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Utility and feasibility of a six-color multiparametric flow cytometry for measurable residual disease analysis in plasma cell myeloma in resource-limited settings with 5-year survival data

1 Department of Hematology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Immunopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission25-Nov-2019
Date of Acceptance18-Jun-2018
Date of Web Publication05-Aug-2021

Correspondence Address:
Man Updesh Singh Sachdeva,
Department of Hematology, Research Block A, 5th Floor, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1027_19

 > Abstract 

Introduction: Treatment of multiple myeloma (MM) has evolved over decades with the introduction of novel therapeutic strategies. Response rates has significantly improved; however, there is a need for more sensitive techniques to study the residual disease other than conventional means. We evaluated the feasibility and utility of a two-tube six color multiparametric flow cytometry (MFC) assay for measurable residual disease (MRD) detection in MM patients on treatment.
Methodology: Pretitrated cocktails containing antibodies against CD38, CD138, CD45, CD19, CD56, CD81, CD27, and cytoplasmic kappa and lambda light chains were used in the combination of two tubes and were acquired on a flow cytometer. Limit of detection was determined through dilution and spiking experiments with a limit of 0.01%.
Results: Of the 62 patients screened, 58 patients were included in the final study cohort (day 100 postautologous stem cell transplant and at the end of induction chemotherapy). Twenty-eight patients (48%) revealed the presence of MRD in bone marrow on MFC (median = 0.12, range = 0.01–5.89%). Out of 28 MFC-MRD positive patients, only 16 patients showed M band on immunofixation-electrophoresis (IFE) (MRD+/IFE+, 57%), and rest of them were IFE negative (MRD+/IFE-, 42%). Patients with MRD positive status at the end of induction chemotherapy or day 100 posttransplant had an inferior overall survival (P = 0.009) and progression-free survival (P = 0.0002) than those with MRD negativity.
Conclusion: We have demonstrated the impact of MRD testing in MM using MFC with a long follow-up data, suggesting its routine incorporation in monitoring the disease independent of the immunofixation status.

Keywords: Minimal residual disease, multiparametric flow cytometry, multiple myeloma

How to cite this URL:
Sharma P, Singh Sachdeva MU, Varma N, Bose P, Aggarwal R, Malhotra P. Utility and feasibility of a six-color multiparametric flow cytometry for measurable residual disease analysis in plasma cell myeloma in resource-limited settings with 5-year survival data. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 6]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=323168

 > Introduction Top

According to the GLOBOCAN/IARC data, multiple myeloma (MM) ranks 23rd among the common cancers in India (1.1%), with 12,923 new cases of MM diagnosed in a year.[1] Although the annual percentage change (APC) of the incidence of MM from 2000 to 2009 is insignificant, the APC in mortality (s722;1.8%) over the same period has been potentially significant due to increased survival from the better modalities of treatment.[2] There is an improvement in the response rates and also in the proportion of patients achieving high-quality categorical responses.[3] The advent of novel therapies in the past decade has further transformed the treatment. In particular, immunomodulatory drugs (thalidomide, lenalidomide, and pomalidomide) and proteasome inhibitors (bortezomib and carfilzomib) when combined as initial therapy for newly diagnosed patients can achieve universal responses and CRs in a significant fraction of patients. Finally, the use of consolidation and maintenance therapy with novel agents can now further increase CR rate and prolong duration of response.[4] In spite of significant advances in the management of the disease, MM remains incurable. The majority of patients with MM have persistent levels of residual disease that is below the sensitivity of bone marrow (BM) morphology, serum protein electrophoresis (SPEP), immunofixation-electrophoresis (IFE), and light chain quantitation, even after attaining CR and will eventually relapse.[5] Measurement of this measurable residual disease (MRD) by more sensitive methods, and the use of these methods as a tool for predicting patient outcomes and guiding therapeutic decisions, has thus become more relevant.

