Assessing Cytogenetic Abnormalities in Myeloma: A Comparative Study of Hi-C, Karyotyping, and iFISH Techniques

Background：Multiple myeloma(MM) is driven by primary and secondary chromosomal alterations (CAs). Identifying these changes is crucial as they significantly affect the patient's prognosis, response to treatment, and survival. Presently, the standard approach for cytogenetic examination involves karyotyping and interphase fluorescence in situ hybridization(iFISH) on bone marrow(BM). Although extensively utilized in diagnostic evaluations, iFISH and karyotyping exhibit moderate sensitivity and limited detection targets, which restricts their capability to detect minor sub-clones vital for prognosis or to track the progression of the disease. Recently, Hi-C, a derivative of the chromosome conformation capture (3C) technique developed to map the 3D configuration of the genome within the nucleus, has demonstrated its effectiveness in identifying chromosomal structural variations in human solid tumor samples. Here, we assessed the potential of Hi-C as a genome-wide assay to detect CAs in MM and compared its sensitivity with karyotyping and FISH.

Methods: A new method, C-MoKa, was developed using the Hi-C technique to detect CAs in MM. In brief, cells were lysed and digested with restriction enzymes. Digested DNA ends were labeled with biotin-11-dATP and ligated using T4 DNA ligase. FA crosslinking was reversed by incubation in 0.3% SDS and proteinase K for one hour at 60℃, followed by AMPure beads purification. The DNA was sheared to 200-600bp fragments and then underwent end repair, dA-tailing, and adapter ligation using a library preparation kit. Biotin-filled DNA fragments were pulled down using Dynabeads MyOne Streptavidin C1 magnetic beads. The DNA product was subsequently amplified, and libraries were sequenced with 30 million read-pairs on a DNBSEQ-T7 (MGI). We simultaneously performed C-MoKa, traditional karyotyping, and iFISH on BM samples from 22 patients with plasma cell neoplasm, including 15 MM, 5 monoclonal gammopathy of undetermined Significance (MGUS) and 2 AL amyloidosis. Pure CD138+ plasma cells (PCs) were isolated from BM mononuclear cells (BMMCs). All iFISH was performed in isolated PCs and all karyotyping in BMMCs. C-MoKa was done in 17 PC samples and 5 BMMC samples to evaluate its limit of detection (LOD).

Results: The results showed that structural variations (SVs) were detected in 3 (13.6%) patients by karyotyping, 4 (18.2%) patients by iFISH, and 9 (40.9%) patients by C-MoKa. This indicates a high sensitivity in identifying SVs by C-MoKa. Additionally, C-MoKa successfully detected all the SVs identified by karyotyping and iFISH. These SVs included +8, t(11;14)(q13;q32) (IGH/CCND1), t(6;14)(p21;q32) (IGH/CCND3), t(4;14)(p16;q32) (IGH/FGFR3), and t(5;19)(q11;q11). Furthermore, by using C-MoKa, SVs were identified in 5 MM samples which had negative results by karyotyping and iFISH. Several extra SVs not covered by the standard probes used in MM iFISH were also detected. These included trisomies (involving chr7, 9,17,18,21), t(8;22)(q24.21;q11.22) (involving CMYC), der(7)t(X;7)(q25;q32.3), and der(1)t(1;11)(q23.3;q13.3). In terms of copy number variations (CNVs), C-MoKa also demonstrated comparative sensitivity in detecting common MM-related CNVs (gain1q21, del13q, del17p) than iFISH, with a positive rate of 31.8% (7/22) by both methods. Moreover, C-MoKa identified several CNVs with potential clinical implications not detected by traditional methods. These included duplication of CCND1and CCND3 genes, and CNVs in unknown locus such as dup(X)(q25q28) and dup(4)(q26). Notably, C-MoKa failed to detect gain1q21 in one case using BMMCs (with only 7% PCs) samples, indicating that a high tumor chimerism percentage or a pure tumor cells isolation strategy is needed when using C-MoKa.

Conclusion:

In conclusion, our report shows that the Hi-C technique-based method C-MoKa effectively detects chromosomal abnormalities in multiple myeloma bone marrow specimens globally. Compared to traditional methods, C-MoKa demonstrates high sensitivity and can detect hidden and complex structural variations, gene fusions at the DNA level, as well as a greater number of duplications, deletions, and other copy number variations.