Clonality Analysis Reveals the Order of Acquisition of Copy Number Alterations in Multiple Myeloma

Multiple myeloma (MM) is characterized by a wide heterogeneity at both clinical and genomic level. Two major subgroups of MM have been identified: hyperdiploid MM (HMM) characterized by the trisomy of eight odd number chromosomes (3, 5, 7, 9, 11, 15, 19, and 21); and the non-hyperdiploid (NHMM) often associated with hemizygous deletion of chromosome 13, translocations involving the immunoglobulin heavy chain gene and copy number alterations (CNA) in other chromosome regions such as 1q, 6q, 8p, and 16q. Although, aneuploidy is a hallmark of MM, the sequence of occurrence of these complex genomic rearrangements during the MM development is unknown. Here we have studied, utilizing large datasets, when and in what sequence does each of these genomic events occurs, in both MM subgroup.

We performed copy number profiling on 336 CD138+ MM cells from newly diagnosed patients and validated the results on another set of 103 samples. All patients were uniformly treated and have clinical data with median 6 years of follow up. We have used both cytoscan array as well as whole genome sequencing, as needed for the analysis. We have analyzed the data using dual quantile normalization, residual variability removal and segmentation using HG19. For each sample than we have extracted kernel smooth over a window of genomically contagious log2 ratio as smoothed signal. Clonality was then assessed by using deviation from two copies. We have also calculated clonality occurrence index which considers number of patients with clonal change for specific chromosome or cytoband.

Our initial results showed that even though copy number gains were usually clonal and early events, while deletions, except 13q, were subclonal and also late events. Focused analysis on HMM samples showed that chromosome 15 is likely the first event with detected clonality in 82% of the cases, followed by occurrence of trisomies involving chromosomes 9 and 19 with clonality observed in 70 and 68% of the cases, respectively. For trisomies involving these three chromosomes, the clonality index was 42, 35 and 35 respectively, suggesting an early hyperdiploid events. Trisomies involving chromosomes 3, 5 and 11 occur subsequently and have clonality index of 25. Finally, chromosomes 7 and 21 trisomies are late events with clonality index of 15. This information is confirmed in an independent data set.

In NHMM deletion 13 is a frequent and early clonal event while the other chromosomal aberrations such as del 1p, del 6q and del 17p are usually subclonal and hence late events. Del 13 is also observed in more than 25% of HMM patients where the clonality for del 13 was higher in the HMM chromosomes (chr 9, 15 and 19) compared to del13 (89% vs 65%, respectively) indicating that HMM occurs before del13 in these patients. Monosomy 14 is observed in 10% of patients and usually occurs at the same time with deletion 13, differently from other deletions such as 16q which is likely to happen after del13. Our analysis also showed that clonal or subclonal events on different regions across the genome actually carries much more information than just the global copy number data, e.g . clonality of chromosome 13 and chromosome 17 carry poor prognosis (p< 0.006, p <0.00026, respectively) while subclonality does not.

Our decomposition analysis shows the potential order of occurrence of aneuploidy in MM and provides a new understand of the development of disease. This information now can be utilized to consider various genes resident within the early and late event chromosomes for their potential role in evolution of the disease.