Sensitive techniques that have been studied with increasing frequency during the past few years are polymerase chain reaction, multi-parametric flow cytometry (MFC), and next-generation sequencing. These techniques have been shown to be valuable in other hematologic malignancies; however, neither of these have become a standard of care in MM.[5] It is possible to detect neoplastic PCs by flow cytometry above the clinically relevant threshold of 0.01%, and this is more informative than conventional assessment.[6] Although not into the clinical practice, studies from the West have established the fact that MRD status in MM patients after achieving CR/very good partial response predicts outcome, guide therapeutic decision, and will become a routine in the near future.[5] However, studies from India related to this context are lacking. In the present study, we evaluated the efficacy of a two-tube 6-color MFC panel for the detection of MRD in patients of MM after chemotherapy/autologous stem-cell transplant (ASCT) along with its impact on the survival of these patients.

 > Methodology Top

Following clearance from the Institutional Ethical Committee, a prospective study was undertaken at a tertiary care institute in North India, during August 2014 to December 2016. Newly diagnosed treatment naïve patients of MM and patients yet to complete induction chemotherapy or within day 100 post-ASCT were included in the study. MRD assessment was performed in these patients irrespective of the clinical/biochemical status or category of response. In addition, marrow samples from few patients were also available for interim disease analysis. After obtaining informed consent, 3–5 mL of BM aspirate sample was collected in EDTA anticoagulated vacutainer tubes. All the samples were processed through Euroflow bulk-lysis technique[7] and were acquired on flow cytometer within 24–48 h of sample collection. Baseline investigations and routinely carried out light microscopic findings of the BM examination were recorded for each of the enrolled patients. The antibody panel for MRD analysis in patients of MM consisted of two tubes, tube 1 having combinations of Cytoplasmic kappa light chain-FITC, Cytoplasmic lambda light chain-PE, CD38 PerCP-Cy5.5, CD45 APC-H7, CD56 APC, CD19 PE-Cy7, and tube 2 containing CD81 FITC, CD138 PE, CD38 PerCP-Cy5.5, CD45 APC-H7, CD19 PE-Cy7 and CD27 APC. At least one million events (in combination of two tubes) were acquired on dual-laser BD FACS Canto II and analyzed using Kaluza software version 2.1 (Beckman Coulter, Inc., Brea, California, USA). For MRD analysis, continuous events were gated on a time plot, which were then plotted on forward scatter-height (FSC-H) versus forward scatter-area (FSC-A) plot for doublet discrimination. For viable cells, the singlets gated in the previous plot were analyzed in SSC-A versus FSC-A. The plasma cells were gated based on CD138 and/or CD38 expression. Further refining of the plasma cells was done on CD45 and CD38 bivariate plot. The gated cells were analyzed on the bivariate plots of all combinations for the expression of various surface markers to identify abnormal plasma cells and for cytoplasmic light chain restriction. Plasma cells were labeled abnormal based on their altered immunophenotype (CD45dim to neg, CD56pos, CD38dim, CD19dim to neg, CD81dim to neg, CD27dim to neg and cytoplasmic light chain restriction). Scatter dot-plots from a patient with abnormal plasma cells indicating residual disease are shown in [Figure 1]. Limit of detection (LOD) was identified using the dilution and spiking experiment. A discrete clustered population of ≥50 events showing abnormal immunophenotype was considered to represent MRD (LOD of 0.01%). These cells were finally recorded as the numerator for MRD calculations. With a detection limit of one neoplastic cell per 10,000 normal cells in MFC, the MRD level of ≥0.01% was considered positive, herewith referred as “MRD positive,” and MRD values <0.01% were considered negative, herewith referred as “MRD negative.”
Figure 1: Scatter dot-plots from a multiple myeloma patient on chemotherapy showing the immunophenotypic profile of plasma cells. The abnormal plasma cells (Red) are CD56pos, CD45neg, CD27dim, CD81neg, CD19dim to neg, CD38dim and cytoplasmic kappa light chain restricted. The normal plasma cells within the sample are highlighted in blue/coarse dots which are polyclonal in nature

