Association Study of Ploidy Category with Mitotic Disruption In Multiple Myeloma
Authors: Dementyeva Elena , Kryukov Fedor , Sevcikova Sabina , Nemec Pavel , Jan Smetana , Greslikova Henrieta , Zaoralova Romana , Buresova Ivana , Penka Miroslav , Kuglik Petr , Hajek Roman 
Published: October 2019

Abstract
Abstract 2952

Background
Centrosome aberrations are common in many types of human malignancies and are associated with aneuploidy. Loss of centrosome duplication control will often create multipolar spindles that in turn could be responsible for incorrect segregation of whole chromosomes leading to aneuploidy. Hyperdiploidy (subtype of aneuploidy) is one of the most frequent cytogenetic abnormalities in multiple myeloma (MM), where molecular changes are among the primary genetic events in disease pathogenesis. But no correlation between centrosome aberrations and aneuploidy in MM has ever been found.

Aims
The objective of our study was to evaluate association of MM ploidy category with centrosome amplification in both B and plasma cells subpopulations and to investigate structural defects (gain/loss) and gene expression changes in genes controlling centrosome numbers.

Methods
Immunofluorescent labeling was used for evaluation of centrosome amplification (CA) in B-cells (CD19+) and PCs (CD138+) of MM patients. Centrin (centrosome protein) copy numbers were used to define presence of centrosome amplification (CA) in cells: cells with more than 4 signals of centrin were considered to be positive. Samples with ≥11% of B-cells or ≥10% of PCs with >4 fluorescence signals of centrin were considered as CA positive. A total of 140 patients were evaluated for CA in PCs and/or B-cells, including 50 patients where both cell types were analyzed. The patient population characteristics were as follows: males/females 67/73, median age of 66 years (range, 40–92 years). Newly diagnosed (52/140) and relapsed (88/140) patients were included in this study; most of them had advanced stage of MM (DS II/III n = 107; ISS II/III n = 92). Interphase FISH with cytoplasmic immunoglobulin light chain staining (cIg FISH), oligonucleotide-based arrayCGH (20 patients) and qRT-PCR (5 CA positive vs 5 CA negative patients) were performed on plasma cells. Hyperdiploidy analysis was done using Multi-Color Probe Panel (LSI D5S23/D5S721, CEP 9 and CEP 15) for chromosome 5, 9 and 15. Only cells with three or more signals from at least two of three investigated chromosomes were classified as hyperdiploid. ArrayCGH and qRT-PCR were focused on chosen list of mitotic genes, according to their role in normal centrosome duplication process.

Results
The frequency of MM cases positive for CA was 35% (35/100) and 39% (32/82), based on the analysis of PC samples and B-cell samples, respectively. Overall, 18% (9/50) of MM patients were double-positive. Presence of centrosome amplification in B-cells of MM patients was established in our previous study. Significant correlation of centrosome amplification in PCs with hyperdiploidy was not found. But association of CA in B-cells with PCs hyperdiploidy using phi 4-point correlation was proven (phi=0.358, p<0.05). In group of newly diagnosed patients (52/140), this correlation was much stronger (phi=0.555, p<0.05). ArrayCGH analysis of genes controlling centrosome duplication did not show correlation between their copy number defects and hyperdiploidy in myeloma cells. As for gene expression analysis, significant differences were found in levels of ARKA and PCNT genes (p<0.05). Relative quantification coefficient R of these genes was two times higher in CA positive patients when compared to CA negative patients. No significant correlation between amount of CA positive PCs and B-cells was found (p>0.05). But after splitting patients based on CA threshold, significant correlation was identified (r=-0,763, p=0.017) in double-positive group.

Conclusion
In our study, we show association of CA in B-cells with PCs hyperdiploidy. This finding relates to the role of B-cell mitotic disruption in MM aneuploidy and cell carcinogenesis. It gives us a possibility to suspect the impact of abnormal B cells in myeloma cells development. B-cells with CA probably enter mitosis but do not finish it properly resulting in aneuploid cells; these cells may represent an aneuploid pool of MM cells.

Acknowledgments
This study was supported by grants MSM 0021622434 and IGA 10207-3 from the Departments of Education and Health of the Czech Republic.

Disclosures:
No relevant conflicts of interest to declare.