Multiple myeloma (MM) is characterized by the accumulation of abnormal clonal plasma cells (PCs) in the bone marrow (BM), which results in BM failure. Treatment includes chemotherapeutic drugs and autologous stem cell transplantation (ASCT), and due to significant advancements in the last decade, most patients achieve a complete response (CR) and high overall survival (OS) rates. However, MM remains an incurable disease, and most patients relapse within five years [1, 2]. Standard monitoring assays such as serum and urine paraprotein (M-protein) detection in the peripheral blood (PB) and the percentage of PCs in the BM are used to define CR [3, 4]. However, these methods are limited in their sensitivity [5, 6] and newer methods have been developed to detect and characterize abnormal PCs including multiparametric flow cytometry analysis (EUROFLOW) (MFC) and polymerase chain reaction (PCR)/next generation sequencing (ASO-PCR and NGS) to detect clonal B cell receptor rearrangements of specific abnormal PC clones . These methods have detection limits of 0.01–0.001% of PC populations and have indicated that minimal residual disease (MRD) is present (defined as abnormal PCs) in the BM of patients with CR, and that this MRD is responsible for clinical relapses [1, 6, 8-11].
These newer methods have their limitations, as they require patient-specific adaptations and BM aspirate samples; additionally, they only assess the PC burden , and thus do not reflect the levels of the total malignant cell population that includes a stem cell component [12-16]. Circulating malignant cells lead to extramedullary disease; thus, monitoring their levels and gene expression profiles may provide a novel tool for assessing relapse and disease progression risk during therapy. A simplified quantitative reverse-transcription PCR (qRT-PCR) approach using PB samples that is applicable in all MM patients may help overcome the limitations of the current methods.
MM is a heterogeneous disease and a common molecular signature has not been established yet; however, we and others have shown a distinct relationship between the number and type of cancer/testis antigens (CTAs) expressed with advancing disease, and suggested a cascade-like expression pattern [17-20]. Additionally, the CTA “melanoma associated antigen C1” (
Our previous studies demonstrated that the CTA “preferentially expressed antigen of melanoma” (
Patient samples were collected and RNA was processed as part of a previous CTA study . Ethics approval for the analysis of these samples was obtained from the Human Ethics Research Committee at the University of Cape Town, Faculty of Health Sciences (HREC REF: 194/2012). Briefly, PB samples (10 mL EDTA) were collected every three months from diagnosis for up to two years during treatment with basic chemotherapy regimens (Table 1). The PB mononuclear cells (PBMCs) were isolated using density centrifugation with Ficoll Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and analyzed via flow cytometry to determine the MAGEC1- positive populations as previously described . The RNA was extracted using the QIAamp RNA blood mini kit (Qiagen, Hilden, Germany), treated with the TURBO DNase-free Kit (Ambion, Waltham, MA, USA) to remove residual DNA, and checked for purity and integrity (quality: 260/280 nm ratios >1.8; RNA integrity number (RIN) values: 7–10 , MIQE guideline compliant ) as per a previous study . The stored RNA from 10 patients was used in the present study. Serum M protein and Sb2M levels were measured by the National Health Laboratory Service (NHLS)/GSH (South Africa) using accredited methodology. We compared our novel monitoring method with standardized hematological monitoring methods. Notably, the monitoring of serum free light chains was not available at our state hospitals during the study period.
Multiplex assays were developed and validated as per MIQE guidelines, including the assessment of amplification efficiency and specificity, and limits of detection. Reverse transcriptase (RT) reactions using 1.5 mg RNA and random hexamers were performed as previously described . The primers for coamplifying
Approximately 2×106 ethanol-fixed PBMCs from two patients prepared during the previous studies [15, 21] were washed twice in 40 mL ice-cold 1× PBS/1% fetal calf serum (FCS), collected via centrifugation (1,500 rpm for 5 min), and re-suspended in 500 mL cold 1× PBS/0.5% bovine serum albumin (BSA) buffer. Approximately 100 mL of this cell suspension was incubated with the following antibody combinations for 30 min at 4°C in the dark: MAGEC1- Alexa488/CD34-PE/CD45-PerCP and MAGEA3-Alexa647 or PRAME-Alexa647 and MAGEA3-Alexa647/PRAME-Alexa488/ CD34-PE/CD45-PerCP; MAGEC1-Alexa488/CD138-PE/CD45PerCP and MAGEA3-Alexa647 or PRAME-Alexa647. All antibodies were used at a final concentration of 5 mg/mL. MAGEC1 (Abcam, clone ab115351), MAGEA3 (Abcam, clone ab38496) and PRAME (Abcam, clone ab135600) antibodies were fluorescently labeled with the Mix-n-Stain Alexa 647 kit (Sigma-Aldrich). The labeled cells were washed with 1 mL cold 1× PBS/0.5% BSA buffer at 1,500 rpm for 5 min; the pellet was re-suspended in 1 mL cold 1× PBS/0.5% BSA buffer and stored in the dark until analysis. The cells were analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using the Cell Quest Pro software, applying parameters and gates that had been previously optimized to minimize spectral overlap and increase sensitivity [15, 21].
