Minimal residual disease analysis in multiple myeloma: A single-center experience :Asish Rath, Tribikram Panda, Jasmita Dass, Tulika Seth, Manoranjan Mahapatra, Seema Tyagi, Journal of Applied Hematology

ORIGINAL ARTICLE
Year : 2023 | Volume : 14 | Issue : 1 | Page : 22--27

Minimal residual disease analysis in multiple myeloma: A single-center experience

Asish Rath, Tribikram Panda, Jasmita Dass, Tulika Seth, Manoranjan Mahapatra, Seema Tyagi
Department of Hematology, All India Institute of Medical Sciences, New Delhi, India

Correspondence Address:
Dr. Asish Rath
Department of Hematology, All India Institute of Medical Sciences, New Delhi
India

Abstract

BACKGROUND: Over the years, with changes in treatment approaches, it has been possible to achieve higher complete response (CR) rates with chemotherapies or chemoimmunotherapies in multiple myeloma (MM). However, a subset of patients in CR still relapse owing to the presence of residual tumor cells in the bone marrow not detectable by conventional methods. Residual disease detection by flow cytometry (FCM) has been proven to be highly sensitive and prognostically significant in a number of clinical studies. AIMS AND OBJECTIVES: In this study, we compared FCM minimal residual disease (FCM MRD) in MM cases post-chemotherapy/autologous stem cell transplant with morphology and biochemical methods. We also tried to correlate the pre-therapy stage of the disease and cytogenetics with MRD. MATERIALS AND METHODS: Twenty eight samples from 26 patients were evaluated for MRD on 6 color 3 tube panel over the period of 2 years. RESULTS: MRD was detectable in 19 samples (67.9%). FCM had a sensitivity of 95% compared to immunohistochemistry (IHC). 100% of cases with MRD positivity had abnormalities in at least three surface antigens. The high risk cytogenetics and high risk stage groups had a higher frequency of MRD positivity compared to the low risk groups. CONCLUSION: FCM MRD analysis is able to risk stratify the patients in CR and stringent CR. Routine use of FCM to detect residual disease posttherapy in MM should be implemented.

How to cite this article:
Rath A, Panda T, Dass J, Seth T, Mahapatra M, Tyagi S. Minimal residual disease analysis in multiple myeloma: A single-center experience.J Appl Hematol 2023;14:22-27

How to cite this URL:
Rath A, Panda T, Dass J, Seth T, Mahapatra M, Tyagi S. Minimal residual disease analysis in multiple myeloma: A single-center experience. J Appl Hematol [serial online] 2023 [cited 2023 Mar 20 ];14:22-27
Available from: https://www.jahjournal.org/text.asp?2023/14/1/22/369843

Full Text

Introduction

Traditionally, response assessment to therapy in multiple myeloma (MM) has been done by evaluation of serum and urine monoclonal protein concentrations by electrophoresis or immunofixation in adjunct to morphological assessment of bone marrow with immunohistochemistry (IHC).[1] Recent advances in treatment strategies have translated into better outcomes with more than 50% of patients achieving complete response (CR).[2] However, IHC has low sensitivity when a low number of abnormal plasma cells (APCs) are admixed with a polyclonal population so creating difficulty in stringent complete response assessment (sCR).[3] Different minimal residual disease (MRD) study groups have concluded the prognostic significance of MRD analysis by flow cytometry (FCM) in both postautologous stem cell transplant (ASCT) and transplant-ineligible patients.[2] MRD analysis in MM has also been studied using allele-specific oligonucleotide polymerase chain reaction and next-generation sequencing.[2] However, FCM has an advantage compared to molecular methods being highly applicable to almost all patients, cost-effective, and less laborious.[2]

In this study, we evaluated 28 posttreatment samples (postchemotherapy/post-ASCT) from 26 patients on a 6-color FCM panel. We compared the results of FCM with morphology, biochemical parameters, and IHC findings. We tried to correlate MRD findings with stage, cytogenetics, and different response assessment groups.

