In the MI-ONCOSEQ study, we aimed to translate high-throughput sequencing into a viable analysis tool for biomarker or mutation-driven clinical trials and, in doing so, addressed important logistical challenges (). First, the study enrolled patients eligible for early clinical trials and completed sequencing efficiently to potentially allow stratification to trial on the basis of the sequencing results. Second, the study addressed ethical implications of genome sequencing through an informed consent process with concurrent input from bioethicists. Third, the study established an STB to deliberate on the clinical value of sequencing results, including those that are unexpected.
Despite these efforts, we anticipate the need for improvements and modifications to the process and procedures used here. Because the pilot study was implemented in a research setting, we did not offer testing as a routine or billable service. Any results that affect clinical decision-making must be validated using a CLIA-certified test. As a next step, we anticipate that the molecular genetics and pathology communities will move high-throughput sequencing toward CLIA certification, which will ultimately reduce costs and improve turnaround time of results. Additionally, declining sequencing costs will make our approach even more practical. The per-patient price tag decreased from $5400 six months ago to $3600 at present. This cost is comparable to routine clinical tests such as OncotypeDx and is financially practical for every patient who is considering clinical trials.
MI-ONCOSEQ has used a combination of DNA and RNA sequencing to reveal a broad view (11
) of an individual's genetic aberrations. Moving forward, we anticipate that incorporation of global epigenetic and small RNA analyses, as well as evolving bioinformatics algorithms, will provide complementary information and enable cross-validation (37
). Alternative strategies that assess a limited panel of genes or genetic aberrations (38
) optimize sensitivity to detect aberrations in clinically informative genes, but are of limited use for basic science research. This trade-off between breadth and sensitivity will be an important consideration in heterogeneous samples with multiple subclones as well as biopsies with low tumor purity. For patients 3 and 4, samples were of acceptable purity and clinically informative variants were captured at substantial depth (fig. S7
), but this does not rule out missed mutations. Therefore, it will be important to develop approaches to assess samples with low tumor purity. Aside from increasing sequencing depth to compensate for low tumor content, one could enrich for tumor-relevant DNA through microdissection, cell-based enrichment, or ploidy-based sorting.
Although others have demonstrated the potential benefits of highthroughput assays for individual patients with cancer (41
), the next logical step is facilitating clinical trials in oncology with biomarker informed therapies. Clinical investigators are increasingly recognizing the importance of patient selection by mutation assessment when using targeted therapeutic agents (42
). The proven effect of this approach in the recent BRAF and ALK phase 1 trials demonstrates the need for molecular stratification (44
). Here, integrative sequencing identified informative oncogenes that would have been missed by standard single-gene clinical assays or approaches with a limited panel of genes. Both patients 3 and 4 had potentially informative aberrations, but these patients did not fit into available trials. Patient 3's CDK8 amplification and NRAS activating mutation provided a good rationale for use of investigational agents such as CDK inhibitors and combined MEK/PI3K inhibition (46
). A phase 1 trial is pending for doxorubicin plus seliciclib (a CDK inhibitor with activity for CDK8) in patients with breast cancer. However, because of the study's limited eligibility for breast cancer, the patient was not eligible (NCT01333423). Similarly, we identified a phase 1 study for a MEK inhibitor in patients with CRC who have BRAF or KRAS, but not NRAS, activating mutations (NCT00959127). This lack of suitable trials for our two patients may be an early warning that we need to restructure the eligibility criteria for trials of molecularly targeted therapies. We envision an array of available mutation and pathway-based trials for targeted therapies, with eligibility based on molecular assessment.
In addition to identifying aberrations in informative genes, integrative sequencing permits discovery research, such as the NEK11 gene fusion (patient 1) and the AURKA alterations (patient 3). Although difficult to interpret at present, these events could plausibly represent rare or “private” drivers or resistance mechanisms. In this context, the sequencing results can serve as a source of correlative data for trials with molecularly targeted therapies. If patients are treated with matching targeted therapies and develop secondary resistance, repeat tumor biopsy and assessment could reveal mechanisms of resistance, for example, the emergence of a resistant subclone. These data can inform the rational combination of targeted therapies to maximize efficacy and response (47
) and minimize resistance. This suggests a future need for the systematic inclusion of tumor biopsies for patients on trials.
Although state-of-the-art technology in genomic sequencing has markedly accelerated biomedical research, translation to the clinical setting has numerous barriers that limit potential benefits. Therefore, we must strive to develop evidence-based, ethically sound guidelines for implementing genomic sequencing in clinical medicine. This multidisciplinary endeavor provides an early road map for translating high-throughput sequencing into biomarker-driven clinical trials in oncology.