Immunophenotypic Markers Associated with Minimal Residual Disease Status and Outcome in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation

Research Article

Ann Hematol Oncol. 2021; 8(8): 1357.

Immunophenotypic Markers Associated with Minimal Residual Disease Status and Outcome in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation

Missassi G1, Ikoma-Colturato MRV2*, Simioni AJ3, Bortolucci CM2, Conte-Spilari JE2, Souza MP3 and Colturato VAR3

¹Multiprofessional Residency in Cancer Attention, Hospital Amaral Carvalho, Jaú, São Paulo, Brazil

²Flow Cytometry Laboratory, Hospital Amaral Carvalho, Jaú, São Paulo, Brazil

³Bone Marrow Transplantation Service, Hospital Amaral Carvalho, Jaú, São Paulo, Brazil

*Corresponding author: Ikoma-Colturato MRV, Flow Cytometry Laboratory, Hospital Amaral Carvalho, Dona Silveria Street, 150, Jaú, Zip Code: 17210-080, São Paulo, Brazil

Received: April 26, 2021; Accepted: May 26, 2021; Published: June 02, 2021


Multiple Myeloma (MM) is one of the most common hematologic malignancies, with a heterogeneous prognosis. Therefore, the recognition of biomarkers can be useful to understand the differences in patient outcomes. Minimal Residual Disease (MRD) has been considered a very important prognostic factor in MM. In parallel, the prognostic value of immunophenotypic markers expressed in MM Plasma Cells (PCs) has also been described. The aim of this study was to assess the impact of CD27, CD28, CD45, CD56, CD117 and β2-microglobulin expressions on the outcome of 154 MM patients undergoing Autologous Stem Cell Transplantation (ASCT). The relation of each marker studied with the Overall Survival (OS) and Progression-Free Survival (PFS) was assessed, alone and in association with pre-ASCT MRD. Scores of good (GPM) and poor Prognostic Markers (PPM) were established, according to their respective survival curves. The expressions of CD27 and CD45 were associated to longer OS (p=0.013 and p=0.00, respectively) and PFS (p=0.00) as well as the absence of CD28 (OS p=0.026; PFS p=0.001) and CD56 (OS p=0.004; PFS p=0.009), in patients with undetectable MRD. The number of GPM showed an inverse correlation with the level of MRD (p=0.04), while a higher number of PPM was observed in patients with higher levels of MRD (p=0.04), which were also significantly associated with OS and PFS.

In conclusion, although pre-ASCT MRD is a powerful prognostic factor in MM, these biomarkers can provide additional prognostic information and be used in the follow-up of MM patients.

Keywords: Multiple myeloma; Minimal residual disease; Prognostic markers; Immunophenotype markers; Autologous stem cell transplantation


Multiple Myeloma is the third most prevalent hematological malignancy. In the last twenty-six years, the incidence of MM has increased, as well as deaths due to the disease [1,2]. MM is a characterized by the expansion of clonal PCs in bone marrow, that can compromise the normal hematopoiesis and systemically affect the patients causing symptoms secondary to elevated calcium levels, renal failure, anemia and osteolytic bone lesions (CRAB) [3,4].

Upfront consolidation with ASCT is one of the most effective therapies for MM in eligible patients, after the first line of chemotherapy [3-5]. ASCT has proven to increase OS and PFS in MM patients [6,7]. However, the majority of MM patients can relapse, despite achievement of Complete Remission (CR) [5,8].

New therapeutic strategies are being introduced and biomarkers have received special attention to identify groups of patients with different prognostic factors, including the cytogenetic classification for risk stratification in MM [4,5,9-15]. Furthermore, MRD has proven to be one of the main predictive factor for relapse in MM patients eligible or not to ASCT [8,13,15-19]. A significant increase in PFS and OS was observed in patients who had undetectable MRD before and after ASCT [13,15]. On the other hand, persistent MRD resulted in reduced PFS and OS after ASCT [15,17,20]. Nowadays, Multiparametric Flow Cytometry (MFC) is one of the most sensitive methods recommended for monitoring MRD to assess efficacy of the treatment [21].

