Micro-classroom No. 18 Professor Luo Jun: The latest exploration of MRD detection and application

The detection of minimal residual disease (MRD) is of great significance for judging the efficacy of multiple myeloma (MM), predicting the prognosis of patients and guiding treatment decisions, and has become a research hotspot
in the field of MM.
In order to understand the application ideas of MRD more systematically and better apply MRD detection to the clinic, Professor Luo Jun of the First Affiliated Hospital of Guangxi Medical University is invited to share the latest exploration
of MRD detection and application.

MRD refers to the state of trace tumor cells that remain in the body after treatment of malignant hematological diseases that achieve complete hematological remission (CR) at any level that cannot be detected by traditional methods such as morphology
.
In the era of new drugs, MM patients can obtain deeper remission, and the past efficacy evaluation standards can no longer meet the latest treatment needs, so MM needs deeper efficacy evaluation standards
.
With the rapid development of detection technology, the sensitivity of MRD detection is getting higher and higher, and the previous cytogenetic method can detect 1 tumor cell from 100 cells (sensitivity 10-2), and the sensitivity of flow cytometry and polymerase chain reaction (PCR) technology has been increased to 10-3-10-5, and the sensitivity of newer techniques can even reach ^10-6
。 The improvement of MRD detection sensitivity is of great significance for the diagnosis and treatment of MM, which is helpful to judge disease residue and patient survival prognosis
.

With the advancement of MRD detection technology and the deepening of related research, more and more MRD detection and application related issues have caused extensive thinking, in order to better apply MRD detection to clinical practice, this issue will start from the following three questions, further thinking and discussion
.
Questions include:

• MRD detection methods are constantly being explored, what are the differences between different detection methods?

• Does MRD need to be evaluated through multiple modes, or one mode?

• In the process of pursuing the efficacy of long-term MRD negative, the clinical guidance value of MRD yin to yang?

MRD detection methods have been constantly being explored, so what is the difference between different detection methods?

In the past ten years, the detection methods of MRD have been increasing, and the technology has been continuously upgraded
.
As early as 2006, traditional molecular modalities such as allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) have been used for MRD detection
.
Subsequently, the first generation of 4-6 color flow cytometry was applied to MRD detection; In recent years, the second generation of 8-color flow cytometry has achieved technological upgrades, gradually replacing the older generation of flow cytometry detection.
Next-generation sequencing (NGS) is also gradually being used more widely in the clinic
.
In 2016, PET-CT was recommended by the International Myeloma Working Group for the evaluation of imaging MRD
.
To this day, the exploration of MRD detection methods has not stopped
.
In general, the current detection methods mainly include microscopic detection methods such as second-generation flow cytometry (NGF) and NGS and overall detection methods
such as PET-CT and magnetic resonance imaging (MRI).

Comparing different MRD assays shows that the principles are different, and there are also differences in the advantages/disadvantages shown in clinical ^{applications1} (Table 1).

Among the microscopic detection methods, the highest sensitivity is NGF and NGS, which can reach ^10-6, and are also the most commonly used MRD detection methods
in clinical practice.
Among them, NGF identifies normal and abnormal plasma cells by detecting the expression of cell surface markers, which has a wide range of applications and is highly automated, but requires fresh samples and is tested within 24-48 hours, which has limitations such as inability to detect clonal evolution
.
NGS uses high-throughput sequencing to detect cloned VDJ gene recombination, which can capture almost all Ig gene rearrangements without immediate sample processing, but relatively speaking, the test takes 1-2 weeks, the detection cost is high, and the data
needs to be interpreted by professionals.

Table 1 Comparison of different MRD detection methods

Perhaps the most obvious limitation is the invasive nature of the need for bone marrow aspiration, which raises doubts about its practical clinical application and its value as a routine test
.
Therefore, new methods for MRD detection through peripheral blood, such as mass spectrometry (MS) detection, have also received more and more attention and exploration
in recent years.
MS testing uses blood samples that are more readily available and less invasive than NGS and NGF using bone marrow samples, while MRD results are accurate
.
It can be seen that MS detection can be used as the future MRD detection exploration direction, and has a good application prospect.

Constantly updated testing methods provide clinicians with more options, but also lead to new thinking: Does MRD need to be evaluated through multiple modalities, or one mode?

Common MRD evaluation modalities include bone marrow, peripheral blood, and imaging (Figure 1), should these three evaluation modalities be performed simultaneously, sequentially, or one or the other? Several studies have explored
this.

Figure 1 Common MRD evaluation modes

A study2 presented at the ²⁰²² EHA Conference compared the sensitivity
of different detection methods.
A total of 33 patients with relapsed/refractory MM (RRMM) who could be detected by serum immunofixation electrophoresis (IFE) or free light chain were included in the study, and serum M protein
was detected by MS before, 28 days and 100 days after infusion of chimeric antigen receptor T (CAR-T) cells.
The results showed that at 28 and 100 days after infusion, patients with negative MS test results had longer
progression-free survival (PFS) than positive patients.
In addition, sero-MS negative was associated with prolonged overall survival (OS) (Figure 2).

