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Circulating Tumor Cell Exome Reflects Mutational Profile of Metastatic Prostate Cancer

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NEW YORK (GenomeWeb) − A multidisciplinary team from MIT's Koch Institute for Integrative Cancer Research, the Broad Institute, and the Dana-Farber Cancer Institute has developed an integrated process for isolating circulating tumor cells, sequencing their exomes, and calling single-nucleotide variants with high fidelity, an approach that could have future clinical applications.

In a proof-of-concept study involving two metastatic prostate cancer patients that was published in Nature Biotechnology last week, the researchers showed that a large percentage of mutations in the primary tumor and its metastases could also be found in CTCs, suggesting that these cells might be useful for non-invasive cancer monitoring.

While mutational profiles of tumors are increasingly being used to guide therapy decisions, biopsies are invasive and often hard to obtain for metastases, for example in prostate cancer, which often metastasizes to the bone. For those cancers, CTCs, circulating free tumor DNA, or exosomes might offer an alternative for monitoring mutations.

But CTCs are usually rare, and often absent, and require complex approaches for isolating and characterizing them. A small number of research groups have embarked on sequencing CTCs, for example Sunney Xie's team at Harvard, which published a study on the exomes of CTCs from lung cancer patients last year.

According to Nick Navin, a geneticist at MD Anderson Cancer Center who has been working on non-invasive cancer monitoring approaches and was not involved in either study, much of the recent research in the field has focused on cell-free DNA, which has the advantage of easy sample storage but also has a lot of normal DNA mixed in. CTCs, on the other hand, represent a pure tumor population. "These papers are bringing the emphasis back to circulating tumor cells," he said.

Both studies essentially came to the same conclusion, Navin said – that a large percentage of mutations in the primary and metastatic cancer can be detected in CTCs. But the two projects used different technical approaches, and last week's study focused on sequencing the complete exome across a population of CTCs, rather than individual cells.

"Up until now, there had not been comprehensive statistical calculations on what was required to accurately call the entire exome, or in the future, perhaps the entire genome of these rare populations of cells," Jesse Boehm, assistant director of the Broad Institute's cancer program and a senior author of the new study, explained.

"We engineered an entire process, which we think is very flexible for addressing and recovering these cells and then identifying the variants in them," said Chris Love, another senior author who is a professor of chemical engineering at MIT and a member of the Koch Institute. "The advance is not a single technology, but rather, it's the entire process that we built and put together, which is very modular in design."

Love and his colleagues devised a two-step method for isolating CTCs from a small vial of blood. It involves the MagSweeper technology – developed by Stanford researchers and commercialized by Illumina – to enrich CTCs expressing the EpCAM surface protein; and nanowell arrays, developed in Love's lab, to visualize individual CTCs and pick them out with a robotic device called CellCelector from Automated Lab Solutions.

They then amplified each CTC's genome by multiple displacement amplification using the RepliPHI kit from Epicentre, which is owned by Illumina.

After that, the researchers performed low-pass whole-genome sequencing, allowing them to predict which CTC sequencing libraries would yield high-quality exome sequencing data.

Researchers at the Broad Institute then sequenced the exomes of these high-quality libraries using the Agilent v2 Human Exon kit and Illumina's HiSeq. As predicted, only a fraction of the total exome could be sequenced in each individual cell, owing to random biases of the single-cell whole-genome amplification process.

But by combining exome data from several independently sequenced CTCs − an approach called census-based sequencing − the scientists were able to cover virtually the entire exome and accurately call single-nucleotide variants using computational methods developed by Gad Getz's group at the Broad Institute.

The goal of the study was not to characterize individual CTCs, Boehm noted, but to sequence as much of their combined exome as possible. "Along the way, we found out that one needed to sequence multiple libraries" for this, he said, which could come from individual cells, as in their study, or from small pools of CTCs.

To test whether mutations found in the CTCs reflect those in the original tumor, the researchers compared the exome data with sequencing data from multiple samples of a prostate cancer patient's primary tumor and one of his metastases.

They found ten single-nucleotide variants that were present in all primary tumor samples and the metastasis, so-called early trunk mutations, nine of which were also present in the CTCs. Fifty-six mutations were present in a least some primary tumor samples, as well as the metastasis, and the CTCs had 41 of these so-called metastatic trunk mutations.

Because such trunk mutations are likely present in most metastases of patients with advanced cancer, sequencing CTCs could have clinical utility, the authors noted.

They also found a large number of mutations in the CTCs that did not occur in the original tumor, Navin pointed out, which might at least in part result from technical errors.

For wider use of the approach, its throughput and cost − currently "many thousands of dollars" because of the need to sequence many libraries for each patient − will need to be improved, Boehm said.

For clinical use in particular, Navin said, the CTC isolation method, which currently requires a lot of hardware, would need to be streamlined and built into "more of a desktop" device. Also, he said, if the sequencing coverage for each individual cell could be increased, fewer cells would need to be sequenced for each patient.

"The designed modularity of our approach can accommodate new emerging technologies for the enrichment and isolation of CTCs, whole-genome amplification, and sequencing platforms," the authors noted.

Also, CTCs in non-prostate cancer do not always express EpCAM, but there might be other CTC-specific markers, or other properties by which CTCs could be isolated, Love said, so the approach could be applied to other cancer types.

Following their proof-of-concept, the scientists would like to apply their process to more patients, both in prostate cancer and other tumor types. "In prostate cancer, we would like to extend this initial work to perhaps several dozen patients," Boehm said, adding that obtaining appropriate samples is challenging.

"We're interested in [asking] some deeper questions about how CTCs relate to different metastatic sites of the body and how those cells might reflect early signs of resistance in the course of therapy," Love said.

In longitudinal studies, for instance, the researchers would like to analyze several blood samples from the same patient over time in order to see whether CTCs can predict drug resistance. "There is no evidence for that yet, but the field is very excited about that potential," Boehm said.

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