International experts work together to discuss the future: the evolution of CLL treatment mode driven by MRD

Chronic lymphocytic leukemia (CLL) is a tumor disease with clonal proliferation of lymphocytes, with an annual prevalence rate of 0.
27 per 100,000 people in China, and given the large base of Chinese, the scale of new onset of diseases per year cannot be underestimated
.
In recent years, with the continuous introduction of new drugs and the improvement of detection methods, the diagnosis and treatment of CLL has been continuously improved and improved
.
The development direction of CLL Minimal Residual Disease (MRD) detection and future diagnosis and treatment has become the focus
of attention in the international blood field.
On September 23, 2022, experts from the China Slow Gonorrhea Working Group (cwCLL) and the European Chronic Lymphocytic Leukemia Research Center (ERIC) conducted an in-depth discussion
on the MRD detection and clinical application value of CLL.

Professor Xu Wei of Jiangsu Provincial People's Hospital and Professor Yi Shuhua of Hematology Hospital of Chinese Academy of Medical Sciences made opening speeches, expressing the hope that through this meeting, they would exchange Chinese and foreign diagnosis and treatment experience, jointly explore the revision and improvement of MRD testing standards, and develop a standardized MRD testing program
for Chinese CLL patients that is both in line with China's conditions and can be in line with international standards.

The significance of MRD detection for the clinical diagnosis and treatment of CLL and prognostic evaluation

Professor Paolo Prospero Ghia has explored the significance of MRD testing in the clinical diagnosis and treatment of
CLL by interpreting multiple studies.

The advent of new targeted drugs has changed the treatment pattern of CLL, and BTK inhibitors (BTKi) can allow patients to achieve sustained and stable deep remission
.
The CLL14 study showed that for patients with initial treatment of CLL, the combined regimen of Venekla and Opturizumab was able to achieve peripheral blood MRD negative in about 3/4 of patients and bone marrow MRD negative in more than 50% of
patients.
The MURANO study showed that in patients with refractory/relapsed (R/R)CLL treated with the Venecra combination regimen, 62% of patients achieved a negative MRD in
peripheral blood.
Both studies showed significantly longer progression-free survival (PFS) in patients who achieved MRD-negative, with greater survival benefit for patients with deeper remission
.

MDACC studies have shown that ibretinib can be treated in combination with Venekla for 1 year to achieve MRD negative in more than 50% of patients, and the remaining patients can gradually achieve MRD negative
in the second year.
Studies suggest that the most appropriate time
to stop may be determined by an MRD-negative level.
The CAPTIVATE study evaluated the possibility of guided treatment based on the MRD negative rate, and the results showed that the 1-year PFS rate of patients who achieved MRD-negative treatment was 95%, indicating the possibility
of replacing ongoing treatment with MRD-guided fixed-term therapy.

Current recommendations for adjusting treatment based on MRD levels and treatment options for patients who do not achieve negative MRD remain to be improved
.
In addition, in the clinic, it is also necessary to pay attention to the realization of MRD-negative is not the treatment goal of all patients, and for some elderly patients, it is possible to control the disease to meet the survival needs
.

In the discussion session, Professor Paolo Prospero Ghia said: (1) The current lack of practical research data to support the clinical application of MRD, now MRD <10-4 as a negative indicator of MRD, the future may be on this basis, such as patients can stop treatment<b12> after MRD <<b10>10-5.
MRD can also be used as an indication
for treatment options.
(2) The treatment goals of patients are not the same, and the elimination of tumor cells needs to be within a reasonable range for CLL patients, so MRD cannot be regarded as a hard treatment goal
.
(3) In clinical trials, the MRD value of patients with R/R is constantly fluctuating, so it takes longer observation to judge the subsequent decision
.

Comparison of the advantages and disadvantages of three different detection protocols of CLL-MRD

Professor Andy Rawstron elaborated
on how to effectively detect and measure MRD levels, as well as considerations during testing.

With the development of medicine, at least half of patients can achieve complete remission (CR) after first-line standard therapy, and the clinical need for more sensitive comparison of the results of different protocols, MRD testing is of great significance
.
Studies have shown poor PFS in patients with high (>1%) levels of MRD after 1 year of treatment, and patients with moderate (0.
01%-1%) and low (<1%) with similar PFS<b11>.
Patients with high and moderate MRD levels after 5 years had poor PFS, and only patients with low MRD levels showed good feedback
.
Therefore, accurate prediction of long-term patient outcomes requires high-sensitivity MRD detection, and short-term results can be predicted using low-sensitivity MRD detection
.
There is substantial clinical evidence that treatment in randomized studies until MRD is negative improves PFS, while differences in overall survival (OS) require longer follow-up
.
When ratifying new drugs for CLL and myeloma, European institutions see MRD as a strong marker, and MRD negative has become the major/secondary endpoint for the vast majority of trials targeting long-term PFS
.