The immunophenotypic profile of clonal PCs is another prognostic parameter that has been investigated in MM patients [9-12]. Antigen expressions on clonal PCs varies according to the stage and biological characteristics of the disease [22]. Considering that more than 90% of plasma MM cells express an aberrant immunophenotypic profile [12,23], the recognition of these specific surface antigens is useful for the evaluation of MRD and can provide additional information on the risk of relapse [15,17,24].

The main immunophenotypic markers with prognostic implications, which were previously described in MM include CD27, CD28, CD45, CD56, CD117 and β2-microglobulin [12,22,25].

CD27, a transmembrane phosphoglycoprotein member of tumor necrosis factor (TNF) family is expressed in normal plasma cells [26,27]. This marker is frequently downregulated in MM plasma cells [28]. Its expression is involved in PCs differentiation [25] and is associated with higher survival rates [28,29]. On the other hand its absence leads to disease progression [30,31].

CD28 is a disulfide-linked homodimer type I glycoprotein, member of the immunoglobulin superfamily, expressed on most T lineage cells, NK cell subsets, and neoplastic plasma cells [32,33]. This marker is a mediator of MM cell survival and chemotherapy resistance [32] and its expression is related with a more aggressive phenotype [27], cytogenetic abnormalities and lower OS and PFS [9].

CD45 is an important regulator of immune cells signaling [34] and it is associated with decreased bone marrow angiogenesis [35]. CD45 expression is also associated with longer OS in MM patients treated with high dose therapy [36]. Regarding CD56, also known as Neural Cell Adhesion Molecule (NCAM), controversial data about its prognostic relevance have been reported. The lack of CD56 on malignant plasma cells has been associated with worse prognosis and a higher incidence of extramedullary disease [37]. While Mateo et al (2008) did not observe a significant influence of CD56 expression on survival [9], other studies reported that the lack of this marker was associated with the presence of chromosome translocation [11,14], associated to poor prognosis [38,39].

CD117 is a receptor involved in cell growth and its presence has been related to higher survival rates and low levels of creatinine, that is, lower risks of renal failure associated with MM activity, which is a factor of poor prognosis [9,40,41].

The other marker already described was β2-microglobulin, whose intensity of expression on the surface of PCs has been associated to greater clinical outcomes of MM patients [42].

Considering the biological diversity of MM, more effective individualized therapies are necessary [43]. In order to identify patients with higher or lower risk of disease relapse, this study investigated the impact of immunophenotypic markers on MM patient survival, including their association with pre-ASCT MRD results.

Materials and Methods

Ethics statement

The study was approved by the institutional research ethics committee (process number: CAAE 17569119.0.0000.5434).

Patients and study design

This retrospective study used the databases of both the flow cytometry laboratory and the bone marrow transplantation service. 154 MM patients who underwent ASCT from June of 2013 to September of 2018 were included in this cohort. Fifty-seven patients (37%) were female and ninety-seven (63%) were male, aged 29 to 73 years. All patients had been previously treated with at least one chemotherapy regimen prior to ASCT. All of them had available the immunophenotypic markers and MRD data before transplantation. The median follow-up was 14 months.

Patients were separated into three groups according to MRD levels (<0.01%, 0.01% to 1% and >1%). During the cohort time, the levels of sensitivity of MRD tests varied from 10-4 (0.01%) by conventional MFC to 10-6 (0.0001%) by Next Generation Flow (NGF). A cut-off of < 0.01% was used to consider MRD as “negative” or indetectable.

The impact of the expression of each marker on diagnosis, such as CD27, CD28, CD45, CD56, CD117 and β2-microglobulin, was individually evaluated regarding the OS and PFS. According to the significant difference in survival curves, these markers were recognized as good (GPM) or Poor Prognostic Markers (PPM).

For each marker, one point was added to the score of either good or poor prognosis. The number of GPM and PPM were scored ranging from 0 to 5 for each patient. Patients were classified according to the number of GPM and PPM (≤2 and >3). The relation of these scores with survival rates were also assessed.

Two groups were defined considering the expression of β2- microglobulin, in order to evaluate its prognostic value, according to previously described [42]: group 1 included patients with low or no expression of β2-microglobulin, and group 2 with high expression of this marker.

