Abhijit Patel, MD, PhD

The release of nucleic acids from tumors into the bloodstream has been well documented in patients with various types of cancer. Recent technological advances are making it possible to analyze these circulating DNA and RNA fragments in ways that are proving to be clinically informative. To better understand the biological features and limitations of circulating nucleic acids, further improvements in assay methods are still needed. Our research efforts are focused on developing more robust analytical methods and on establishing the clinical utility of these methods.

ASSAY DEVELOPMENT:

Circulating tumor DNA

Tumor-derived DNA fragments in the circulation can be distinguished based on the presence of cancer-specific mutations. However, these mutant DNA copies are often obscured by a relative excess of wild-type background DNA in the plasma. In patients with early stage tumors or minimal residual disease following treatment, the fraction of mutant tumor-derived DNA in plasma can be well below 1 in 1,000. Detection of such low-abundance DNA variants poses a significant technical challenge, especially if one has no prior knowledge of the tumor’s mutation profile. We have developed methods to enrich mutation-prone regions of plasma DNA by highly multiplexed PCR. We use next-generation DNA sequencing technologies to oversample thousands of copies of these mutation-prone genomic regions, allowing rare sequence variants to be identified and enumerated. However, the sensitivity of this approach is limited by sequencer errors and PCR misincorporations, both of which might be mistaken for true mutant copies. To overcome this limitation, we have devised molecular and computational error-suppression strategies that enable ultrasensitive detection of rare mutant copies with broad mutation coverage.

High-throughput RNA profiling

Tumors are known to shed RNA molecules into blood. These circulating RNAs are surprisingly stable when bound to serum proteins or when encapsulated within tiny vesicles called exosomes. Such tumor-derived RNAs are showing great promise as biomarkers for cancer diagnosis and treatment monitoring. To facilitate analysis of RNAs from many patient samples in a high-throughput, low-cost manner, we have developed a method called META RNA profiling (for “modular early-tagged amplification”). The method is able to quantify a broad panel of microRNAs or messenger RNAs simultaneously across a large number of samples. It uses a next-generation sequencing readout, but demands far less sequence depth than existing digital RNA profiling approaches. An up-front quantitative tagging scheme allows all samples to be pooled, simplifying downstream processing steps and reducing inter-sample variability. While this approach can be applied to RNA samples derived from a variety of biological sources, we are using it as a tool to analyze gene expression in tumors and in clinical biofluids.

CLINICAL APPLICATIONS:

Liquid biopsy

The ability to sample tumor-derived nucleic acids in blood provides an opportunity to analyze a tumor’s mutations and gene expression signatures without requiring an invasive procedure to obtain a tissue specimen. Such information can be used to personalize cancer therapy, as drug choices are often guided by knowledge of a tumor’s genomic profile. Moreover, because tumors are known to be heterogeneous, a tissue biopsy taken from a single tumor site might miss critical genetic features present in other metastatic sites within the same patient. Because blood contains a sampling of nucleic acids derived from all tumors in the body, it is likely to enable a more comprehensive assessment of genomic heterogeneity. We are testing these concepts using clinical samples from patients with various types of cancer.

Assessment of treatment response

As is true for many protein biomarkers, quantitative changes in the levels of circulating nucleic acids appear to be correlated with treatment response. This may prove useful in assessing the response of tumors for which good serum protein markers are not available. We are also investigating whether unique aspects of nucleic acid specificity and kinetics can be exploited to complement information obtained from protein markers.

Emergence of treatment resistance

Genomic profiles of tumors are known to evolve over time. This can be especially important when a therapy leads to the selective proliferation of tumor cell populations containing mutations that confer treatment resistance. Therapy can be modified if the emergence of resistant clones is documented by repeating a biopsy, but such invasive procedures can be risky and impractical. We are evaluating whether nucleic acids in blood can be used to noninvasively track genomic and transcriptomic changes in tumors over time. Might we be able to predict treatment resistance before it becomes clinically evident?

Early detection of new and recurrent cancer

It has long been one of the grand challenges of oncology to detect tumors at their earliest stages, when treatment is more likely to be curative. However, efforts to develop blood tests for early cancer detection have had limited success because of the difficulty in identifying serum protein biomarkers that are sufficiently cancer-specific. Circulating tumor DNA may be a better-suited marker for cancer screening because tumor-specific somatic mutations can be used to distinguish tumor-derived DNA molecules. Since cancer-associated mutations should rarely be found in the plasma of healthy individuals, false-positive results are expected to be extremely uncommon. Furthermore, because there is no physiologic background, a small amount of mutant DNA released from an early-stage tumor should be detectable if the technical background of the assay can be minimized. Similar principles apply to the detection of small amounts of residual or recurrent disease following curative-intent therapy. We are keenly interested in evaluating whether our ultrasensitive ctDNA assay can be used for early detection of new or recurrent tumors.