In contrast, no association
with PFS or OS was observed with a negative serum IFE or NGF test.
It can be seen that compared with IFE and NGF, MS detection can identify M protein in all cases with high sensitivity and correctly label treatment response
.

Fig.
2 Correlation between MS test results and PFS and OS at 28 or 100 days after infusion

Compared with bone marrow testing, MRD analysis by peripheral blood provides a systemic assessment, avoids the pitfalls of bone marrow sampling heterogeneity, and enables detection and monitoring of extramedullary lesions
that may be missed by bone marrow testing.

Another ^study3 of newly diagnosed MM (NDMM) patients who did not undergo autologous transplantation, presented at the 2022 EHA Conference, evaluated MRD agreement
between bone marrow NGS testing and peripheral blood MS testing.
The results showed that the consistency between early NGS and MS detection was low, and the consistency between NGS and MS detection in the later stage was increased (Figure 3).

Therefore, MS detection by peripheral blood and NGS assessment by bone marrow can play complementary roles
.

Figure 3 Consistency between MS and NGS detection

The 2022 EHA Conference also presented a ^study4 comparing the accuracy of NGF and functional imaging in MRD assessment and exploring the guiding significance
of MRD in consolidation therapy.
A total of 102 patients with MM were included in the study, including 57 patients with NDMM and 45 patients with RRMM, all of whom were detected using the NGF method, and 78 of whom underwent PET-CT or MRI to assess MRD
after NGF evaluation (median time to 1 day).
。 The results showed that 45% of patients achieved MRD negative (double negative) on both NGF and functional imaging; For some patients, functional imaging is the only technology that can detect MRD; Functional imaging positive in patients with NGF-negative (≥4-line) is more common
in patients with posterior line than in patients with NDMM.
The study suggests that combining NGF with functional imaging can help improve the accuracy of
MRD detection.
In addition, the study also found that PFS in patients who started consolidation therapy under MRD-positive guidance was significantly better than PFS
in patients with standard lenalidomide maintenance therapy.
MRD-based consolidation therapy helps improve PFS
in post-transplant MRD-positive patients.

From the above studies, it can be seen that there is good complementarity between different MRD detection methods, and how to combine different MRD detection methods in the future is worth further exploration
.
At the same time, more and more research is exploring how to guide clinical treatment decisions
based on MRD status.
So, in the process of pursuing long-term MRD negative, what is the clinical guidance value of MRD yin to yang?

Studies have shown that negative conversion from MRD to positive may indicate disease recurrence
.
A study ⁵ of 23 patients with relapsed MRD assessment showed that MRD-negative conversion occurred before clinical relapse (median time to 9 months).

Whether MRD should be used as an indication for treatment has also attracted attention and discussion
.
The REMNANT ^{Study 6} is a multicenter, open-label, randomized phase II/III study that includes 391 transplant-eligible NDMM patients, and after receiving first-line therapy, 176 patients who achieve CR or better remission (≥CR) and are MRD-negative will enter the second part of the study, in which patients are randomly assigned to group A/B to start D-Kd (daratumumab+) when MRD relapses or disease progression, respectively Carfilzomib + dexamethasone) (Figure 4).

This study aimed to assess whether PFS and OS
can be prolonged after first-line therapy with MRD recurrence as the time node compared with starting the second line of treatment with the occurrence of disease progression as the time node.
At present, the study is still ongoing, and whether MRD yin to yang can be used as a therapeutic indication, it is expected that the study will give an answer, and it is worth continuing to pay attention
to as the future exploration direction of MM treatment.

Figure 4 REMNANT study design

summary 

With the continuous improvement and improvement of MM diagnosis and treatment, MRD detection has also become a research hotspot in the field of MM, and this micro-class mainly discusses and explores hot issues related to MRD:

• As an important evaluation tool for MM treatment, MRD has different detection methods and technologies being explored, NGS and NGF are still the most commonly used MRD detection methods in clinical practice, and non-invasive detection methods such as MS analysis are gradually receiving clinical attention.
  

• Studies have shown that the combination of MRD evaluation with different modes can improve the accuracy of MRD detection, and more evidence is still expected on how to jointly evaluate multiple methods in clinical applications in the future.
  

• The value of guiding the treatment of clinical MM patients according to MRD status has attracted much attention, and in the continuous discussion, the latest research is exploring whether MRD yin to yang can be used as an indication to start treatment as soon as possible, further effectively improve the prognosis of patients, and maximize the benefits of patients, and look forward to the release
  of more research data in the future.