By Monica Heger
Sequenta, a molecular diagnostics company focused on developing assays based on immune repertoire sequencing, presented data from several collaborations using its technology at this week's American Society of Hematology meeting in San Diego.
The company demonstrated that its immune cell receptor sequencing technique, which it has developed on Illumina's HiSeq, can detect residual disease in cancer patients at levels undetectable by conventional methods such as flow cytometry, real-time PCR, and positron emission tomography scans.
The technique could be used to monitor patients' response to therapy and diagnose the risk of relapse earlier, in order to help prevent disease progression and determine which patients need more aggressive therapeutic intervention, according to Tom Willis, the company's CEO.
Willis told Clinical Sequencing News that the company is planning to launch assays, initially for cancer but later other diseases, as lab-developed tests and that it is seeking CLIA certification for its lab.
The company, which last year changed its name from MLC Dx, raised $13 million in a Series B financing round last December to fund development of its tests (IS 12/14/2010).
The technology is "exquisitely sensitive" and can "detect reliably a single cell in the presence of one million other cells," Willis told CSN.
At this week's conference, the company presented data from five different projects with collaborators at Stanford University and St. Jude Children's Research Hospital, showing its technology could detect residual disease and predict clinical relapse in childhood and adult patient samples of acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and mantle cell leukemia.
The company's platform relies on proprietary multiplex PCR assays that can amplify immune cell receptor rearrangements in T-cells and B-cells from blood and tissue for subsequent sequencing. The company is currently developing universal assays for the IgH locus; Itk cells; and TCR beta, delta, and gamma cells, Willis said.
"With a universal PCR assay, we can amplify all the rearrangements at the genome level in those cells and put them on a standard sequencing platform," he said.
Currently, the company is using the Illumina HiSeq for sequencing, but Willis declined to disclose whether it would use that platform to launch its diagnostic assays. He also did not disclose the timeline for commercialization or CLIA certification.
Using the HiSeq, Sequenta generates between one and five million reads per sample, allowing it to "reconstruct the frequencies of each of the immune cells in the sample," Willis said.
Monitoring Response and Relapse
At this week's meeting, Sequenta presented data from five different collaborations that used its assays in more than 100 patients at multiple time points.
The results demonstrated two main purposes of the assays in cancer care: to monitor patients' response to drugs and to serve as a more sensitive predictor of residual disease, which could be used to flag patients at risk for relapse.
Clinicians currently use methods such as flow cytometry and PET scans to try and diagnose residual disease in leukemia patients who have received a bone marrow transplant. But flow cytometry is not very sensitive and requires bone marrow as opposed to blood, while PET scans yield a lot of false positives and subject patients to radiation, Willis said.
Nevertheless, physicians continue to use these methods because they are the only alternatives for detecting residual disease, which, if detected early, can lead to earlier intervention and better outcomes.
In one of the studies presented this week, researchers from Sequenta and Stanford used the technology to retrospectively analyze 11 mantle cell lymphoma patients that had been determined to be negative for disease by PET scan at six months and one year post-treatment.
In two patients that had relapsed despite being diagnosed as disease-free, Sequenta's technology was able to detect low levels of disease.
Similarly, in a collaboration with other Stanford researchers, the technology was used to retrospectively analyze 37 samples from 14 patients with chronic lymphocytic leukemia. The Sequenta method was compared to a PCR-based method, which diagnosed 30 of those samples as disease free and was inconclusive in the remaining seven.
It has previously been shown that patients are likely to relapse if leukemic cells are present at a level of one in 10,000 in their blood following a bone marrow transplant. Since Sequenta's sequencing method can detect one cell out of a million, the company set out to determine whether it was sensitive enough to improve on the PCR-based assay.
The comparison found that the two methods were concordant in just 14 of the 37 samples and that the sequencing-based approach was more highly associated with survival and relapse than the PCR-based diagnostic.
For example, there were five patients in which the sequencing test came back positive for disease and the PCR test came back negative, and all of these patients relapsed an average of 10 months later. For seven patients deemed disease free by both methods, one relapsed.
The team is now looking at nearly 300 additional samples from 42 CLL patients to validate the findings. So far, the team has found that nearly 60 percent of the samples that had been diagnosed as negative by PCR are predicted as positive for disease using sequencing, and that the test is "highly predictive" of relapse.
In a separate collaboration with St. Jude Children's Research Hospital, the company developed an assay to amplify and sequence the IgH locus for acute lymphocytic leukemia patients. The detection of aberrant immunophenotypes is highly predictive of outcome in these patients. Currently, most methods use either flow cytometry to detect aberrant immunophenotypes or PCR amplification of patient-specific antigen-receptor patients.
While the PCR method has proven effective, Willis said that sequencing will allow for a "universal assay that doesn't have to be customized to each patient." In addition, he said, it is a "couple orders of magnitude more sensitive than flow cytometry and PCR."
The St. Jude team first demonstrated that the method could detect leukemic clonotypes in a dilution of one cell per million. They then tested the method in 10 patient samples whose disease level had been measured by both real-time PCR amplification of IgH genes and flow cytometry.
The sequencing method found disease in five patients that the other two methods also scored as positive. In one sample that both PCR and flow cytometry predicted as disease free, Sequenta's sequencing method detected very low levels of disease. All three methods predicted the remaining four samples to be disease free.
Willis said that the company is now following up on all of these studies to validate the findings in larger patient cohorts and across more disease states. While these initial studies are focusing on cancer, and leukemias in particular, he said the approach will be applicable to a wide range of diseases.
"The immune system is involved in so many diseases," he said. "We have a pipeline of clinical programs that we think will add value in the future."
The company has not yet settled on a price for its assays, but Willis said that he is "certainly targeting the test to be more cost effective than competing technologies such as PET scans."
Additionally, he expects that insurance companies will be likely to cover the tests once the company demonstrates their cost effectiveness.
While the assays will initially be launched as LDTs under CLIA regulations, Willis said that because the US Food and Drug Administration has indicated an interest in regulating LDTs, the company is "planning to have the kinds of datasets that will be necessary for [FDA] approval."
Have topics you'd like to see covered by Clinical Sequencing News? Contact the editor at mheger [at] genomeweb [.] com.