Flow cytometry

Bone marrow samples collected in EDTAK2 tubes were processed within 24 hours of collection. Euroflow standard operating procedures were used for instrument setup, sample preparation, data acquisition and analysis as previously described [44]. For the diagnosis of MM, the samples were stained with an 8-color combination of monoclonal antibodies (MoAb) in 2 tubes as previously described [45]: (1) CD45HV450 (2D1), CD138PO(B-A38), CD38FITC(HB7), CD56PE (MY31), β2-microglobulin PerCPCy5.5 (TÜ99), CD19PECy7 (J3119), cytoplasmic (cy)Kappa APC (C022), cyLambda APCH7(1- 155-2), and (2) CD45HV450(2D1), CD138PO (B-A38), CD38 FITC (HB7), CD28PE (L293), CD27PerCPCy5.5 (L128), CD19PECy7 (J3119), CD117 APC (104D2), CD81APCH7 (JS-81). Pre-ASCT MRDs were evaluated using the conventional 8-color MFC, from June 2013 to September 2014. Next Generation Flow (NGF) were the methodology used from that date. The samples used to evaluate NGF-MRD were prepared using bulk-lysis prior to staining with MoAb, to allow recovery of > 107 cell events in most samples [46]. 8-color MM MRD combinations included 2 tubes: CD19PECy7 (J3119), CD27BV510 (O323), CD38FITC (HB7 or CYT-38F2), CD56PE (MY31), CD45PerCPCy5.5 (HI30), CD138BV421 (MI15) in both tubes, used as backbone markers [46]. They were combined with cyKappa APC (C022) and cyLambda APCH7 (1-155-2) in the first tube and CD117APC (104D2) and CD81APCH7 (JS-81) in the second tube, according to Euroflow recommendations [44,46]. A minimum of 1x106 cell events per tube was acquired for diagnostic samples and for conventional MFC-MRD samples, while at least 5x106 events per tube were acquired for NGF-MRD assessments, using a BD FACSCanto II 8-color flow cytometer/3lasers (BD Biosciences, San Jose, CA) and the FACSDiva software (BD Bioscience). The MRD detection and quantification limits were 10-4/10-5 for conventional MFC and 10-6 for NGF-MRD. InfinicytTM software (Cytognos SL, Salamanca, Spain) was used for MFC data analysis.

Statistical analysis

PFS was defined as the time from ASCT to progression or death from any cause and OS as the time from ASCT to death. Kaplan–Meier method and the two-sided log-rank test for each group were used to plot and compare OS and PFS. A Cox proportional hazard regression was used to evaluate the prognostic value of immunophenotypic markers. Pearson’s coefficient was used to calculate the correlations between MRD and the number of GPM and PPM. The tests were performed in the R 3.4.2 [47] environment software, with a statistically significant predictive value of p<0.05.


Prognostic value of immunophenotypic markers

The presence or absence of most immunophenotypic markers had an impact on patient survival. The isolated expressions of CD27 and CD45 were significantly related to higher OS (84% vs 29%, p= 0.013 for CD27; 84% vs 18%, p= 0.00 for CD45) and PFS (66% vs 0% for CD27; 38% vs 18% for CD45; p= 0.00 for both) when compared with cases without expressions of these markers (Figure 1A-B and E-F). On the other hand, the expressions of CD28 and CD56 were significantly associated to reduced survival rates: OS = 42% vs 61% (p= 0.026) and PFS= 0% vs 40% (p= 0.001) for CD28; OS=34% vs 86% (p= 0.004) and PFS 20% vs 44% (p= 0.009) for CD56 (Figure 1C-D and G-H). No differences were noticed in OS and PFS regarding CD117 (OS, p= 0.43; PFS, p= 0.25) and β2-microglobulin (OS, p= 0.41; PFS, p= 0.82) expressions (Figure 1 I-L). Although the high intensity of membrane β2-microglobulin has been associated with better SG rates [43], our data did not show a prognostic value for this marker, since patients with different intensities of β2-microglobulin expression had survival rates similar.