Keywords
multiple myeloma - flow cytometry - next-generation flow - measurable residual disease
Introduction
Multiple myeloma (MM) is a hematological malignancy characterized by the proliferation
of clonal plasma cells (PC) in the bone marrow (BM) and presence of monoclonal protein
(M protein) in serum and/or urine and lytic bone lesions. Though the development of
novel therapeutic strategies has improved the overall prognosis, a substantial number
of patients relapse despite achieving good clinical response. Following the current
guidelines of response assessment in MM, over two-thirds of the patients achieving
complete remission (CR) relapse within 2 years.[1]
[2] Thus, in order to refine the response assessment in MM, International Myeloma Working
Group (IMWG) included the measurable residual disease (MRD) assessment as additional
response criteria in 2016.[3] MRD in MM is defined as the residual small number of cancer cells surviving after
treatment that is not identifiable by routine clinical and laboratory parameters.
IMWG recommends either intramedullary or extramedullary MRD detection based on BM
aspirate (BMA) samples and imaging techniques respectively.[3] Extramedullary MRD detection includes imaging techniques, such as positron emission
tomography with computed tomography using 18F-deoxyglucose or magnetic resonance imaging.[4]
Earliest evidence of MRD assessment in MM came in late 1980s when an attempt was made
to detect residual PC in patients' BM biopsies using immunohistochemistry, though
the technique was too crude to estimate MRD.[5] Following this, researchers in the initial years estimated MRD in MM by qualitative
polymerase chain reaction (PCR) followed by quantitative allele-specific oligonucleotide
PCR (ASO qPCR).[6]
[7] Use of multiparametric flow cytometry (MFC) for MRD detection in MM was attempted
in 1999, when Almeida et al explored the role of flow cytometric immunophenotyping
(FCMI) and DNA ploidy status for the investigation of residual neoplastic PC in MM
patients.[8] Since then, MFC has contributed significantly to the understanding of the aberrant/clonal
PC (APC) compartment and has now found a place in the diagnosis, prognosis, and in
treatment monitoring of MM. In this review, we will critically appraise the role of
MFC for MRD detection in MM and make an attempt to identify the caveats present in
MM MRD estimation by FCMI.
Flow Cytometry in Myeloma MRD—Transitioning from Conventional Flow Cytometry to Next-Generation
Flow cytometry
Flow Cytometry in Myeloma MRD—Transitioning from Conventional Flow Cytometry to Next-Generation
Flow cytometry
MFC has become the most valuable tool to monitor MRD and evaluate the depth of response
in MM patients. MFC offers an edge over other techniques of MRD detection, that is,
ASO qPCR and next-generation sequencing (NGS) due to its rapid turn-around time, wider
applicability, and cost-effectiveness. Furthermore, it plays a crucial role in treatment
monitoring and response assessment in oligo-secretory and nonsecretory myeloma where
monoclonal protein estimation has no role. FCMI has also shown a promising role in
demonstrating rare phenomenon like clonal switch and light chain escape. The very
first step in the FCMI for MRD evaluation is optimal panel design and validation before
using it routinely in clinical diagnostics.[9] Four set of markers are required for a panel to precisely determine MRD:
-
A. Gating marker—CD38, CD138, and CD45
-
B. Markers of aberrancies—CD19, CD56, CD27, CD81, CD117, CD20, CD28, etc.
-
C. Markers to identify clonality—cyt kappa and cyt lambda
-
D. Markers for adequacy—CD117. The major limitation of four to six color panels is lack
of incorporation of all sets of markers in a single tube that led to the transition
to higher versions with added benefits of increased sensitivity.