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Response assessment to therapy was performed using the modified International Myeloma Working Group (IMWG) consensus criteria for response assessment in MM.[8] The Kolmogorov–Smimov test was used to confirm the pattern of distribution of the data. Percentage of BM PCs yielded a skewed data while all the other parameters were normally distributed. Continuous data were represented as mean ± standard deviation. For the comparison of the means between various parameters, in case of continuous data, having normal distribution, paired t-test was applied. For skewed distribution, the Mann–Whitney test was used. Progression-free survival (PFS) was defined as the time from the diagnosis of the MM to first-documented disease progression/death after a therapy and overall survival (OS) from the time of initial diagnosis to death from any cause. The statistical analysis was performed using the MS Excel 2007 and GraphPad Prism version 8.0 for Windows, GraphPad Software, San Diego, California, USA. Survival data were studied using the Kaplan–Meier analysis and log-rank (Mantel-Cox) test. All tests were two tailed and were statistically significant at P < 0.05.

 > Results Top

Of the 62 patients who underwent BM examination during the study period for morphologic evaluation of disease status during the course of treatment, 58 patients were included in the final cohort. The group comprised of males and females in the ratio of 1.6:1. The mean age of the patients was 53.5 years. Among them, 20 patients were evaluated for MRD at day + 100 of ASCT, and 35 patients were evaluated at the end of induction phase of chemotherapy. Three patients were evaluated during the maintenance/during the interim course of treatment for the suspicion of a relapsed disease. Twenty-eight patients (28/58 patients, 48%) revealed the presence of residual disease in BM on multiparametric flow cytometric analysis (median = 0.12%, range = 0.01%–5.89%). 20/28 patients with MRD positivity on MFC were said to be in morphological remission (<5% plasma cells on BM).

Serum IFE was positive showing a residual monoclonal “M” band in 18/58 (31%) patients. All of these 18 patients, except two, had measurable disease detected by MFC as well. Out of 28 MFC-MRD-positive patients, only 16 patients showed M band on IFE (MRD+/IFE+, 16/28, 57%). Rather, 12 patients with MFC-MRD positive results showed no M band on IFE (MRD+/IFE-, 12/28, 42%). Out of all patients showing an M band, SPEP+/IFE + results were noted in 13 patients and SPEP-/IFE + results in five patients.

Evaluation of statistical correlation between MRD status and various hematological and biochemical parameters revealed PC percentage in BM aspirate smears to be significantly higher in the MRD-positive group (median = 2, range = 1–27, P = 0.0076). However, no significant difference was noted between the MRD status and age and sex distribution, mean values of hemoglobin, platelet count, total leukocyte count, serum creatinine, serum calcium, and serum albumin.

Kaplan–Meier survival characteristics for OS and PFS were studied between the two groups, [Figure 2] with a follow-up range of 6–103 months. The median OS of patients in the MRD positive and MRD negative sub-group was 47 months and 57.5 months, whereas the median PFS among the sub-groups were 27 months and 52 months, respectively. There was a significant difference between the OS (P = 0.009) and PFS (P = 0.0002) among the MRD positive and MRD negative subgroup. Patients with MRD positive status at the end of induction chemotherapy or day 100 posttransplant had an inferior outcome than those with MRD negativity. The complete data of clinical, biochemical, and hematological parameters of the study cohort are summarized in [Table 1].
Figure 2: Overall survival and progression free survival of measurable residual disease negative and measurable residual disease positive sub-group

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Table 1: Summary of clinical, biochemical and hematological parameters of the study cohort

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 > Discussion Top

The study is based upon the data obtained from 58 Indian patients of MM on treatment (chemotherapy/transplanted), demonstrating the role of MFC in detecting the MRD and predicting the PFS and OS in these patients. We have shown that a two tube six-color antibody panel is well suitable for MRD detection with adequate sensitivity and definite clinical implications. Furthermore, MFC-MRD positivity at the end of induction chemotherapy/day-100 posttransplant confers a significant inferior impact on both OS and PFS of MM patients. Extensive literature search of existing Indian literature identified a few studies on the role of minimal residual disease in MM [Table 2].[9],[10],[11],[12],[13] There was a marked heterogeneity among these Indian studies in the methodology and criteria for MRD detection and reporting, reflecting the results of a recent survey conducted across various institutions of the United States of America.[14]
Table 2: Summary of studies from India incorporating MFC for MRD analysis

