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IRON-II Study Uses 454 Sequencing to Investigate New Markers for Hematological Malignancies

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A consortium of mostly European laboratories is in the midst of investigating new markers for hematological cancers using amplicon deep sequencing.

The research study, called IRON-II, for Interlaboratory Robustness of Next-generation sequencing, is supported by Roche and uses the 454 platform. A follow-on to an earlier study to test the technical performance of amplicon deep sequencing in different laboratories, the new study is investigating panels of one to 25 genes in a total of 6,000 to 8,000 retrospective samples covering five hematological malignancies.

While the study, which kicked off earlier this year, is purely scientific in nature, individual labs may translate the results into routine diagnostic use eventually.

The original IRON-I study, which involved 10 laboratories from around the world, demonstrated the robustness of 454 amplicon deep sequencing in a clinical environment. Results from the study, which analyzed mutations in three genes – TET2, CBL, and KRAS – in 18 chronic myelomonocytic leukemia patient samples using the 454 GS FLX, were published in Leukemia last year.

Munich Leukemia Laboratory, a large German diagnostic lab that spearheaded the project, has since been using these markers routinely in diagnostics, and Roche recently commercialized a primer set for the three genes, as well as another one for the RUNX1 gene, also developed in conjunction with MLL, for research use only on the GS FLX or GS Junior sequencers.

According to Alexander Kohlmann, head of next-generation sequencing and microarrays at MLL, the goal of the second, larger phase of the IRON study is to test a variety of new genetic markers, either on their own or in panels, in five disease types: acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, as well as myelodysplastic syndromes and myeloproliferative neoplasms.

Mutations in many new genes have recently been discovered for various types of hematological malignancies through large-scale sequencing efforts like the Cancer Genome Atlas, the Genomics of Acute Myeloid Leukemia project at Washington University, and the St. Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project. These markers now need to be tested in larger patient cohorts in order to determine their incidence in different patient groups, and whether mutations occur in hotspots, Kohlmann said.

In addition, some more advanced markers need to be studied in larger numbers of patients with clinical follow-up data in order to establish their diagnostic or prognostic relevance, he said.

While Kohlmann acknowledged that the trend in next-gen sequencing-based cancer diagnostics is toward larger and larger gene panels, he said it is difficult to ensure consistent quality for large panels, especially with non-PCR enrichment and library prep methods. "So for the hematological malignancies, we focus on what we would call actionable mutations today, because there is some information available for either classifying, diagnosing, or providing prognostic information about a disease type," he said. "That's why I also think that exome sequencing for routine cancer diagnostics is still many years in the future. For the foreseeable future, it will be panels."

More than 25 laboratories from 14 countries are participating in the research study, which is organized into five working groups by the five disease types.

Each working group has been selecting markers to be included in the study, and Roche has been manufacturing primers in 96-well plates to amplify these genes, which the company is providing to the study consortium. "This way, we ensure standardized measurements of amplicons," said Kohlmann, adding that they also plan to use the same data analysis pipeline across the study.

As of now, about 20 plate designs are available, and most of these have been shipped to participants after quality checks at MLL. While some plates test for a single gene, others, for example for MDS, include panels of up to 25 genes.

Participants have already sequenced individual markers in several thousand cases using either the GS FLX or GS Junior, and the entire study will involve between 6,000 and 8,000 patients, Kohlmann said. The majority of the labs have their own 454 sequencer available, he said.

For Roche, the study provides an opportunity to establish its 454 platform further in clinical research. Besides the production of "several thousand" primer plates, Roche is providing personnel to coordinate the study as well as technical support for participants. "The robust data we generated in the IRON-I study convinced us also to move into IRON-II with the goal not only to answer open questions in research of hematological malignancies but also to develop and confirm new markers for the research setting," Thomas Schinecker, president of 454, told CSN in an e-mail message.

And although Roche is quick to point out that the study is solely focused on research, depending on the results and the need for the redesign of markers, individual labs may choose to start using the plates in routine diagnostics in the future, as MLL has already done with the TET2/CBL/KRAS and RUNX1 plates. "Technically, I don't see hurdles where this approach would not be feasible. In contrast, we have had very good experience using the RUNX1 and TET/CBL/KRAS plates day after day in our accredited routine operations," Kohlmann said.

The consortium plans to present first data from the study at the American Society of Hematology annual meeting in December, as well as at the European Hematology Association annual congress next summer.