Conventional 4–6 Color Panel
Though the conventional four to six-color flow-MRD is applicable in majority of patients
(⩾95%), the sensitivity of conventional flow cytometry remains lower (<10−4) than that of molecular techniques, namely ASOqPCR (<10−5) and NGS (<10−6).[10]
[11]
[12] Due to the limited sensitivity of four to six color assays, there was high likelihood
of missing the low APC burden. Limitations of such limited panel assay are high likelihood
of missing the low APC burden and failure to combine immunophenotypic aberrancies
with light chain restriction leading to difficulty in the characterization and differentiation
of APC from non-neoplastic normal PC (NPC). Thus, overall, the four to six-color panel
was not suitable for MRD assessment in MM and the transition to higher version was
inevitable.
Conventional 8-Color Panel
A step forward in disease monitoring was conventional eight-color flow-MRD assays
with an increased sensitivity of less than 10−4 to less than 10−5, leading to a significantly improved prediction of outcome across different studies.[9]
[13]
[14] However, the two major limitations of the conventional eight-color antibody panel
were limited sensitivity and lack of standardization in terms of variation in antibodies,
number of cells evaluated, and lack of unanimous cutoff for MRD levels. To offset
this challenge, standardization efforts were made by the Euroflow consortium for highly
sensitive and standardized detection of MRD in MM using the NGF approach.[15]
Euroflow 2-Tube-8-Color Panel
Euroflow standardization study for NGF included an optimized 2-tube eight-color antibody
panel, acquisition of more than or equal to107 cells/sample using bulk lysis procedure and construction of novel software tools
for combined analysis of both the tubes for automated PC gating. In this multicenter
evaluation of NGF for myeloma MRD, one-fourth (∼25%) patients classified as MRD-negative
by conventional eight-color flow cytometry were found to be MRD-positive by NGF. This
resulted in identification of a flow MRD-negative subset of patients within the CR
group who had significantly superior progression-free survival (PFS). However, in
the real-life clinical settings especially in the resource constraint countries, the
inherent challenges associated with Euroflow two tube panel are as follows:
-
A. High sample demand with wastage: Two eight-color tubes with the acquisition of more than 1 million cells per tube
require a higher volume of BMA that may not be always available due to the primary
fibrotic nature of MM. Furthermore, it also leads to wastage of precious samples which
can be utilized for cytogenetic and molecular studies.
-
B. Turnaround time: Comparatively longer due to increased sample processing and data acquisition time
-
C. Wastage of antibodies: Common gating markers in both the tubes
-
D. Increased technical demand: Heavy demand on sample quality control, and data storage
-
E. Analysis: Time-consuming with inferential reasoning between the tubes. As a result, investigators
started looking for a better, cost-effective, and efficient panel for MRD evaluation
in MM.
Single-Tube-10-Color Panel
In an attempt to develop less expensive, low sample requisite method to detect MRD
with equivalent sensitivity to that of Euroflow-NGF, single-tube-10-color panel was
designed across few centers.[16]
[17] In a study by Sato et al,[17] the analytical ability of the10-color panel was compared to that of Euroflow-NGF
that demonstrated a good correlation between the two methods. This study also demonstrated
a comparable performance of 10-color-MFC and Euroflow-NGF in terms of median percentages
of total PC (0.2148 vs. 0.2200%, r = 0.950) as well neoplastic PC (0.0012 vs. 0.0007%, r = 0.954). Thus, as per the published literature, the single tube10-color-panel is
a promising alternative to Euroflow two tube eight-color panels for MRD detection.
Beyond 10-Color Panel in MM
In constructing the panel for MRD detection, the next relevant question is the need
of additional immunophenotypic markers for MRD evaluation. In a recent study, fair
discrimination between normal and clonal PC was demonstrated in most of the plasma
cell proliferative disorders (PCPD) samples (∼99.5% of samples) using eight markers
(Gating:CD38, CD138, CD45; aberrancy:CD19, CD56, CD27; clonality: Cy-kappa & Cy-lambda).[18] This confirmed the high efficacy of single tube-10-color-panel in APC identification
and thus, MRD detection. Our in-house data for 13-color panel with addition of CD20,
CD28 and CD3 (unpublished) showed a good correlation between single-tube-10-color
panel (median TPC: 0.98%, range: 0.02–5.24; median APC: 0.59%, range: 0.009–4.72;
median neoplastic plasma cell index [NPCI]: 54.25%, range: 8.16–99.11) and single-tube-13-color
panel (median TPC: 1.12%, range: 0.03–4.28; median APC: 0.48%, range: 0.01–4.39; median
NPCI: 60.55%, range: 1.65–98.46; [Fig. 1]) with no added benefits of more than 10 color panel in MRD analysis. Nonetheless,
a higher version of panel can be explored for ancillary immunophenotypic studies,
namely B-cell subset analysis; immune cell profiling in MM patients owing to the benefits
of additional markers. [Supplementary Table 1] (available in the online version) shows various antibody panels used for flow cytometry
based MRD assay in MM.