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Out of 28 MRD-positive patients, 16 patients had positivity in IFE (MRD+/IFE+) and only 12 patients had an M band on SPEP. Despite MFC being more sensitive than IFE, two of our patients had MRD negative/IFE positive results. Similar results have been noted previously by Paiva et al.[15] and also by Rawstron et al.[16] probable reasons not limited to the facts that there may be a longer half-life of some of the immunoglobulins, nonrepresentative BM obtained for MFC, presence of residual plasma cells outside BM, or patchy persistence of residual disease. Interestingly, when these groups were evaluated by Paiva et al. for OS and PFS, MFC-negative/IFE positive patients had a very good outcome, similar to MRD negative/IFE negative patients, significantly better than MFC positive/IFE negative ones.[15] Of the MRD-negative/IFE-positive patients in the present study, five patients did not show an M band on SPEP, which may be explained by the fact that the sensitivity of IFE for detecting a clonal band is more by a multitude times than that of SPEP.[17] Hence, studies recommend that MRD should be performed in all the patients on therapy regardless of the status of the M protein response and the same has been incorporated into the IMWG MRD criteria for response assessment in MM.[8]

We demonstrated that 42% (12/28) of patients after postinduction chemotherapy/ASCT had a detectable disease after achieving IFE negative CR. Rawstron et al. demonstrated the same in 14.5% of patients after ASCT.[16] The same group demonstrated MRD in 27% of IFE-negative CR patients earlier[18] and Paiva et al. demonstrated MRD in 36% of patients in CR, treated with conventional chemotherapy and ASCT.[15] All the studies have shown that MRD positivity is an independent factor for influencing the PFS and OS of MM patients regardless of the IFE status.

Most of the patients who had residual disease identified by MFC belonged to the group in whom MRD was evaluated after chemotherapy (22/38 patients who received chemotherapy only). Whereas 14/20 (70%) patients who underwent a BM transplant proved to be MRD negative. Patients, who underwent ASCT, have less chances of having minimal residual disease assessed by MFC, when compared to the patients receiving chemotherapy alone. San Miguel et al. compared the efficacy of high-dose chemotherapy followed by ASCT (n = 47) with that of conventional chemotherapy (n = 40) by using a four-color MFC and found that a significantly greater reduction of neoplastic PCs and better recovery of normal PCs occurs in patients who underwent ASCT than in patients who had received conventional chemotherapy.[19] Rawstron et al. and others have also shown that ASCT remains a highly effective component of myeloma therapy.[15],[18]

 > Conclusion Top

Considering the fact that India being one of the low-to-middle income countries, it has limited availability of advanced flow cytometry centers, lack of awareness and specialized training, economical factors including low health insurance coverage and lesser affordability of the patient toward health care; we designed a two tube 6 color-panel for MRD testing in MM using MFC with reasonable sensitivity and long follow-up showing significant impact on prediction of OS and PFS among these patients. Much larger studies are needed in this regard including clinical trials incorporating therapeutic interventions based on MRD status for better understanding of the disease and improved monitoring and treatment of MM in India. We also suggest the use of this technique in routine prognostication of myeloma patients on treatment, complementing other routine biochemical investigations, in order to predict/achieve longer OS of the patients.

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Conflicts of interest

There are no conflicts of interest.

