A Comprehensive Set of In Vitro Transcribed RNAs as Controls for Leukemia Molecular Diagnostics.

Introduction: Leukemia is one of several cancers in which genetic sub-types are differentially responsive to specific chemotherapeutic agents, with success rates being directly proportional to early detection and treatment. Detection of specific fusion transcripts from chromosomal translocations (sub-typing) is required for rapid differential diagnosis and initiation of treatment strategies. Since leukemia assays are often performed in small test runs in many facilities, a pooled control that minimizes kit usage and cost by supplying multiple markers in a convenient single-tube format is optimal. As a means to better assess the accuracy and robustness of leukemia assays, we have developed a simple yet flexible external control comprised of in vitro transcribed RNA fusion transcripts of leukemia-associated chromosome translocations. These transcripts include b2a2, b3a2, e1a2, E2A/PBX1, TEL/AML1, AML1/ETO, PML/RARa (Long and Short forms), MLL/AF4 (e10e4 and e9e5) and inv16 (A and D types). To accompany this positive control is translocation-negative, HL-60 cell-line RNA. It serves as a negative control to assess assay background levels.

Materials and Methods: The RNA fusion transcripts include about 150 nucleotides flanking each side of the transcript fusion point. Additionally, we fused an MS2 bacteriophage operator to the 3′ end of the transcript sequences to provide a binding site for MS2 coat protein dimers for eventual conversion into Armored RNA Quant™, a stable synthetic quantitative standard that can be utilized upstream in the workflow to run alongside patient samples at the collection and RNA extraction steps. Once all the sequences have been verified for their accuracy, the transcripts were manufactured individually and formulated into a mixture with HL-60 total RNA to mimic leukemic cell extract conditions. The HL-60 total RNA was also tested as an external negative control.

Results: We demonstrated that these controls can be used with the Signature LTx assay for simultaneous detection of twelve leukemia-related fusion transcript targets in a single well. We anticipate that these control transcripts will be compatible with current conventional RT-PCR or real-time PCR platforms as well.

Conclusion: The twelve transcripts in a single-tube pooled positive control plus the accompanying negative control will provide clinical laboratories with an invaluable tool for tracking day-to-day performance for both multiplex and simplex leukemia molecular diagnostic assays.