To investigate whether monitoring
An example of this is shown in Fig. 1 (MM4), where the patient had stage1 disease at therapy initiation; the serum M levels were always within the normal range (<8.9 g/dL) and the Sb2M levels decreased to clinically normal levels (<1.8 g/dL) after therapy. While
Even in cases with stable Sb2M and serum M levels following therapy for a significant period of time (Fig. 1 MM6), monitoring the patient with qRT-PCR revealed that their condition deteriorated before clinical changes were observed; a 2.6-fold increase in
Although the frequency of
The relative expression of
The co-expression of multiple CTAs in this patient group indicated a cascade-like pattern as disease progressed, as previously inferred [17-20]. However, as qRT-PCR was performed on enriched PBMCs and not isolated cell populations, it was not possible to establish whether cells were undergoing sequential clonal changes, or whether different clones, or even different cell types, expressed single CTAs. Using limited residual ethanol-fixed PBMCs from two patients from the initial studies (MM3 and MM4) [15, 21] where MAGEC1-expressing cells have been extensively characterized, we additionally analyzed the expression of PRAME and MAGEA3 antigens in the matched samples (MM3 and MM4); co-expression of
The ability to predict and prevent relapse in MM is hindered by insufficient knowledge about the role of circulating malignant stem cells in extramedullary disease and disease progression, and clonal evolution leading to treatment resistance. Despite recent advancements, MRD monitoring techniques have limitations; affordable, non-invasive novel molecular tools that can monitor circulating disease, and can be easily applied to all patients in a standard clinic/hospital environment are needed. BM sampling for monitoring residual disease is limited in the following ways: residual disease is non-uniformly distributed in the BM, and thus, MRD can easily be missed based on the sampling site; circulating malignant cells that can lead to extramedullary disease cannot be detected. The CTAs
We included patients who were not eligible for ASCT and were treated with limited chemotherapy regimens. A significant proportion of South African patients with MM in the state health sector did not undergo ASCT because of advanced age and disease at presentation (>65 yr and stage 3 disease), and comorbidities (heart disease, diabetes, and liver damage) which limit the use of myeloablative induction therapies. While most patients achieved a CR during treatment, all relapsed with extramedullary disease, and additional therapy was administered. Due to lack of novel agents for MM treatment in South African state hospitals, patients were treated primarily with cycles of cyclophosphamide and dexamethasone. Relapse in these cases was thus not unexpected. In this scenario, we aimed to assess whether qRT-PCR analysis of CTA expression could help in predicting relapse at an earlier time, allowing earlier intervention. Our previous study  showed that
Detecting potential disease-related changes earlier than at the symptomatic level may be important in predicting response to salvage therapy at the time of relapse. Therefore, we investigated the expression of
Clonal evolution of the “Myeloma cells” in MM is thought to be responsible for relapsed and treatment-refractory disease [16, 31, 32]. CTAs may be involved in this clonal evolution, leading to the development of sub-clones with higher proliferative capacity, more resistance to apoptotic signals, and refractory to inflammatory control. Although the function of many CTAs is unclear at present, members of the MAGE family are known to be involved in stimulating cell cycle progression (via cyclin activity), reduction of tumor suppression activity (P53 activity), and p21 activity via increased ubiquitination [7, 33-35]. Many CTAs are reported to be involved in overcoming cell cycle blocks via different pathways . Based on our results, we may hypothesize that these clones may be highly responsive to proliferation stimuli (or may proliferate without stimuli). New pathways may be activated/inhibited by the co-expression of multiple CTAs, which may influence the effectiveness of salvage therapy, depending on the drug class (i.e. proteasome inhibitors). As the cumulative effects of the CTA cascade become clear in the future, the CTA expression profiles of patients at relapse may help to guide drug selection.
Overall, this small study has provided evidence that a coordinated expansion of stem cell clones progressively expressing more CTAs may occur during disease progression in MM (Fig. 4). We also demonstrated that a multiplexed CTA qRT-PCR assay combining
CTA biomarkers linked to advanced disease may allow for the monitoring of the clonal evolution of the disease (not possible with current MRD methods), which signals the development of more aggressive disease and may in future guide treatment choices as the effect of these proteins on cellular processes is established.
*This study was supported by a grant from National Health Laboratory Research Trust, AstraZeneca Research Trust and South African Medical Research Council.
No potential conflicts of interest relevant to this article were reported.