Cytogenetic testing by fluorescence in situ hybridization is improved by plasma cell sorting in multiple myeloma

Accurate detection of cytogenetic abnormalities has become more important for improving risk-adapted treatment strategies in multiple myeloma (MM). However, precise cytogenetic testing by fluorescence in situ hybridization (FISH) is challenged by the dilution effect of bone marrow specimens and poor growth of plasma cells ex vivo. It has been suggested that FISH should be performed in combination with plasma cell enrichment strategies. We examined cytogenetic abnormalities in newly diagnosed MM and compared the efficacy of three different enrichment modalities for FISH: direct FISH (n = 137), fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) technique (n = 224), and a plasma cell sorting FISH with fluorescence-activated cell sorter (FACS) (n = 132). FISH disclosed cytogenetic abnormalities in 38.0% of samples by direct FISH, 56.3% by FICTION, and 95.5% by FACS-FISH, and the percentage of cells with abnormal signals detected by FISH was significantly higher by FACS-FISH than direct FISH or FICTION. Our results suggest that the efficacy of FISH is dependent on the plasma cell enrichment modalities and reveal that plasma cell sorting FISH with FACS enables better detection of cytogenetic abnormalities in diagnostic MM samples.


Results
Baseline patient characteristics. Among BM aspirates of 493 NDMM patients, 137 samples were tested by direct FISH, 224 samples were assessed by FICTION, and 132 samples were analyzed by FACS-FISH. There were no significant differences in baseline patient characteristics in terms of age, gender, isotype, serum creatinine, serum hemoglobin, serum calcium, percentage of bone marrow plasma cells, and international staging system (ISS) among these patients ( Table 1). The positive rate of FISH differed according to the method (Fig. 1).   (Fig. 3). The median percentage of cytogenetically abnormal cells by FACS-FISH was 67.8%, which was significantly higher than 30.0% by FICTION (P < 0.001) (Fig. 3). The proportion of cases in which abnormal cells were detected in a low fraction of less than 10% was highest in order of FACS-FISH (18.1%), FICTION (10.0%), and direct FISH (9.8%).

Discussion
In this study, we analyzed the efficacy of direct FISH, FICTION, and FACS-FISH in detecting cytogenetic abnormalities in BM samples from a large number of MM patients. We demonstrated the importance of FACS-FISH in improving the sensitivity of FISH for accurate detection of cytogenetic abnormalities. The overall detection rate of cytogenetic abnormalities by FISH reported in previous work differs depending on the number of probes and cells analyzed and the difference in analysis capability. 6,7 For example, even if the same enrichment method is used, when 5 to 6 probes were used, the overall detection rate of cytogenetic abnormalities was 64.2 to 69.0%, and when 10 probes were used, the overall detection rate was reported as 96% using MACS. [8][9][10] However, the most important variable influencing FISH testing is indeed the enrichment modality. 11 The FICTION method was described in 1992 by Weber-Mat-thiesen et al. as the first technique to combine immunophenotyping and FISH, enabling evaluation of immunophenotype and hybridization signals simultaneously under a fluorescence microscope. 12 In the present study, chromosome abnormalities were identified in 56.3% (126/224) of MM patients using FICTION, similar to that reported (53%-69%) in previous studies. 13,14 The detection rate of cytogenetic abnormalities with FICTION was significantly higher than direct FISH of 36.6% (52/142) shown in this study. However, FICTION requires an additional laborious cytomorphologic selection process of plasma cells, which are frequently interfered with by background signals overlapping target signals. 15 In this regard, plasma cell enrichment processes such as MACS or FACS are employed to eliminate the need to manually identify plasma cells, resulting in higher throughput. Plasma cell targeting FISH using MACS was reported to show detection rates as high as 64.2% (120/187, 5 probes) to 69.0% (257/373, 6 probes), which is significantly higher than that of direct FISH. 8,9 However, MACS can be unsuccessful if the percentage of plasma cells in the sample is very low or when plasma cells are dim or negative for the MM plasma cell surface marker CD138. 16,17 In this work, we demonstrated that chromosomal abnormalities were identified in 95.5% (126/132) of cases of MM patients by FACS-FISH, consistent with the study finding more than one chromosomal abnormality in 96% of patients using FACS-FISH. 18 Furthermore, we found that positivity of FISH by direct FISH and the FICTION method A limitation of this study is that the two methods could not be compared in parallel. It cannot be excluded that differences in the characteristics of the patient groups, especially differences in severity, or used FISH probes influence the comparison of positive rates. However, considering that the severity of the patients was rather high during the direct FISH test period with a low positivity rate, the difference cannot be due to the difference in patient groups. In the case of FISH probes, the internal verifications of analytical performance were conducted whenever the manufacturer or lot was changed, so it was judged that the impact on the results would not be significant.
Taken together, we show that the FISH results are highly dependent on the enrichment method and demonstrate that FACS enables improved FISH sensitivity regardless of the plasma cell abundance in BM samples from MM patients. Our work supports target enrichment using FACS to minimize false-negative results of FISH, especially in MM patients with a low tumor burden.