Fig. 1 Flow cytometric dot plots showing PC quantification using 10- and 13-color panel,
respectively (1.1 and 1.2); Bland-Altman correlation analysis showing linear positive
correlation between 10-color versus 13-color panel (1.3). APC, aberrant PC; NPC, normal
PC; PC, plasma cell.
Preanalytical Considerations for MFC-Based MRD Detection
Preanalytical Considerations for MFC-Based MRD Detection
The basic step in the MRD monitoring of MM is to ensure optimal sample quality till
the processing of sample ([Supplementary Fig. 1], available in the online version).
Sample Type and Collection
BMA sample is the prerequisite for MRD estimation; however, peripheral blood sample
has been explored as an alternative for circulating PC identification and quantification
using sequencing studies across the literature.[19]
[20]
[21] The major drawbacks associated with the use of bone marrow sample for MRD detection
are use of quasi-invasive BM aspiration procedure, inadequate PC representation due
to the inherent marrow fibrosis associated with MM, and procedural hemodilution. The
first two issues can be addressed with the use of non-BMA-based alternate strategy
to detect MRD, that is, estimation of M-protein and imaging.[22]
[23] Hemodilution is one of the major challenges in MRD estimation and can be reduced
using first pull BMA sample for MFC assay as shown across various studies.[24]
[25]
[26] For sample collection, ethylenediaminetetraacetic acid (EDTA) is the anticoagulant
of choice. Although heparin can be used as an alternative to EDTA in MRD estimations
in other hematolymphoid tumors, its use in MM is best avoided as it interferes with
assessments of CD138 that is a key gating marker in myeloma MRD. The use of sodium
citrate is also avoided as it reduces the cell viability.[27]
Sample Storage and Transportation
Apart from the type of anticoagulant used, the handling and transportation of the
specimen are critical. The peripheral blood and BM specimen must be insulated from
the external temperature. While transportation, the age of the specimen also plays
an important role, therefore, the specimens must be labeled with the date and time
of collection. A 24-hour cutoff is considered appropriate for sample processing to
ensure PC viability for accurate MRD determination.[9] However, for all practical purposes a 4 to 6-hour cutoff from sample collection
to sample processing is preferred. The viability of the specimen should be assessed
with the flow cytometric evaluation and the samples with less than 85% viability should
be reported stating that the viability of the sample was suboptimal for testing.[28] Factors indicating a compromised specimen are hemodilution, clots, small BMA volume,
and cold or excessively warm samples.
Sample Processing
BMA sample for MRD detection should be preferably processed within 4 to 6 hours of
collection to get maximum PC yield; however, it may be stored for 2 days at 4 to 8
degrees. Lysing the sample before staining is preferred especially in samples for
MRD analysis as this allows the delivery of maximum number of cells per tube in the
panel. However, this method also has a drawback that certain antibody-fluorochrome
conjugates become unstable. Cell fixation post-staining is recommended for resolving
such issues.[29] The samples can be gently prelysed by incubating with ammonium chloride-based lysis
solution. Another method is used is lysis of the sample post-staining, but it is associated
with lower stability issues and decreased sensitivity due to lower number of cells.
