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Sequenta, Chronix Pursue NGS Tests for Minimal Residual Disease in Cancer Patient Blood Samples

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At recent clinical meetings, two firms — Sequenta and Chronix Biomedical — presented data illustrating the distinct next-generation sequencing-based strategies each is taking to monitor minimal residual disease in cancer patients.

At this year's American Society of Clinical Oncology meeting in Chicago, for instance, Chronix researchers co-authored a poster showing that it was possible to track MRD after surgery in women with breast cancer using tumor markers found in blood samples taken prior to surgery.

That work hinged on an enrichment and sequencing scheme that Chronix uses to find distinct, patient-specific tumor markers in circulating cell-free DNA from individuals with various solid tumor types — a method that firm eventually hopes to advance into the companion diagnostics arena.

For Sequenta, the ASCO meeting and other recent clinical conferences are affording an opportunity to highlight the firm's ongoing collaborations with researchers using its immune cell receptor-focused platform, known as LymphoSight (see CSN 8/15/2012, CSN 12/14/2011), to test for MRD in individuals with various blood cancer types.

"In the last couple years we've been doing a lot of clinical validation work in all sorts of different hematological malignancies," Sequenta CEO Thomas Willis told Clinical Sequencing News.

In particular, he noted that the LymphoSight platform has compared favorably with standard methods for MRD detection such as flow cytometry, allele-specific PCR, protein profiling, or radiographic imaging. The approach has also shown promise for predicting patient outcomes and finding those at risk of relapse.

Sequenta secured CLIA certification for its San Francisco-based lab this past October (CSN 10/24/2012) and began offering a clinical assay known as ClonoSight in February of this year. The ClonoSight test is centered on Sequenta's LymphoSight platform and is initially being offered for individuals with acute lymphocytic leukemia, chronic lymphocytic leukemia, and mantle cell lymphoma.

Sequenta is currently in talks with insurance payers regarding reimbursement for its existing clinical test.

In poster presentations at ASCO, meanwhile, teams led by investigators in Spain and Japan reported on the utility and prognostic potential of using Sequenta's sequencing-based LymphoSight platform to monitor MRD in multiple myeloma.

At a European Hematology Association meeting kicking off later this week in Stockholm, Sequenta's collaborators also plan to present data on the use of LymphoSight for monitoring MRD in follicular lymphoma.

Such studies represent "the latest in a long series of clinical collaborations," Willis noted.

"We will continue to publish clinical validation studies over the next year to demonstrate the power of this technology to identify patients at risk of relapse," he said.

In a study led by Hospital Universitario 12 de Octubre researcher Joaquin Martinez-Lopez, for example, investigators tried their hand at using LymphoSight to find MRD markers in blood samples from 68 individuals with multiple myeloma enrolled through Spanish Myeloma Group trials.

The group, which included researchers from Sequenta, used LymphoSight to track down cancer-related immunoglobulin gene rearrangements in diagnostic samples from 59 of the 68 patients.

When they followed those individuals over time after treatment, Martinez-Lopez and colleagues found that patients who did not have LymphoSight-detectable MRD after treatment showed an uptick in both progression-free survival and overall survival rates.

Similar studies involving larger groups of multiple myeloma patients are already in the works, Martinez-Lopez told CSN in an email message.

As a group, such studies support the notion that there's a prognostic benefit to picking up increasingly lower levels of residual disease, Willis noted, adding that there are "more and more options by which patients at risk of relapse from leukemia and lymphoma can be treated."

Because the LymphoSight approach focuses on sequences within immunoglobulin genes, Willis said the platform's clinical and research applications could theoretically extend beyond MRD detection in cancer into other areas where immune profiling is beneficial.

"Residual disease testing is both an important, near-term clinical problem and one in which the clinical validation could be done relatively expeditiously, so it's the first product we're bringing to market," he said. "But we have follow-on programs in several other disease states as diverse as autoimmune disease, profiling of immunotherapy response, and other [diseases]."

In its search for signs of MRD in individuals with various solid cancer types, meanwhile, San Jose, Calif.-based Chronix has been developing MRD detection methods that couple next-generation sequencing with enrichment for cancer-specific signatures associated with double-stranded DNA breaks.

In a collaboration with other investigators at Chronix, the University of Texas MD Anderson Cancer and Vanderbilt University, for instance, Chronix CEO Howard Urnovitz used this method to track MRD in cell-free circulating DNA from 16 women before and after surgical treatment for breast cancer. The team described findings from that pilot study in a poster at the recent ASCO meeting.

The work is paving the way for a larger study of at least 100 women receiving neoadjuvant treatment for breast cancer — a move toward validating the Chronix MRD detection scheme for use as a neoadjuvant companion diagnostic.

If successful, the product would be the first cancer-related companion diagnostic test offered by Chronix, Urnovitz noted. He told CSN that the firm also hopes to develop similar companion diagnostic tests for prostate and ovarian cancer in order to distinguish between cases that are successfully resolved with primary treatments and those that require follow-up interventions.

