Clinical oncology is in the midst of transition from a treatment paradigm dictated primarily by the anatomic site of tumor origin to one in which genetic and/or molecular characteristics play a decisive role in guiding choice of therapy. Moreover, the proliferation of targeted agents in development and clinical practice necessitates concomitant implementation of companion diagnostic approaches that enrich for subpopulations most likely to respond to a drug. New diagnostic approaches are therefore needed to profile any tumor for pivotal genetic mutations in multiple cancer genes simultaneously, in contrast to most existing tests that focus on single genes (or proteins).
In this study, we adapted genotyping-based mutation profiling for the characterization of both frozen and FFPE-derived tumor specimens spanning 12 cancer types. We robustly detected cancer gene mutations that direct clinical use and predict resistance to existing agents such as tyrosine kinase inhibitors (e.g., EGFR and KRAS mutations). We also identified multiple mutations that may guide the use of emerging agents. Finally, in a specific investigation of pediatric low-grade astrocytomas, we demonstrated the special value this platform may have for rare “orphan” cancers by identifying mutations that may inform molecular classification as well as new therapeutic avenues for children with these malignancies.
Diagnostic interventions that successfully introduce tumor mutation profiling to clinical practice must circumvent several technical and logistical hurdles. Chief among these is the attainment of robust performance in samples derived from FFPE and/or archival tumor material. We found that the OncoMap platform achieved nearly 100% specificity in both fresh/frozen and FFPE-derived tumor DNA, indicating that false positive mutation calls are likely to be relatively rare with this approach. It should be noted, however, that achieving this level of specificity required the implementation of an analytical sequence in which raw genotyping data is subjected to automated base calling followed by manual review of candidate mutations and validation of all candidates using alternative genotyping chemistries. Thus, clinical implementation of OncoMap must incorporate both genomic data generation and bioinformatic analytical expertise into a molecular pathology or clinical diagnostic setting.
The sensitivity of OncoMap is influenced by inherent technological parameters, individual mutation assay performance characteristics, and the quality and purity of tumor tissue. The 89–94% assay sensitivity observed in this study is sufficient for many translational and clinical applications; however, there are of course circumstances where even higher assay sensitivities will be desirable. Enrichment of tumor cells using core needle dissection or laser-capture microdissection prior to mutation profiling may offer one avenue to enhance sensitivity, particularly in tumors where the stromal or inflammatory content is high. At the same time, the sensitivity of OncoMap vastly exceeds that of Sanger sequencing, which remains the gold standard for many genetic diagnostic approaches. Furthermore, the breadth of cancer genes and specific mutations interrogated by OncoMap—supported by the aforementioned rigorous sensitivity and specificity determinations—substantially exceeds that of existing commercial mass spectrometric-based genomic profiling approaches
Genomically guided therapies may play an especially prominent role in rare tumors where large randomized trials are often impractical. To test the ability of the OncoMap to identify uncommon and/or sample-limited tumors that may benefit from specific classes of therapeutic agents, we profiled a panel of pediatric low-grade gliomas for common cancer gene mutations. Knowledge of the genetic abnormalities present in pediatric LGAs is limited, though recent studies have identified BRAF
translocations, chromosomal duplications, and occasional base mutations in low-grade astrocytomas
, as well as diverse mechanisms for activating the ERK/MAPK pathway in pilocytic astrocytomas 
. These findings suggest that the small molecule inhibitors of BRAF
already in adult trials may also represent promising therapeutics for subsets of these tumors. Our results indicate that the frequency of BRAF
point mutations in pediatric LGAs as a whole may be higher than previously reported, and specifically that gangliogliomas possess BRAFV600E
mutations at very high frequency. This observation may also aid in diagnostic identification of these tumors. Gangliogliomas, which tend to be indolent tumors, may therefore share some properties with cutaneous nevi, whose melanocyte precursors also derive from the nervous system (neural crest), exhibit indolent growth, and where the BRAFV600E
mutation is also highly prevalent. We also identified several mutations not previously reported in pediatric astrocytoma, some of which (EGFR
) represent potentially actionable targets. In concordance with previous reports 
we observe that mutations in genes frequently observed in adult anaplastic astrocytomas and/or glioblastomas, such as TP53
, are only rarely encountered in pediatric pilocytic and low-grade diffuse astrocytomas.
Although our findings support the clinical feasibility of high-throughput tumor mutation profiling, we recognize that the mass spectrometric genotyping approach has certain limitations that may preclude its implementation as a definitive cancer diagnostics platform. These include the finite number of specific point mutations that can be assayed (designated a priori within a subset of cancer genes), difficulties in designing genotyping assays that identify small insertions or deletions (“in-dels”) larger than ~50bp in size, an inability to detect most TS gene mutations (which may occur anywhere within the gene, not just “hotspot” regions) or additional genomic alterations such as high-level gene amplifications or deletions that may also affect key cancer genes, and the somewhat labor-intensive nature of manual review and orthogonal assay validation. Over the long term, the adaptation of new genomics technologies such as second generation sequencing may offer a unifying approach to comprehensive tumor mutation profiling. However, the OncoMap platform may offer one immediate avenue by which systematic mutation profiling might be initiated to guide clinical trial design as well as use of existing targeted agents across many cancer types.
In summary, this study represents the first large-scale application of the OncoMap platform for tumor mutation profiling in the clinical and archival setting. These results therefore enliven a framework wherein systematic tumor profiling might emerge as a widely feasible means to guide patient stratification for rational cancer therapeutics. Despite the inherent complexity of cancer, incorporating the growing knowledge of the molecular basis of cancer into both large-scale molecular epidemiologic studies and, ultimately, clinical decision making should ultimately speed the advent of more effective anticancer therapies.