Euroflow consortium also supports the prelysis method for MRD detection with high
cellular yield in MM and the same method can be used for PC enumeration in other PCPDs.[30]
Analytical Considerations for MFC-Based MRD Detection
Analytical Considerations for MFC-Based MRD Detection
Determinants of Reproducible MRD Enumeration
Multicenter data has demonstrated that the level of 0.01% is of clear prognostic value
for MRD monitoring in MM.[31] With the advancement in the technology, the assay sensitivity has increased to 10−5 to 10−6 and with the improvements in therapy, the current minimum acceptable sensitivity
is 0.001% and the recommended threshold for abnormal PC in the determination of MRD
ranges from 20 to 100 cells.[32]
[33] A number of studies have demonstrated that a cluster of 20 cells is a conservative
value for the smallest number of a homogenous and clustered population of cells that
can be reliably detected by an experienced Hematopathologist.[9]
[15]
[17] The limit of blank (LOB), the limit of detection (LOD) and the lower limit of quantitation
(LLOQ) are further determined to establish the sensitivity and reproducibility of
the flow cytometric assay for MRD assessment.[9] LOB is calculated as LOB = mean of blank values plus 1.645 x (Standard deviation
[SD] of blank values).[34] The LOD is calculated as LOB plus three times SD of blank values.[34] LOD determined using the values from the LOB experiment may further be validated
with a dilution and spiking experiment. In the experiment for LLOQ determination,
spiked and diluted cells with the same concentration (number) of neoplastic PC are
processed and acquired multiple times separately to determine the minimum number of
clonal PC present in the sample that can provide assay results with CV less than 20%.[16] Next critical determinant in MRD calculation is assigning appropriate numerator
and denominator for MRD estimation. For all practical purposes, numerator is clonal
PC showing light chain restriction with/without aberrant immunophenotype designated
as APC in the subsequent sections. The denominator used for MRD studies is total viable
cells that are total events after removal of doublets and debris and thus, MRD = clonal
PC/ total viable cells. Another parameter that has been shown to be relevant in MRD
estimation in MM by our group is total PC. The use of total PC as denominator is based
on the premise that in a hemodiluted BM, both the normal and neoplastic PC are equally
diluted and thus this statistic offsets the effect of hemodilution to some extent.
Thus, using clonal PC as numerator and total PC as denominator, we defined NPCI, as
NPCI = clonal PC/total PC.[35]
Data Acquisition and Analysis Strategy
A number of clinical studies have supported the fact that the total number of events
acquired is a key step in the interpretation of the specimen quality for MRD negative
cases with the acquisition of at least two million cellular events as the acceptable
minimum number in the absence of MRD.[9] If MRD is not detectable and the total cellular events acquired are fewer than two
million, both LOD and a qualifying statement as to the decreased level of sensitivity
should be mentioned in the final MRD report.[28] Data acquisition is considered to be accomplished when the acceptable total cell
collection is achieved, and the specimen meets the criteria of quality. For samples
in which MRD is not detected, the quality of the specimen must be checked by an assessment
of normal cell population such as mast cells, B cell progenitors, NPC, nucleated red
blood cells, and/or myeloblasts ([Fig. 2]).[26]
[36]
[37] After ensuring BMA adequacy and acquiring enough events, the next critical step
is the gating strategy for identification of the PC compartment. The backbone gating
markers for the PC analysis strategy are CD38, CD138, and CD45.[38]
[39] However, in the era of targeted therapy especially in the setting of use of anti-CD38
monoclonal antibody in the treatment protocols, a search for alternate gating markers
for PC continues. In quest of newer markers for PC identification, Pojero et al carried
out a detailed analysis of several PC-associated markers and demonstrated that CD54
and CD319 had limited utility for PC identification because of significant overlap
of the staining on PCs and other myeloid cells in the sample.[40] Furthermore, CD229 was found to be reliable marker for the identification of PCs
in patients undergoing anti-CD38 or anti-CD138 therapy. Recently, Broijl et al explored
the role of CD38-multiepitope antibody and the VS38c antibody for MRD assessment in
MM patients and concluded that both antibodies allow reliable MRD detection with 100%
concordance with added benefits of VS38c in daratumumab-treated patients due to its
intracellular expression.[41]
[Fig. 3] demonstrates the gating strategy for PC identification using four gating markers
and shows that best discrimination between PC and non-PC compartment is achieved with
the combination of CD38 and CD138. The utility of CD229 as a gating marker for PC
identification reaffirms its role in MRD detection in patients receiving anti-CD38
therapy.[42] Overall, the combination of CD38, CD138, and CD45 remains the mainstay for PC gating
and additional markers may help in specific circumstances.[39]
Fig. 2 Flow cytometric dot plots showing various components used to measure adequacy of
BM in MRD assessment. BM, bone marrow; MRD, measurable residual disease.
