We have shown that the CTC-chip reproducibly isolated circulating tumor cells in sufficient quantity and with sufficient purity to allow molecular analyses. Circulating tumor cells were readily identified in all patients in numbers that were higher than those identified with previously available methods by a factor of approximately 100.6–9
The use of the allele-specific SARMS assay to identify EGFR mutations in rare cell populations was made possible by the recurrent nature of these mutations, with only 2 of 31 patients carrying mutations identified by sequencing that were absent from the assay panel. Together with the CTC-chip, the SARMS assay may allow for non-invasive genotyping in patients with non–small-cell lung cancer, which could be repeated at therapeutic decision-making points during a patient’s course of therapy. Genotyping of circulating tumor cells appeared to be more sensitive than analysis of free plasma DNA (P = 0.009), and the concomitant quantification of circulating tumor cells provided an important context in which to interpret genotyping results.
The analysis of circulating tumor cells commonly identified the T790M drug-resistance mutation in a majority of patients who had clinical tumor progression while receiving therapy with tyrosine kinase inhibitors. Unexpectedly, use of the highly sensitive allele-specific assay showed that a subgroup of patients with the EGFR
mutation harbor rare T790M alleles both before exposure to tyrosine kinase inhibitors and during clinical response. T790M is thought to emerge through selective pressure during therapy, although it has been reported in rare cases in patients without previous drug exposure,25,26
and the mutation confers additional transforming properties when combined in cis
with the more common EGFR
Thus, T790M may initially arise by virtue of its oncogenicity and may rapidly emerge as a dominant allele after treatment. The presence of rare T790M alleles in pretreatment tumor specimens did not preclude dramatic responses to tyrosine kinase inhibitors but did have a significantly adverse effect on progression-free survival. We speculate that this molecular marker may be a way of distinguishing patients who are likely to have a prolonged response to erlotinib or gefitinib from those whose response is short-lived and who may be appropriate candidates for second-generation, irreversible tyrosine kinase inhibitors or combination targeted-therapy regimens. Although amplification of the c-met proto-oncogene (MET
) has recently been reported as a second mechanism of acquired resistance to tyrosine kinase inhibitors,19,20
we did not detect it in pretreatment tumor-biopsy specimens or in circulating tumor cells collected during therapy using a quantitative PCR assay (data not shown).
The most unexpected observation in our study was the emergence of additional activating EGFR mutations in circulating tumor cells during therapy. Although the SARMS assay cannot determine whether two different mutations are present on the same or on separate alleles, the patient for whom we had sufficient DNA for direct sequencing had mutually exclusive mutations in the original tumor and in circulating tumor cells (). We therefore presume that the identification of different EGFR activating mutations represents the emergence of different tumor clones. In some patients, additional mutations emerged during tumor progression after chemotherapy. Therefore, such mutations may be associated with drug-induced shifts in tumor-cell populations, reflecting clonal selection during treatment.
The mutation of EGFR
is thought to be an early molecular event in the genesis of non–small-cell lung cancer in nonsmokers on the basis of the transforming potential of these mutations in vitro and in mouse models.27–30
Although most non–small-cell lung cancers appear to have a single clonal EGFR
mutation that is evident at multiple sites of metastatic disease, rare cases of bronchoalveolar cancer may present with multifocal tumors, each harboring different EGFR
Circulating tumor cells may be derived from multiple disease sites with different responses to therapy and be associated with an evolution in tumor genotypes that cannot be appreciated by a single tissue biopsy performed at the time of presentation. The absence of treatment-induced genetic change may explain why recent clinical trials of first-line tyrosine kinase inhibitors in patients with EGFR
mutations have shown a high concordance between tumor genotype and clinical response,21,31,32
as compared with earlier second-line and third-line studies, in which multiple courses of chemotherapy separated the diagnostic tumor specimen from the clinical evaluation of responsiveness to tyrosine kinase inhibitors.33,34
Therefore, genotype-directed clinical trials of molecularly targeted agents may benefit from “real time” tumor genotyping, either in the form of coincident tumor biopsy or the analysis of circulating tumor cells.
Direct sequencing of an EGFR
mutation indicates that the captured circulating tumor cells represent a largely pure tumor-derived cell population. Further characterization of such precursors of metastasis35
may provide important opportunities for diagnostic and therapeutic interventions. However, optimization and automation of the device for capturing circulating tumor cells for high-throughput processing will be required to allow large-scale clinical trials that use this novel technology.