Patients and Methods

This is a prospective observational study approved by the Institutional Ethics Committee. A total of 28 BM samples from 26 patients were run for MRD analysis (in a single patient MRD was done at three time points). Patients completing induction chemotherapy or post-ASCT were recruited.

Study procedure

Bone marrow aspirate smears were stained with Jenner-Giemsa stain and evaluated for PCs. Concurrent serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), serum/urine-free light chain (FLC), and FISH for high-risk cytogenetics (CTG) data were obtained from patients' medical records. For all patients, bone marrow biopsy samples were stained with hematoxylin and eosin as well as CD138 (rabbit monoclonal, Clone-EP 201, PathInsitu, CA, USA), kappa (polyclonal, Dako, CA, USA), and lambda IHCs (polyclonal, Dako, CA, USA).

First, pull BM aspirate samples for MRD were received in EDTA anticoagulant and processed within 12 h. An ammonium chloride based bulk lysis/prelysis protocol (RBC lysis buffer, Biolegend, San Diego, CA) was used for all the samples. A 3-tube 6-color antibody panel was used for immunophenotyping [Table 1]. For cytoplasmic light chain staining, permeabilizing solution (BD Perm/Wash, BD Biosciences, San Jose, CA) was used after surface staining. In all washing steps, the aspiration of supernatant was done in place of simple decantation of the tube to minimize cell loss. A minimum of 1 million events were acquired on BD FACSCanto II 3-laser flow cytometer (BD Biosciences, San Jose, CA) immediately after sample processing in all the cases. A sequential gating strategy was adopted [Figure 1]. Mast cells, hematogenous, and NK cells were also evaluated to assess sample dilution.{Table 1}{Figure 1}

Calculation of lower limit of blank, lower limit of detection (LLOD), and lower limit of quantification (LLOQ) for MRD analysis were done as per published literature. Classically, 20 events and 50 events are considered the minimum events for LLOD and LLOQ in rare event analysis.[4],[5] The LLOD was further confirmed by a dilution experiment with a known chronic myeloid leukemia peripheral blood sample. For LLOQ, 50 events were considered in 1 million events.

Statistical analysis

The data entry was done in the Microsoft Excel spreadsheet, and the final analysis was done with the use of Statistical Package for Social Sciences (SPSS) software, IBM manufacturer, Chicago, IL, USA, version 21.0. For statistical significance, P < 0.05 was considered statistically significant.

Results

The patients (male/female, 22/4) were treated with different lines of chemotherapy alone or chemotherapy followed by an ASCT. BM samples were analyzed for residual disease after chemotherapy or post-ASCT. Patient characteristics are shown in [Table 2].{Table 2}

Number of acquired events in minimal residual disease cases

In 20 samples (71.4%), one million events could be acquired in each tube. In 5 samples (17.9%), <0.5 million events could be acquired due to the dilution of the sample. In rest 3 samples (10.7%), at least 0.5 million events could be acquired in each tube.

Residual disease in immunohistochemistry versus flow cytometry-minimal residual disease

Along with FCM MRD, simultaneous IHC for CD138, kappa, and lambda were done on bone marrow biopsy. The comparison between IHC and FCM MRD is shown in [Table 3].{Table 3}

Sensitivity and specificity of immunohistochemistry and flow cytometry to detect abnormal plasma cell in biochemical parameter (serum protein electrophoresis/immunofixation electrophoresis/free light chain) positive cases

Sixteen samples out of 28 samples analyzed were positive for residual disease by SPEP/IFE or FLC. When compared with the biochemical response (SPEP/IFE/FLC), FCM-MRD had a higher sensitivity of 95% in comparison to IHC (42.11%) to detect residual disease. One case though was in very good partial remission (VGPR) with an M-spike of 0.3 g/dL, both IHC and FCM showed only polyclonal expansion of PCs.

Plasma cells by morphology and flow cytometry in minimal residual disease cases

Total PCs obtained from FCMPC were lower in number compared to BMPC with mean/median of 4.03%/0.47% and 10%/4%, respectively.