Based on discussions at ASCO, Chronix is potentially interested in collaborating with pharmaceutical companies working on new cancer treatments, too, Urnovitz noted, since the ability to pick up MRD after treatment could offer a peak at specific patient responses to burgeoning therapies.

While it's possible to profile an individual's original tumor from cell-free circulating DNA using next-generation sequencing to pick up glitches or fusions within specific panels of genes, Urnovitz argued that approaches focused on predetermined sets of genes or markers might miss later stages of disease involving clonal derivatives that have shifted in their genetic profiles since the time of diagnosis.

To get around that issue, he and his colleagues have based their cell-free tumor DNA profiling method around a sequence signature associated with double strand DNA breaks in cancer — a proprietary "insert" sequence that they identified using around 1,000 cell-free circulating DNA samples from individuals with breast, prostate, or colorectal cancer.

Generally speaking, the approach unearths around 100 candidate cancer markers per patient, according to Urnovitz, which theoretically leaves room for clonal shifts in the tumor without losing track of its DNA in the blood.

"The more markers you have, the higher the probability that you're tracking the original tumor, even though it's losing them step by step," he said. "That's why we think this is going to have clinical utility — because we pick so many biomarkers."

In their ASCO poster, Urnovitz and colleagues from Chronix, MD Anderson, and Vanderbilt University applied the cell-free DNA enrichment and sequencing scheme to blood samples from 16 women with breast cancer, looking at whether tumor markers identified in each patient's blood prior to surgery were present in blood samples taken a few weeks later.

For that study, the team generated around 40 million reads for each of the pre- and post-treatment samples, on average, using Illumina's HiSeq 2000 platform. Post-treatment blood samples were collected between one and four weeks after surgery — well beyond the apparent turnover time for cell-free DNA in the blood, Urnovitz noted.

To ensure that the candidate markers identified in each woman's blood samples coincided with the presence of cancer, the researchers compared these sequences to those found in tumor samples obtained during surgery and to patterns in each woman's cancer-free white blood cell samples.

Indeed, profiles in the tumors matched those predicted from cell-free DNA in pre-surgery blood samples, the team reported, suggesting that it should be feasible to develop a test for tumor DNA that's entirely blood-based.

In the post-surgery samples, researchers did not detect MRD in cell-free circulating DNA from 13 of the women. But blood samples from the other three patients contained some or all of the tumor-associated markers identified prior to surgery, hinting that at least some of the cancer clones had persisted after surgery.

For Chronix, the next step toward a clinical MRD test will be a larger study of women with breast cancer.

In conjunction with collaborators at MD Anderson, Chronix researchers have secured institutional review board approval for a trial involving 100 women receiving neoadjuvant treatment for breast cancer, Urnovitz said. He anticipates still larger trials of between 300 to 600 patients before the test is fully clinically validated.

"We were tasked with building a test for this kind of minimal residual disease," Urnovitz said. "We think the first application will be to help [breast cancer] patients who are going through neoadjuvant therapy."

Using blood samples collected before treatment and after each round of chemotherapy, the researchers plan to explore the possibility of using the Chronix enrichment and sequencing method to track levels of remaining breast cancer following neoadjuvant chemotherapy.

Ideally, the approach would identify women successfully treated with chemotherapy alone, Urnovitz said. If so, that could spare an estimated 30 percent of the patients from unnecessary surgery, he noted, while paving the way for a neoadjuvant chemotherapy companion diagnostic test based on his company's methods.

In parallel with that research, Chronix is taking the first steps toward setting up a CLIA-certified lab this week. The firm is pushing for CLIA certification by around this time next year with the goal of offering a clinical test targeted to breast cancer patients receiving neoadjuvant treatment by around the third quarter of 2014.

Such a test would be done entirely in house at Chronix, Urnovitz noted. The firm is aiming to offer the blood-based tumor biomarker profiling service for around $4,000 to $5,000.

Follow-up blood tests to look for markers identified in initial patient profiling should be much cheaper — in the $100 to $200 range — once appropriate PCR primers are designed to match an individual's tumor markers.

"Once we know the exact locations of double-stranded breaks and the Chronix insert, it's a PCR test … you can monitor the patient by blood test," Urnovitz said.

"This is what we think is the breakthrough, is the ability to do this without the tumor and to do it cost effectively — with an initial setup charge followed by cost-effective PCR," he said.

Chronix plans to take a crack at validating Life Technologies' Ion Proton as it sets up its CLIA-certified lab in the hopes of bringing the turnaround time for initial sample testing down to around one day. With the Illumina HiSeq 2000-based protocol the group currently uses, Urnovitz said the test takes around two weeks.

Sequenta's Willis said that his firm is continuing to use the Illumina HiSeq and MiSeq instruments for research studies of the LymphoSight platform and for in-house clinical testing at its CLIA lab. The ClonoSight assay is currently offered with seven-day turnaround time between the patient's blood draw and return of results to his or her doctor.

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