S1) because alterations in these genes have been associated with mTORC1 dependence in preclinical models (2). Sequencing of both genes in a second cohort of 96 high-grade bladder cancers identified five additional somatic TSC1 mutations, whereas no additional NF2 mutations were detected (fig. S1). Although the NF2 mutation was uncommon in bladder cancers, knockdown of NF2 expression in TSC1-null bladder cancer cells was associated with enhanced sensitivity to mTORC1 inhibition (fig. S1).

Fig. 1

(A) Computed tomography images of the index patient demonstrating complete resolution of metastatic disease (arrows). (B) Somatic abnormalities in the outlier responder’s genome included (from outside to inside) copy number alterations; mutations at ~10-Mb resolution; regulatory, synonymous, missense, nonsense, nonstop, and frameshift insertion and deletion mutations (black, orange, red, green, and dark green); and intra- and interchromosomal rearrangements (light and dark blue). (C) Best overall response of 14 sequenced trial patients. Negative values indicate tumor shrinkage (red line, threshold for partial response). Gradient arrow, patient with rapid progression in bone.

Because TSC1 is mutated in a subset of bladder cancers (3), we explored whether TSC1 mutation is a biomarker of clinical benefit from everolimus therapy in this disease. We thus analyzed 13 additional bladder cancer patients treated with everolimus in the same trial with a targeted deep sequencing assay designed to interrogate the coding exons of ~200 genes commonly mutated in human cancers [(1) and fig. S1]. This analysis revealed three additional tumors harboring nonsense mutations in TSC1, including two patients who had minor responses to everolimus (Fig. 1C; 17 and 24% tumor regression). A fourth patient with 7% tumor regression had a somatic missense TSC1 variant of unknown functional consequence. In contrast, tumors from eight of the nine patients showing disease progression were TSC1 wild type. Patients with TSC1-mutant tumors remained on everolimus longer than those with wild-type tumors (7.7 versus 2.0 months, P = 0.004) with a significant improvement in time to recurrence (4.1 versus 1.8 months; hazard ratio = 18.5, 95% confidence interval 2.1 to 162, P = 0.001).

These results suggest that mTORC1-directed therapies may be most effective in cancer patients whose tumors harbor TSC1 somatic mutations (4) and demonstrate the feasibility of using whole-genome and capture-based sequencing methodologies in the clinical setting to identify previously unrecognized biomarkers of drug response in genetically heterogeneous solid tumors. Although single-patient anecdotes are often dismissed as failing to provide meaningful clinical evidence, this example illustrates the potential for such cases to inform future clinical development of drugs in molecularly defined populations.

Supplementary Materials

Materials and Methods

Fig. S1

Table S1

References (521)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: The authors would like to acknowledge C. Sawyers for critical reading of the manuscript and G. Getz and K. Cibulskis for sharing a prepublication version of the MuTect algorithm. This work was supported in part by the Michael and Zena Wiener for Therapeutics Program in Bladder Cancer, a Stand Up to Cancer Dream Team Translational Research Grant, and a Program of the Entertainment Industry Foundation (D.B.S.); Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center, the Geoffrey Beene Cancer Research Center at Memorial Sloan-Kettering Cancer Center, and an American Society of Clinical Oncology Young Investigator Award (G.I.); and the David H. Koch Foundation (B.S.T.). Funding for the phase II clinical trial of everolimus in advanced urothelial cancer was provided by Novartis Pharmaceuticals. D.F.B. is a paid consultant for Novartis, which markets everolimus. M.F.B. is a paid consultant for Foundation Medicine, a company specializing in personalized cancer medicine. The compressed binary Sequence Alignment/Map files (BAM) have been deposited in dbGaP (accession no. phs000535.v1.p1).
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