Clonal Heterogeneityand Evolutional Paths in Multiple Myeloma Revealed By QM-FISH in Single Cell of Sequential Analysis

Background: Clonal heterogeneity is a hallmark of many solid and hematologic malignancies. Clonal heterogeneity may foster clonal evolution and affect personalized care strategies in multiple myeloma(MM). In order to grasp this ubiquitous phenomenon and its effect on MM patients’ prognosis, we report on sequential analysis by quantitative multicolor-fluorescence in situ hybridization (QM-FISH) in single cell of myeloma patients who were longitudinally followed.

Methods: Bacterial artificial chromosomes (BAC) clones that contain target genes including TP53, Rb1, CKS1B, CYLD and cIAP, which belonged to secondary genetic variation of MM and were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP and dCTP to target genes by nick translation. We performed QM-FISH in single cell of 42 specimen from 17 myeloma patients who were longitudinally followed and compared the difference between myeloma patients at initial diagnosis and progression of disease.

Results: Sequential samples revealed that MM may follow three clonal evolution patterns in the course of disease progression. In these 17 patients, myeloma clone in 5 patients had no changes over the time, 6 patients acquired one or more molecular abnormalities and only one predominant clone over the course, 6 patients had more than 1 clone in the course and all clones competing and tiding, or acquired one or more molecular abnormalities resulted in new clones emerging and all involved clones waxing and waning. Furthermore, we analyzed clone architecture in samples at initial diagnosis and 15/17(88.2%) cases had multiple subclones architecture at initial diagnosis. 3 patients of the first group (3/5, 60%) had more than 1 subclone, and all patients of the latter two groups had multiple subclones at initial time. Clonal heterogeneity may contribute to clonal evolution

Conclusions: The outcome of tracking clonal heterogeneity and evolutional paths by QM-FISH is effective and feasible. Myeloma patients may follow 1 of 3 pathways to progress. Clonal heterogeneitymay foster lonal evolution in the course of disease progression.