Minimal residual disease percent

MRD% in MRD-positive cases ranged from 0.0028% to 9.7% with mean ± standard deviation (SD) of 0.705% ± 0.020% and a median of 0.0255%. Only a single case had MRD detectable just above LLOD and all other cases had MRD above LLOQ.

Abnormal antigen expression in minimal residual disease cases

CD19 was abnormal in 100% of cases, followed by CD45, CD81, and CD27 showing abnormal expression in 17 (89.4%) cases each. CD56 was abnormal in 12 (63.1%) cases. CD117 helped detect APCs in only 4 (21%) cases [Table 4].{Table 4}

Number of abnormal antigens in minimal residual disease-positive cases

100% of MRD cases had ≥3 abnormal antigen expressions with 18 cases (94.7%) showing abnormalities in at least four antigens [Table 5].{Table 5}

Light chain restriction in multiple myeloma minimal residual disease cases

Only in two cases, a light chain restriction could not be elicited due to a lower number of acquired events (<0.5 million in both cases) and possibly cell loss. However, both cases showed surface antigen abnormalities in four antigens each.

Comparison of CD138 and CD38 median fluorescence intensity (MFI) in abnormal plasma cells versus normal plasma cells

CD38 MFI was significantly lower in APCs in MRD cases in comparison to normal plasma cells (NPCs) (P = 0.006). NPCs were defined by the polyclonal nature of the cells. However, there was no significant statistical correlation between CD138 MFI of APCs and NPCs (P = 0.327).

Normal plasma cells s in minimal residual disease cases

NPCs in MRD cases ranged from 0% to 3.7% with a mean ± SD of 0.352% ± 0.717% and a median of 0.105%. In cases with MRD positivity, NPCs ranged from 0% to 1.032% with a mean of 0.196% ± 0.295% and a median of 0.1%.

Distribution of minimal residual disease cases

MRD cases (n = 19) included five cases of relapse/refractory MM (RR). All the RR cases had a residual disease in BM detected by FCM. In the rest of the cases, MRD was positive in 14 cases (60.9%).

Minimal residual disease positivity in high-risk cytogenetics and standard-risk cytogenetics

100% of high-risk CTG cases (n = 8) showed MRD positivity, however, only 50% of cases with standard CTG (n = 16) showed MRD positivity. The frequency of MRD positivity was significantly higher in high-risk CTG in comparison to standard-risk CTGs (P < 0.05) [Table 6].{Table 6}

Distribution of minimal residual disease cases in different staging and response groups

The MRD positivity rate was higher (85.9%) in the higher stage (RISS3/ISS3) group compared to the lower stage (RISS2/ISS2/ISS1/RISS1) (50%). However, the difference was not significant statistically (P = 0.106). About 90.9% and 100% of cases in VGPR (n = 11) and PR (n = 3), respectively, showed MRD positivity. Four cases (33.3%) out of all cases in sCR (n = 12) showed MRD positivity.

Minimal residual disease positivity in chemotherapy only and post-autologous stem cell transplant groups

Out of seven cases post-ASCT, six cases showed MRD positivity. One case was suspected of relapse and two cases had high-risk CTGs. Two cases did not have a baseline CTG report.

Out of 21 samples in postchemotherapy only group, 13 cases were MRD positive. Out of 8 cases which were MRD negative, 7 had a lower stage (RISS2/1 and ISS2/1), and no case had high-risk CTGs.

