Genetic abnormality is an important factor for risk stratification in MM [6], as well as for predicting survival and defining a treatment strategy. It has been reported that almost all MM patients possess cytogenetic abnormalities, sometimes during the course of the disease [7]. However, PCs occur at the final stage of differentiation, which have a low proliferation rate and long cell cycle. The viscosity of bone marrow fluid in MM patients is high, which often causes specimens to coagulate during extraction. As a result, cytogenetic studies of MM after conventional short-term culture methods are hampered by difficulty in or even failure to obtain good metaphases for analysis. Furthermore, the abnormal detection rate of karyotypes is also low. In previously reported studies [8–10], only 20–40% of abnormal karyotypes could be detected by traditional metaphase cytogenetic analysis.
To improve the detection of chromosomal abnormalities in MM, the application of different cytokines was explored. IL-6 has been recognised as the major growth factor for PCs both in vitro and in vivo. It has been used in combination with haematopoietic factors such as GM-CSF and IL-3, as these growth factors increase the responsiveness of IL-6 for supporting PC growth. However, controversial results have been reported. Lai[4], Facon[2], and Cuneo [1] have suggested that adding different combinations of GM-CSF, IL-6, and IL-3 to the culture medium and prolonging culture time to 6 d offers an advantage over unstimulated overnight cultures. In contrast, Smadja [11] and Brigaudeau [12] found that compared to adding growth factors, the abnormal detection rate of a 3-day unstimulated culture was comparable or higher.
Based on previous studies, IL-6 (10 ng/mL) and GM-CSF (40 ng/mL) were added to the culture medium in this study and prolonged the culture time to 6 d to improve the abnormal detection rate of karyotypes in MM patients. As a result, the number of metaphases and the quality of chromosomes improved significantly in most samples compared to ordinary CCA. However, abnormal karyotypes were detected in samples 10 and 17 under improved culture conditions, while the conventional short-term culture method detected normal karyotypes. The number of karyotypes was lower after culture in improved conditions. This may be because the proliferation of non-PCs is faster during short-term culture, and PC proliferation inhibits that of non-PCs after the addition of IL-6 and GM-CSF.
Compared with conventional culture, culturing in improved conditions increased the karyotype and abnormal karyotype detection rates of MM patients. The abnormal detection rate reached 40.9%, which was similar to that reported in the literature[2, 4, 10].
It is now recognised that there are two broad genetic subtypes of MM, as defined by chromosome number. Hyperdiploid myeloma (48–74 chromosomes) accounts for about 50% of cases and are mostly trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. This subtype rarely involves IgH translocations at the locus 14q32. Non-hyperdiploid myeloma (< 48 or > 74 chromosomes) is associated with the presence of IgH translocations [13]. This study confirms and expands upon prior reports, identifying that 2 cases of hyperdiploidy and 7 cases of non-hyperdiploidy were detected in improved culture conditions. Further, 88.9% (8/9) had both numerical and structural abnormalities. This suggested that the chromosomal abnormalities in MM were often complex. The abnormality involves all chromosomes except chromosomes 16 and 22, as abnormal hyperdiploid MM typically involves chromosomes 2, 4, 5, 7, 9, 10, 11, 12, 13, 17, 19, and 21. Three cases of non-hyperdiploid MM were associated with IgH translocation and 2 cases with structural abnormalities involving 19p13.
A considerable proportion of MM patients have only a small proportion and uneven distribution of PCs in the BM. Therefore, determining a means by which to accurately identify PCs under a microscope without the interference of normal cells is the primary issue to be addressed. The International Myeloma Working Group and the European Myeloma Network recommend that FISH detection in MM should not be conducted without first concentrating the PCs or employing a means for identifying PCs, ensuring that only these cells are scored [14] To fulfil this recommendation, PCs need to be sorted by CD138 immunomagnetic bead-based sorting or by labelling the cytoplasmic immunoglobulin light chain (cIg).
However, enriching PCs via CD138 immunomagnetic bead-based sorting requires at least 20 mL [15] to satisfy the detection requirements, specimen acquisition is difficult, and the sorting process is labour and time-intensive. Cytoplasmic light chain immunofluorescence combined with FISH (cIg-FISH) requires identification of the light chain types of clonal PCs before detection, ensuring the correct light chain antibody is selected. This is not easily incorporated into the routine workflow of a cytogenetic laboratory. Another proposed technique is the use of an initial Giemsa stain with PC mapping by image analysis software, followed by subsequent FISH analysis of the previously mapped PCs [16]. However, this technique relies on expensive image analysis software, which inevitably limits its clinical application.
Unlike the techniques mentioned above, when used in combination with simultaneous immunofluorescence (IF) using CD138 and FISH, FICTION does not require a large number of PCs, nor prior knowledge of the restricted light chain involved. Further, specialised procedures such as cell sorting or image analysis are unnecessary for this method. This makes it possible to recognise PCs under a fluorescence microscope and interpret genetic changes in PCs with cytoplasmic staining. FICTION can be implemented in laboratories that routinely perform FISH analysis, which is suitable for both fresh BM specimens and archived BM smears [16].
In the present study, FICTION detected a higher percentage of abnormal cases than FISH. In one CR sample (No. 21), the short-term conventional culture method, CCA with the improved culture system, and FISH did not detect abnormalities, but FICTION detected a low proportion of positive signals. These results suggested that the patient may be in the early stages of relapse. Compared to conventional FISH analysis, FICTION is more sensitive and could detect abnormalities when the proportion of PCs was low. FICTION is also more specific, detecting a higher proportion of abnormal cells compared with FISH when the proportion of PCs was the same.
Hanamura [17] showed that the 1q21 amplification rate of newly diagnosed MM patients is less than 20%, while the detection rate of relapsed patients can increase to 44%. Patient prognosis is worse when increased amplification signals are detected. In the current study, 4 1q21 amplification signals were detected by FICTION in patient No. 8 54 months after diagnosis. FICTION detected 60% positive cells, while FISH detected only 18%. Two months after the detection of 4 amplified signals in 1q21, the patient discontinued treatment due to septic shock, paroxysmal atrial fibrillation, cardiac insufficiency, decompensated respiratory alkalosis, coagulation abnormalities, and electrolyte disturbance.
A partial deletion of chromosome 13 is the first genetic abnormality found in MM and is related to drug resistance. In terms of prognosis, 13/13q abnormalities are often attributed to a poor outcome, regardless of whether they receive conventional chemotherapy or autologous stem cell transplantation [18]. The positive rate of RB1 and D13S319 detection by FISH was higher in this study than those reported in the literature [17]. This may be related to different methodologies and was also affected by the small sample size.
This study determined that combining CCA with improved culture conditions and FICTION could increase the abnormality detection rate to 92.3%. Combining CCA with improved culture conditions and FICTION could improve the sensitivity of cytogenetic risk stratification in MM patients, allow clear interpretation of the results of numerical critical sample detection, and provide more accurate information for personalised medicine. In future, the number of samples will be expanded to confirm the difference in detection quality between the two methods.