Circulating nucleotides in plasma have been previously quantified and sequenced in studies involving 65 patients with multiple myeloma (
Acta Onc 39, 789 2000
). RT-PCR revealed RNA incorporating a 709-nucleotide sequence (GenBank #AFO18254) with 99.7% homology with a non-coding ALU sequence of a type capable of retro-transposition and disease linked mutation. Levels of this circulating ALU RNA correlated with myeloma disease status. Circulating DNA, devoid of cellular material was also noted as part of these studies. We now describe the molecular analysis of this circulating DNA before, during, and after therapy. Based upon prior findings, direct PCR was performed utilizing primers with a 3′ Alu sequence linked to a variety of 5′ sequences. Detailed studies including serial evaluations were conducted in one patient. 167 total clones were analyzed and categories set to compare under and over-representation in comparison of active myeloma samples versus samples from healthy controls. 96 selected clones fitted into 10 homologs. Homolog #2, which proved to be reflective of myeloma disease activity, incorporated only non-coding DNA regions. One homolog sequence, CNA Clone #2, matched one GenBank entry in the human genome (GenBank #AC020734; chromosome 4), incorporating only repetitive elements and interspersed DNA. The percentage occurrence rates for different types of repeat elements in CNA Clone 2 were: LINEs 25.2%; MaLR 5.5%; HERVs 4.8%; ALUs 4.8%; MERs 3.8%; MiR 2.1%; microsattelites 1.1% and A-T rich regions 1.0%. Using a combination of the 10 homologs containing ALU-attached sequences, distinct patterns of clonal change occurred over several months of follow-up in the patients studied. Initial clones associated with active disease disappeared with response. Transiently, expressed different clones emerged with intervening infection and disappeared with resolution of infection. Electron microscopy has revealed that circulating nucleotides are present in the form of exosomes or membrane vesicles released into the extra-cellular environment upon exocytic fusion of multivesicular endosomes with the cell surface membranes.Conclusions: Our studies revealed several important aspects. Firstly, specific types of circulating DNA and RNA accompany the presence of active myeloma. Secondly, the predominant types of RNA and DNA are repetitive elements, especially SINEs, LINEs and ALUs. Thirdly, that these repetitive elements are typically linked to other non-coding sequences derived from interspace gene (intergenic) “hotspots”, which may be linked to particular patterns of disease and/ or treatment responses. The serial testing of DNA patterns has the potential for personalized molecular patient monitoring. Further studies are ongoing to assess these findings in a randomized trial setting and in comparison with proteomic and other markers.