Discussion

The study demonstrated the role of FCM in MRD analysis in MM as well as the feasibility and utility of a 6-color MRD analysis in resource-constraint settings. We have shown the 3-tube 6-color panel to be adequately sensitive to detect most of the residual disease posttherapy which poses definite clinical implications. The studies based on FCM MRD in MM from Indian subcontinent are scarce and ranged from 4-color panels to 10-color panels.[6] There is a marked heterogeneity in MRD assessment protocols, panels as well as the MRD negativity frequencies in these studies. The MRD negativity rate varied from 37.3% to 60%.[6] In our cohort of patients analyzed for MRD, 67.9% (n = 19) of samples were positive for residual disease. Paiva et al., Li et al., and Rawstron et al. found MRD positivity of 70% (4-color FCM), 74.7% (6-color FCM), and 81% (6-color FCM), respectively, in postinduction chemotherapy samples.[7],[8],[9] Multiple studies have got 36% to 46% MRD in post-ASCT samples by 4–6 color FCM.[7],[10],[11] In contrast, Campbell et al. and Dold et al. found MRD positivity in 72% and 74%, respectively, post-ASCT.[12],[13] Although we got a higher percentage of MRD positivity (85.7%) in post-ASCT samples, smaller sample size and the presence of high-risk CTG may be contributing factors.

Although FCM is more sensitive than biochemical response parameters, a single patient in our study had MRD-negative/IFE and SPEP-positive results. Similar findings have been reported in the literature by Sharma et al. and Rawstron et al.[6],[9] The possible reasons for this can be a patchy persistence of residual disease, extramedullary residual disease, or a longer half-life of monoclonal proteins.[6]

Goicoechea et al. in an evaluation of 390 cases by next-generation FCM (NGF) showed high-risk CTG cases with a higher frequency of MRD positivity compared to standard risk (67% vs. 51%).[14] Similarly, Paiva et al. had shown a higher unsustained CR rate in patients with high-risk CTG and advanced stage at baseline.[10] In the PETHEMA/GEM2012MENOS65 trial on 390 cases, there was significant higher number MRD positivity in high-risk CTG cases 63% compared to 51% with standard-risk CTG (P = 0.04).[15] In our series, cases with high-risk CTG (n = 8) were MRD positive in 100% of cases whereas standard-risk CTG cases (n = 16) showed MRD positivity in only 50% of cases (P < 0.05). The higher MRD percentage in our cohort can be attributed to a smaller number of cases compared to most of the studies cited.

Paiva et al. in their study found 87 cases (36%) of patients with sCR with positive MRD similar to what we have observed (33.3%).[7] Twelve patients were in sCR in our cohort, of which four cases (33.3%) showed residual disease in the marrow. FCM showed a higher sensitivity as compared to IHC to detect APCs in this study (95% vs. 42.11%). This finding highlights the importance of FCM MRD even if the morphology, biochemical, and radiological parameters show a stringent response.

Similar to diagnostic immunophenotyping studies,[3],[16] we found CD19 abnormal under-expression in 100% of cases with MRD-positive cases. Flores-Montero et al. had shown similar findings with 96% CD19 abnormality in their cohort of 110 posttreatment cases of MM.[17]

All the cases in our cohort had ≥3 abnormalities in surface antigens. Similar observations were also obtained by Gupta et al. who showed at least three antigenic abnormalities in 90.7% of cases analyzed for MRD.[18]

CD38 MFI was significantly low in APCs versus NPCs in MRD-positive cases (P = 0.006). Flores-Montero in their study by NGF showed 77% cases with dim CD38 expression compared to NPCs.[17]

Conclusion

FCM MRD is able to detect a higher percentage of cases with the residual disease compared to traditional investigations. It is useful to detect residual disease in a cohort of patients who are otherwise in sCR by IHC. APCs in almost all cases of MM posttreatment show three or more than three surface antigenic abnormalities and hence a panel with multiple number of surface antigens is required for MRD assessment. Posttherapy MRD analysis by FCM should be incorporated in the management of MM cases as it can guide treatment strategies. A larger prospective trial should be done for further validating FCM-MRD with higher sensitivities.

