International Research Studies Underscore Utility, Robustness of 454 Sequencing Systems for Characterizing Blood Cancers

The research studies used the Genome Sequencer FLX and GS Junior Systems from 454 Life Sciences to perform targeted resequencing of known cancer-related genes, such as KRAS, RUNX1, EZH2, and TET2, in order to detect genetic mutations associated with the disease. By sequencing these genomic regions in large numbers of leukemia samples, the researchers were able to successfully identify novel mutations, as well as stratify individuals into unique disease risk subtypes.

Blood cancers such as leukemia and other myeloproliferative disorders cause rapid, abnormal growth of blood cells and are known to consist of a broad spectrum of subtypes. Identifying the unique molecular genetic profile of an individual’s cancer is critical for disease diagnosis and prognosis, as well as detection of reemergence of resistant clones during or post therapy. Currently, a variety of techniques are available to characterize leukemia types, including traditional Sanger capillary sequencing, cytogenetics, and immunohistochemistry, but are expensive, time-consuming and, in some instances, fail to offer the depth of analysis or sensitivity enabled by next-gen sequencing. “The number of molecular markers used to categorize myeloid neoplasms is constantly increasing. Amplicon-based next-generation sequencing is a suitable method to accurately detect and quantify the variety of molecular aberrations with high sensitivity (1),” explained Alexander Kohlmann, Ph.D., Head of the Next-Generation Sequencing Group at the MLL Munich Leukemia Laboratory, and coauthor of 10 studies presented at this year’s conference.

The MLL Munich Leukemia Laboratory is leading the way in adoption of next-generation sequencing to profile blood cancers. “Through our extensive research and development work using 454 Sequencing Systems, we’ve found that the technology has potential for mutation analysis in daily routine operations, and we have developed assays targeting known leukemia-associated biomarkers such as RUNX1 or TP53 for detection of molecular aberrations” explained Prof. Torsten Haferlach, co-founder and CEO of MLL.

The MLL is also leading an international research study to investigate the reproducibility of 454 Sequencing System assays across sites, findings of which were outlined at the conference. The IRON study (Interlaboratory RObustness of Next-Generation Sequencing) investigated the use of 454 System targeted amplicon resequencing to identify mutations in known disease-associated genes from chronic myelomonocytic leukemia (CMML) samples. An international consortium of 10 laboratories from six countries sequenced and analyzed the commonly mutated TET2 gene in the same 18 samples with the GS FLX System. Results showed a high-concordance in mutation detection across all sites, including the robust detection of novel variants which were undetected by Sanger sequencing. The sensitivity to detect rare variants present in as low as 1-2% frequency, compared to the 20% threshold for Sanger-based sequencing, together with the systems’ high-quality long reads and speed, make it particularly interesting for future clinical applications.

“We are pleased by the initial results of this research study, which reiterate the findings of robustness and sensitivity from previous multi-site studies on HLA genotyping and HIV variant detection,” said Christopher McLeod, President and CEO at 454 Life Sciences. “With the rapid uptake of the GS Junior System in the market, we are pleased to work together with the growing community of researchers to develop a menu of target-specific assays for a variety of future medical applications.” Early next year, the company plans to launch its first target-specific assay for Human Leukocyte Antigen (HLA) genotyping on the GS Junior System and GS FLX System.