Fig. 3 Flow cytometric dot plots showing gating strategy; maximum PC yield with CD38 versus
CD138 (E) followed by CD38 versus CD45 (F); CD229 show overlap with non-PC compartment
(D) and (H) reconfirming CD38 and CD138 as PC specific marker. PC, plasma cell.
Characterization and Discrimination of Normal from Aberrant PC
After ensuring the optimal gating strategy, the next critical step in MRD evaluation
by MFC is the discrimination of nonmalignant polyclonal NPC from the malignant APC.
There is no single surface marker that would enable 100% discrimination of benign
PCs from myeloma PCs, highlighting the need of multiple markers to differentiate NPC
from APC. A major challenge in the immunophenotypic characterization of APC is the
presence of a polyclonal reactive PC that closely mimics the immunophenotype of neoplastic
PC. With the progressive increase in the number of markers and events analyzed, we
are increasingly becoming aware of the existence of polyclonal PCs with immunophenotypic
aberrancies and therefore, an in-depth understanding of the immunophenotypic heterogeneity
of the background NPC is essential. Recent studies have shown evidence of wider immunophenotypic
variation in NPC than earlier studied, thus, revealing a partial overlap between the
NPC and APC.[43]
[44] Immunophenotypic aberrancies in polyclonal PCs have demonstrated for all the markers
using 10-color panel, signifying that an aberrancy of immunophenotypic marker alone
does not define malignancy in otherwise polyclonal population of PCs.[16] Thus, in view of the high frequency of immunophenotypic aberrations observed in
polyclonal PCs, presence of aberrant immunophenotype alone can no longer be used to
define clonal APC and, thus antigen aberration must be supported by light chain restriction
for correct assessment of MRD.
Impact of Therapy on MRD Assessment in MM
Data on immunomodulation of the PCs subsequent to antimyeloma therapy is sparse.[45]
[46] However, it may be noted that the discriminative power of an individual antigen
for delineating neoplastic from non-neoplastic PC varies from case to case and is
not stable during the disease course of treatment. Almost a decade ago, we reported
immunophenotypic changes in clonal APC following antimyeloma therapy in as many as78%
cases.[47] In this context, it is important to note that the high degree of immunomodulation
renders the baseline diagnostic immunophenotype redundant for the purpose of MRD assessments
in MM. CD45, CD38, and CD138 are gating markers and thus change in their expression
is likely to affect the MRD as recently described.[16] Thus, more prospective studies in a larger cohort of patients may unravel the mechanism
of post-therapy immunomodulation under the aegis of various treatment modalities and
lines of therapy administered.
Caveats and Pitfalls of Flow-Based MRD Assessment in MM
Caveats and Pitfalls of Flow-Based MRD Assessment in MM
Since MRD emerged as one of the strongest and independent prognostic indicators relating
to long-term treatment outcomes across various studies, accurate and precise determination
of MRD is crucial for disease monitoring.[48] There are multiple factors influencing the MRD status in MM patients on therapy.