Acknowledgment

We would like to acknowledge technologists and staffs in the Department of Hematology, AIIMS, New Delhi.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1	Rajkumar SV, Harousseau JL, Durie B, Anderson KC, Dimopoulos M, Kyle R, et al. Consensus recommendations for the uniform reporting of clinical trials: Report of the international myeloma workshop consensus panel 1. Blood 2011;117:4691-5.
2	Jelinek T, Bezdekova R, Zatopkova M, Burgos L, Simicek M, Sevcikova T, et al. Current applications of multiparameter flow cytometry in plasma cell disorders. Blood Cancer J 2017;7:e617.
3	Kumar S, Kimlinger T, Morice W. Immunophenotyping in multiple myeloma and related plasma cell disorders. Best Pract Res Clin Haematol 2010;23:433-51.
4	Arroz M, Came N, Lin P, Chen W, Yuan C, Lagoo A, et al. Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting. Cytometry B Clin Cytom 2016;90:31-9.
5	Stetler-Stevenson M, Paiva B, Stoolman L, Lin P, Jorgensen JL, Orfao A, et al. Consensus guidelines for myeloma minimal residual disease sample staining and data acquisition. Cytometry B Clin Cytom 2016;90:26-30.
6	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 Cancer Res Ther 2021;17:1515-20.
7	Paiva B, Martinez-Lopez J, Vidriales MB, Mateos MV, Montalban MA, Fernandez-Redondo E, et al. Comparison of immunofixation, serum free light chain, and immunophenotyping for response evaluation and prognostication in multiple myeloma. J Clin Oncol 2011;29:1627-33.
8	Li H, Li F, Zhou X, Mei J, Song P, An Z, et al. Achieving minimal residual disease-negative by multiparameter flow cytometry may ameliorate a poor prognosis in MM patients with high-risk cytogenetics: A retrospective single-center analysis. Ann Hematol 2019;98:1185-95.
9	Rawstron AC, de Tute RM, Haughton J, Owen RG. Measuring disease levels in myeloma using flow cytometry in combination with other laboratory techniques: Lessons from the past 20 years at the Leeds haematological malignancy diagnostic service. Cytometry B Clin Cytom 2016;90:54-60.
10	Paiva B, Gutiérrez NC, Rosiñol L, Vídriales MB, Montalbán MÁ, Martínez-López J, et al. High-risk cytogenetics and persistent minimal residual disease by multiparameter flow cytometry predict unsustained complete response after autologous stem cell transplantation in multiple myeloma. Blood 2012;119:687-91.
11	Puig N, Sarasquete ME, Balanzategui A, Martínez J, Paiva B, García H, et al. Critical evaluation of ASO RQ-PCR for minimal residual disease evaluation in multiple myeloma. A comparative analysis with flow cytometry. Leukemia 2014;28:391-7.
12	Campbell L, Panitsas F, Basu S, Anyanwu F, Lee S, Ferry B, et al. Serological normalisation as a surrogate marker for minimal residual disease negativity in multiple myeloma. Br J Haematol 2019;185:775-8.
13	Dold SM, Riebl V, Wider D, Follo M, Pantic M, Ihorst G, et al. Validated single-tube multiparameter flow cytometry approach for the assessment of minimal residual disease in multiple myeloma. Haematologica 2020;105:e523.
14	Goicoechea I, Puig N, Cedena MT, Burgos L, Cordón L, Vidriales MB, et al. Deep MRD profiling defines outcome and unveils different modes of treatment resistance in standard- and high-risk myeloma. Blood 2021;137:49-60.
15	Paiva B, Puig N, Cedena MT, Rosiñol L, Cordón L, Vidriales MB, et al. Measurable residual disease by next-generation flow cytometry in multiple myeloma. J Clin Oncol 2020;38:784-92.
16	Tembhare PR, Yuan CM, Venzon D, Braylan R, Korde N, Manasanch E, et al. Flow cytometric differentiation of abnormal and normal plasma cells in the bone marrow in patients with multiple myeloma and its precursor diseases. Leuk Res 2014;38:371-6.
17	Flores-Montero J, Sanoja-Flores L, Paiva B, Puig N, García-Sánchez O, Böttcher S, et al. Next generation flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. Leukemia 2017;31:2094-103.
18	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.