Methods
Patients and samples. This study was approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea (IRB No. 4-2020-1383). Informed consent was waived for this retrospective study that evaluated anonymized samples and data and involved no potential risk to patients. We obtained BM aspirates from 493 newly diagnosed MM (NDMM) patients from February 2010 to March 2020 (Table 1). The percentage of bone marrow plasma cells were enumerated from 400-cell differential count on bone marrow aspirate smears. The ISS stages were defined as the 2005 International Myeloma Working Group (IMWG) recommendations. 19 Since the subject of our study is about tools that detect genetic abnormalities, we used the ISS instead of R-ISS 20 to eliminate the influence of the technique on the patient's stage. For cytogenetic analysis, BM aspirates were collected into sodium heparin-coated tubes (BD Biosciences, San Jose, CA). All methods were performed in accordance with the relevant guidelines and regulations.   Fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION). After post hybridization wash steps, slides were washed with PBS and air-dried.
Fluorescein isothiocyanate (FITC)-conjugated polyclonal rabbit antibodies against human kappa and lambda light chains were applied to stain plasma cells. After incubation and washing, slides were counterstained with DAPI, and 200 plasma cells were assessed for each analysis.
Fluorescence activated cell sorter (FACS)-FISH. About 3 mL of BM aspirate collected in a sodium heparin tube was centrifuged at 2100 rpm for 5 min. Then about 2 mL of buffy coat was isolated from BM aspirates was incubated with erythrocyte lysis buffer. After incubation at room temperature for 20 min, samples were centrifuged at 2100 rpm for 5 min. The cell pellet was then washed three times with PBS, and the cell concentration was adjusted to 1 × 10 6 -4 × 10 7 cells/mL. Antibody staining was performed using anti-CD38-FITC and anti-CD138-PE (Beckman Coulter, CA, USA), and plasma cells were sorted with the BD FACSMelody™ (BD Biosciences, San Jose, CA, USA) or S3e™ Cell Sorter (Bio-Rad Laboratories, Hercules, CA, USA). Cell gating and acquisition was done with FlowJo™ (BD Biosciences) or ProSoft™ softwares (Bio-Rad Laboratories). To achieve high purity (target: ≥ 90% of sorting efficiency), the event rate was adjusted to 1,200 to 1,700 cells per second. The median of sorted events in samples of NDMM patients was 62,146 (range, 12,007-270,962). The median sorting efficiency was 90% (range, 84%-100%). Cytospin preparations were prepared by centrifugation of 220 µL of cell suspension at 800 rpm for 4 min in an Epredia™ Cytospin™ 4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical analysis.
The Shapiro-Wilk test was conducted to assess whether continuous variables followed the normal distribution. Age, creatinine, hemoglobin, calcium, and the percentage of bone marrow plasma cells among total nucleated cells were not normally distributed. The Kruskal-Wallis test and Mann-Whitney-Wilcoxon test were performed among the three groups or two groups with different methods, respectively. Categorical variables were expressed as percentages and were compared by the chi-square test. Post hoc Tukey's test was used to find the pairwise significance between groups. Logistic regression was performed to investigate the association of plasma cell percentage with FISH positivity. A two-sided P of less than 0.05 was considered statistically significant. Statistical analyses were performed with R version 4.0.0 (R Foundation for Statistical Computing, Vienna, Austria).