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Repeated Whole-Exome Sequencing of Tumor Sheds Light on Sensitivity, Resistance to Treatment

NEW YORK (GenomeWeb) – Researchers from the Dana-Farber Cancer Institute and elsewhere reported using whole-exome sequencing to uncover two previously unknown mutations that appear to be responsible for a cancer patient's unexpected response to a drug and for her subsequent resistance to that drug.

The patient, a 57-year-old woman, has metastatic anaplastic thyroid cancer, a disease that typically is fatal within five months. But after receiving everolimus as part of a clinical trial, the patient experienced a near-complete response for 18 months, though her disease then progressed.

This case study, reported today in the New England Journal of Medicine, highlights the role that repeated sequencing of tumors might have in directing patient treatment and understanding drug sensitivity and resistance.

By sequencing the patient's pretreatment tumor and drug-resistant tumor, the researchers found a nonsense mutation in TSC2, a negative regulator of mTOR, in her pretreatment tumor that could explain her response to everolimus, an allosteric inhibitor of mTOR. Meanwhile in her drug-resistant tumor, the researchers uncovered an mTOR mutation that leads to resistance to allosteric mTOR inhibitors, though not to mTOR kinase inhibitors, suggesting an avenue for further treatment,

"This is personalized, precision medicine at its best," senior author Jochen Lorch from Dana-Farber said in a statement.

The team performed whole-exome sequencing on the patient's pretreatment tumor, drug-resistant tumor, and blood to a mean depth of coverage of between 300x and 375x to suss out indels and copy-number alterations.

The pretreatment tumor, Lorch and his colleagues reported, harbored a somatic nonsense mutation in TSC2. This truncating mutation, they added, inactivates the protein, which in turn activates the mTOR pathway. This makes, they noted, some cancers sensitive to mTOR inhibition, but such a mutation had not been previously reported in thyroid cancer.

They also uncovered some 317 SNVs and 44 coding indels, including a TP53 mutation and a frameshift mutation in FLCN, a tumor-suppressor gene that is also involved in TSC2 and mTOR signaling.

In addition to the TP53, FLCN, and TSC2 mutations, the drug-resistance tumor also had a somatic mTOR mutation.

The researchers calculated that while the TP53, FLCN, and TSC2 mutations were present in between 98 percent and 100 percent of cancer cells from both the pretreatment and drug-resistance tumors, the mTOR mutation was in none of the pretreatment tumor cells and about 96 percent of the drug-resistance tumor cells.

Such an mTOR mutation, which the researchers said was localized to the FKBP–rapamycin binding (FRB) domain, had not been previously linked to everolimus resistance nor seen in patients.

The homologous mutation has been reported in fission yeast, Lorch and his colleagues said. Through a yeast two-hybrid, they noted that the yeast protein homolog of mTOR with the mutation couldn't bind FKBP-rapamycin. This suggested to the researchers that the human cancer mTOR mutation could cause resistance to allosteric mTOR inhibition by preventing binding.

In cell lines, they found that that appeared to be the case.

Kidney cells expressing mutant mTOR were more resistant to rapamycin inhibition as compared to cells expressing wild-type mTOR. Additionally, though at baseline, cells expressing mutant mTOR and wild-type mTOR had similar levels of phospohorylated S6K1, a downstream target of mTOR. However, after rapamycin treatment, only mutant mTOR-expressing cells still had phosphorylated S6K1.

As this mTOR mutation is localized to the FRB domain and not the active site, Lorch and his colleagues hypothesized that it would still be sensitive to inhibition by a direct mTOR kinase inhibitor. In cells, they found, it was.

"Because we could show that an mTOR inhibitor that is using a different mechanism could overcome resistance in anaplastic thyroid cancer, these findings could provide a rationale for treatment once resistance to everolimus occurs," Lorch said.

A new mTOR kinase inhibitor is entering clinical trials, according to Dana-Farber, and the patient described in this case study is to receive it.

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