 > References Top

The Global Cancer Observatory; 2018. Available from: http://gco.iarc.fr/today/data/factsheets/populations/356-india-fact-sheets.pdf. [Last accessed on 2019 Nov 20].  Back to cited text no. 1
Kazandjian D. Multiple myeloma epidemiology and survival: A unique malignancy. Semin Oncol 2016;43:676-81.  Back to cited text no. 2
Barlogie B, Shaughnessy J, Tricot G, Jacobson J, Zangari M, Anaissie E, et al. Treatment of multiple myeloma. Blood 2004;103:20-32.  Back to cited text no. 3
Munshi NC, Anderson KC. Minimal residual disease in multiple myeloma. J Clin Oncol 2013;31:2523-6.  Back to cited text no. 4
Hart AJ, Jagasia MH, Kim AS, Mosse CA, Savani BN, Kassim A. Minimal residual disease in myeloma: Are we there yet? Biol Blood Marrow Transplant 2012;18:1790-9.  Back to cited text no. 5
Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H, et al. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica 2008;93:431-8.  Back to cited text no. 6
Theunissen P, Mejstrikova E, Sedek L, van der Sluijs-Gelling AJ, Gaipa G, Bartels M, et al. Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia. Blood 2017;129:347-57.  Back to cited text no. 7
Kumar S, Paiva B, Anderson KC, Durie B, Landgren O, Moreau P, et al. International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma. Lancet Oncol 2016;17:e328-e346.  Back to cited text no. 8
Gupta R, Bhaskar A, Kumar L, Sharma A, Jain P. Flow cytometric immunophenotyping and minimal residual disease analysis in multiple myeloma. Am J Clin Pathol 2009;132:728-32.  Back to cited text no. 9
Gupta R, Kumar L, Dahiya M, Mathur N, Harish P, Sharma A, et al. Minimal residual disease evaluation in autologous stem cell transplantation recipients with multiple myeloma. Leuk Lymphoma 2017;58:1234-7.  Back to cited text no. 10
Mukesh P, Lalit K, Atul S, Ritu G, Sreenivas V, Ramavath Devendra N, et al. Prospective evaluation of minimal residual disease in multiple myeloma following autologous stem cell transplantation (ASCT) and consolidation therapy. Journal of Clinical Oncology. 2018;36:e20033.  Back to cited text no. 11
Subramanian PG, Chatterjee G, Ghogale S, Gokarn A, Gudapati P, Deshpande N, et al. Standardization of high sensitivity minimal residual disease monitoring in multiple myeloma: An experience in tertiary cancer centre. Clin Lymphoma Myeloma Leukemia 2019;19:e357.  Back to cited text no. 12
Yanamandra U, Mittal BR, Reddy A, Sachdeva M, Prakash G, Khadwal A, et al. Is 18F-FDG-PET/CT a Good MRD marker in patients with multiple myeloma? Comparison and correlation with biochemical markers/flow cytometry. Clin Lymphoma Myeloma Leukemia 2017;17:e113-4.  Back to cited text no. 13
Flanders A, Stetler-Stevenson M, Landgren O. Minimal residual disease testing in multiple myeloma by flow cytometry: Major heterogeneity. Blood 2013;122:1088-9.  Back to cited text no. 14
Paiva B, Vidriales MB, Cerveró J, Mateo G, Pérez JJ, Montalbán MA, et al. Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation. Blood 2008;112:4017-23.  Back to cited text no. 15
Rawstron AC, Child JA, de Tute RM, Davies FE, Gregory WM, Bell SE, et al. Minimal residual disease assessed by multiparameter flow cytometry in multiple myeloma: Impact on outcome in the Medical Research Council Myeloma IX Study. J Clin Oncol 2013;31:2540-7.  Back to cited text no. 16
Tripathy S. The role of serum protein electrophoresis in the detection of multiple myeloma: An experience of a corporate hospital. J Clin Diagn Res 2012;6:1458-61.  Back to cited text no. 17
Rawstron AC, Davies FE, DasGupta R, Ashcroft AJ, Patmore R, Drayson MT, et al. Flow cytometric disease monitoring in multiple myeloma: The relationship between normal and neoplastic plasma cells predicts outcome after transplantation. Blood 2002;100:3095-100.  Back to cited text no. 18
San Miguel JF, Almeida J, Mateo G, Bladé J, López-Berges C, Caballero D, et al. Immunophenotypic evaluation of the plasma cell compartment in multiple myeloma: A tool for comparing the efficacy of different treatment strategies and predicting outcome. Blood 2002;99:1853-6.  Back to cited text no. 19


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