Hemodiluted Bone Marrow Aspirate
This is the major pitfall in assessing MRD across laboratories as it limits the minimum
number of neoplastic cells required to detect MRD. To increase the challenge, high
throughput techniques like NGF require high amounts of starting material. Several
measures have been taken in the past to overcome this challenge including use of first-pass
sample for MRD assessment, bulk lysis as the optimal preanalytical procedure, and
evaluating sample cellularity (e.g., by quantification of erythroblasts more than
5% and mast cells by flow cytometry or smear) before proceeding with MRD evaluation.
However, none of these methods found to be beneficial in all cases. Thus, recently
we demonstrated the utility of NPCI, that is, the percentage of clonal APC of the
total PC in for MRD evaluation as a parameter to document MRD.[35] Since both neoplastic and NPC are proportionately reduced in a hemodiluted BMA,
NPCI may prove to be a better parameter to assess MRD and may be explored further
in larger cohorts.
Multiple Institutional and Nonstandardized Protocols
This is one of the major pitfalls in the MRD detection in MM. A survey of multi-institutional
practices for MFC based MRD detection in MM revealed that MRD estimation suffers from
major heterogeneity with considerable variation in the number of BM cells analyzed
(events) and the number of APC needed to identify the MRD.[49] Nonetheless, the variation also existed in the definition of APC by MFC affecting
ability to differentiate normal from neoplastic PC. To overcome this challenge, the
College of American Pathologists in 2014 recommended to include the LOD and the LLOQ
for flow-based MRD assays in the final diagnostic report along with the documentation
of methods for MRD assay's LOD measurement.[50] Furthermore, the International Clinical Cytometry Society, European Society for
Clinical Cell Analysis, and the Euroflow Consortium recommend the harmonized use of
different reagents, antibody panels, sample processing protocols, instrument standardization
and LOD to improve the sensitivity and reproducibility of MFC-MRD detection in MM.[28]
[30] Recently, an international harmonized approach for data analysis has been evaluated
across seventeen participants from thirteen countries using a consensus gating protocol
to reduce inter-laboratory variation in MRD reporting.[51] Taken together the precise definition of LOD and LOQ is mandatory for reliable and
reproducible MRD reporting.
Optimal Timing to Assess MRD
There is no data to depict exact frequency of MRD monitoring. In this context, it
is important to mention that sustained MRD negativity has survival benefits over MRD
negative status documented at single time point. In a recent study, Sharma et al demonstrated
that MM patients with MRD negativity had survival benefit at the end of induction
chemotherapy or at day 100 post-transplant in terms of PFS (p = 0.0002) and overall survival (OS; p = 0.009).[52] San-Miguel et al also showed that patients with newly diagnosed multiple myeloma
who achieved sustained MRD negativity lasting more than or equal to 6 and more than
or equal to 12 months had deep remission and improved clinical outcomes.[53] Suggested time points for MRD assessment in MM based on literature review are as
follows:
-
A. At first CR—to evaluate the depth of response.
-
B. Post-therapy—after a predefined number of chemo-cycle
-
C. Prior to autologous stem cell transplant (ASCT)—to assess transplant eligibility.
-
D. Post-ASCT—at day 100 (most of the literature on MRD is based on this time point)
-
E. During maintenance therapy—at periodic intervals to assess duration of response.
Of all these time-points day 100 post-ASCT is the most studied in clinical trials
and demonstrated superior survival of MRD-negative patients on day 100 post-ASCT as
compared to MRD positive patients. Recently, we too have demonstrated that day 100
post-transplant MRD positive status is associated with poor survival outcome compared
to MRD negative status with progressive worsening of both PFS and OS with increase
in MRD levels.[35]
Depth of MRD Monitoring
Latest IMWG guidelines define the MFC-based MRD negative status as the absence of
phenotypically aberrant clonal PC by NGF on BMA using the Euroflow standard operative
procedure (or validated equivalent method) with a minimum sensitivity of 1 in 10−5.[53] Thus, IMWG recommends minimal sensitivity of 10−5 or higher for the BMA-based MRD testing. A few publications in the literature correlated
the log reduction in MRD assessment with survival where researchers studied MRD level
as a continuous variable determined by flow cytometry and demonstrated survival benefits
in MM patients per log depletion in MRD.[54]
[55] Likewise, our group also demonstrated that gradual fall in MRD levels from 0.1 to
0.001% is predictive of superior survival outcomes[35] ([Fig. 4]). In order to get an optimal sensitivity for clinical relevance more prospective
studies needed to define the depths precisely since disease progression and relapse
evident in MRD negative subsets as well.
