By Monica Heger
The HudsonAlpha Institute for Biotechnology has launched a project that aims to sequence the immune repertoires of 10,000 individuals representing 100 diseases, including cancers, autoimmune diseases, inflammatory diseases, and infectious diseases.
The goal is to find personalized biomarkers in the immune system that will lead to better and earlier diagnostics. By sequencing an individual's "personalized immunorepertoire, we can take a snapshot of the health and disease status," said Jian Han, a HudsonAlpha faculty investigator who is leading the project.
Han expects to complete the effort — dubbed the Repertoire 10K, or R10K, Project —within five years, and has begun a pilot study to sequence 30 patients diagnosed with either breast, lung, or colon cancer, as well as eight healthy controls.
HudsonAlpha plans to establish an international consortium comprised of academic and industry groups to carry out the study. Han said he is in discussions with several instrument vendors and other firms regarding involvement in the consortium but said it was still too early to disclose potential participants.
So far, Han has secured $500,000 in funding from HudsonAlpha, and expects another $3 million of in-kind support from industry participants. He said the total project should cost around $5 million. The researchers are also finalizing a scientific advisory board, which will include representatives from academia and industry.
While the bulk of the sequencing will be done at HudsonAlpha, some of it will be contracted out or done by the different industry or academic partners.
The plan is to sequence each individual's T-cell repertoire on the Illumina HiSeq and to sequence the B-cell repertoire on Roche's 454 GS FLX, said Han. "We need reads of about 300 base pairs for the B-cell repertoires," said Han, in order to span the entire recombinant region. For the T-cell repertoire, that region is much smaller, only about 100 base pairs, he said.
They will use a sample-prep method developed by Han's company iRepertoire, which involves amplicon rescued multiplex PCR, or arm-PCR, which is designed to amplify small amounts of material in an unbiased way. The iCubate system, also designed by Han's team, will be used to automate the process.
Samples will then be pooled. Up to 25 samples can be multiplexed into one run of the HiSeq, said Han, although the team is still working out the optimal number of patients to multiplex. In the pilot study, they are multiplexing 20 samples.
The main bottleneck will be access to the 10,000 samples. Many of them will be purchased from commercial sources, and a significant amount will come from collaborators in the project. Patient information will be anonymous and results will not be returned to the patients, he said.
Han said that there are four main criteria for choosing the diseases to be studied in the project. They should all have either a high incidence or high death rate, so that a diagnostic would significantly improve patient care. The diseases should also currently lack good diagnostics and should also have a link to immune function. And finally, the development of a diagnostic should be able to make an immediate clinical impact.
R10K is the second large-scale immune sequencing project to kick off in recent months. Last month, researchers from the Fred Hutchinson Cancer Research Center and Hutch spinout Adaptive TCR began a proof-of-principle project to sequence the T-cells and B-cells of 100 volunteers on the Illumina Genome Analyzer, looking for marks of exposure to 17 different pathogens (CSN 4/12/2011).
Additionally, the company Sequenta is developing immune repertoire sequencing-based diagnostics (IS 12/14/2010).
Focusing on the immune repertoire offers a look at not only how genetics impact diseases, but also the role of environmental factors, said Chris Gunter, research director at HudsonAlpha and R10K's project coordinator.
One problem with the multitude of whole-genome sequencing studies is that "they're not able to factor in environmental responses," she said. "Immune cells are shaped both by the genes you have and your responses to the environment. … Having the sequences [of the immune cells] is one way to start teasing out how much has been contributed from the environment and how much from genes."
Despite the potential of sequencing immune repertoires to study disease, there are challenges. For one, there is so much diversity even within an individual that sequencing an individual's immune cells at different time points will yield different results. Also, it is difficult to distinguish rare clonal types from sequencing errors (CSN 3/22/2011).
Nonetheless, Han and others think that immune repertoire sequencing will prove to be vitally important for understanding disease. Han said that in unpublished work that is part of the R10K pilot project, he has found significant clonal expansion of some CDR3s in a breast cancer patient that are not found in normal patients, colon cancer patients, or lung cancer patients, which he thinks may serve as a "personalized biomarker." Additionally, he has found that a specific clone, unique to breast cancer, can be traced in both the patient's cancer tissue and peripheral blood, suggesting that the clone could be used to monitor the patient, Han said.
Han and his team have are also developing a new measure of immune cell diversity — the D50 test — which will provide a quantitative measure of how diverse a person's immune repertoire is. Han likened the D50 test to measuring a person's white blood cell count. Rather than diagnosing a person with a specific disease, it evaluates the amount of immune repertoire diversity in order to determine whether the patient is healthy or sick.
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