Fig. 4 Kaplan–Meier survival curves displaying sequential improvements in PFS and OS for
each log depletion in MRD level. OS, overall survival; MRD, measurable residual disease;
PFS, progression free survival.
MRD to Make Therapy Decisions
Prognostic value of MRD evaluation in predicting long-term survival has been demonstrated
in major clinical trials. A meta-analysis by Munshi et al demonstrated the strong
prognostic impact of MRD negativity in MM patients and confirmed the utility of MRD
as a relevant surrogate for PFS and OS in MM.[56] However, acceptance of MRD as a surrogate endpoint for treatment requires correlation
with PFS and OS benefit in larger cohort of patients in multiple trials.
Future Perspectives
With the growing evidence of MRD as the strong and independent predictor of survival
in MM, it is recommended to evaluate MRD more precisely and accurately to benefit
the patients in the long run.
Role of Flow Cytometry in Clonal Evolution
Though the phenotype and antigenic variability of malignant PC can be evaluated by
flow cytometry, there is limited evidence of the identification of intratumoral heterogeneity
by FCMI. Intratumoral heterogeneity can be described as the heterogeneous distribution
of genetically distinct MM clones and subclones within a single patient and may complicate
diagnostic and prognostic groups posing a significant challenge for disease monitoring.
However, FCMI can identify multiple subclones of malignant PC within the same patient
based on differential expression of various surface markers on different subpopulations
of PC ([Fig. 5]). This highlights the potential role of FCMI in identifying clonal evolution in
MM cases when studied at multiple time points and needs to be explored further in
clinical trials to see the impact of these clones on treatment response to explore
if a particular subclone is treatment refractory or is responsible for possible relapse.
Fig. 5 Flow cytometric dot plots showing multiple subclones of APC (major myeloma clone:
orange events; subclone I: magenta events; subclone II: green events; subclone III:
blue events) in a case of NDDM. APC, aberrant plasma cell; NDMM, newly diagnosed multiple
myeloma.
Emerging Techniques for MRD Evaluation
NGS-based approach for MRD investigation has drawn the attention of researchers in
the recent past, however, limited by the costing, labor-intensive technique with analytic
expertise. A few studies have also investigated the role of gene expression profiling
in myeloma MRD estimation and revealed the role of differentially regulated genetic
pathways in MRD landscape of MM.[57]
[58] Furthermore, considering the patchy nature of disease and spatial heterogeneity
in MM, BMA sample may not always be the representative for MRD assessment by MFC or
molecular techniques. Nonetheless, in a substantial proportion of extramedullary disease/relapse,
BMA is not the ideal sample and may lead to false-negative MRD status. Imaging studies
for residual disease estimation may be useful in such scenarios, however, limited
by failure to identify the disease present at microscopic/submicroscopic levels. In
this regard, utilizing peripheral blood for MRD evaluation has been investigated recently
using cell free DNA (cfDNA).[23] In a long-term study on blood based MRD monitoring in MM, cfDNA detection of VDJ
rearrangement by ASO-qPCR was used to demonstrate its utility as a prognostic marker.[59]
Conclusion
In conclusion, flow cytometry offers a promising role in MRD estimation in MM. However,
it suffers from the inherent challenge of standardization and expertise in analysis.
Thus, adoption of a unanimous harmonized approach for MRD analysis by flow cytometry
as well as integration of MRD reporting in clinical trials is the need of the hour
to utilize MM MRD as a surrogate clinical endpoint.