In the specific practice process, clinicians need to make multiple choices and measurements
.
In terms of specimen selection, several studies have shown that bone marrow is more sensitive than peripheral blood, but in most cases MRD of peripheral blood can be used to determine whether bone marrow testing
is required.
In terms of detection methods, if only to determine whether the MRD is less than 0.
1%, flow cytometry (FCM) and real-time PCR (qPCR) can be done; However, neither is suitable when studying circulating tumor DNA, and high-throughput sequencing (NGS) is more feasible; Molecular detection is required to determine the specific level of MRD, and NGS detection is recommended, which can achieve a sensitivity of ^10-6
.
In terms of testing time, there is consensus that MRD testing in patients treated with a fixed course of treatment should be consistent with the assessment of remission and should be performed at least once every 2 months after the end of treatment; Patients with ongoing treatment should have MRD testing
when the optimal clinical response is achieved.

During the discussion session, Professor Andy Rawstron said: (1) Overall, FCM is easier to operate, less costly, more suitable for clinical practice, and can also consider the use
of multiple technologies in combination.
(2) It is theoretically feasible to increase the sensitivity to ^10-6, but in practice it does not need to be demanding, and FCM can be used to guide
subsequent operations.

Molecular analytical methods and recommendations for molecular-level analysis of MRD

Professor Kostas Stamatopoulos elaborated
on the problems and solutions that may arise during molecular detection.

For MRD detection at the molecular level, immunoglobulins expressed on the surface of mature B cells should be used as amplification targets
.
When analyzing polyclonal profiles, immunoglobulin gene differences are very pronounced, with each one varying
in length.
When evaluating these rearrangement lengths, a bell graph is obtained, but when there is a single clone in the context of polycloning, the length corresponding to the amplified clones increases sharply, and most of the cells in the comparison of real samples are derived from the same clone, and the IgG rearrangement is consistent, and the peak is very obvious
.
Depending on the patient's situation, different pedigree plots
will correspond.
Professor Kostas Stamatopoulos elaborated on the pedigree characteristics
of patients in different situations, citing specific cases.

At present, the ERIC and the European NGS working group are validating a standard process that can ensure that the results of gene sequence amplification are stable and reliable, and then the sequencing results are annotated
through the IMGTA database.
Professor Kostas Stamatopoulos proposed that the adoption of a unified approach can improve the consistency of analytical results across different laboratories, which will further promote the standardization
of quantitative testing in the future.

During the discussion session, Professor Andy Rawstron stated that (1) oligoclonal or diconal amplification usually means that the level of disease is very low, mostly chronic
.
But one clone in an invasive disease overrides another clone and may have an impact on the disease and requires special attention
.
(2) From the perspective of cell detection, the analysis of methods and practical problems needs to select different samples according to different treatment options, and the number of cells used for detection must be sufficient
.

MRD detection and clinical application in CLL patients in China

Professor Wu Yujie introduced the selection and improvement
of MRD detection protocol for CLL patients in China.
At present, FCM commonly used in clinical practice mainly uses CLL immunophenotypic characteristics to identify, which is sensitive, fast and simple, and is suitable for almost all patients
.
The effective CLL-MRD protocol is mainly a four-color and six-color standardization protocol, in addition to ERIC has also introduced an eight-color single-tube standardization protocol, none of which involves light chain, and the core antibodies are CD19, CD81, CD5, CD43, CD0 and CD789b
.

In view of the problems of flow cytometry standardization in China, Professor Wu Yujie conducted relevant research
.
Through the comparison of detection schemes with or without light chains, 257 cases were detected, and only 12.
4% had inconsistencies
.
A deeper examination of inconsistent results found that the combination without light chains was more
sensitive.
The reason for the analysis may be that 1/4 of the kappa/lambda negatives in the CLL cannot be detected, and factors such as detection processing steps and samples have a great
influence on the light chain.
The results suggest that light chain detection is non-essential
.

At the same time, Professor Wu Yujie improved the eight-color scheme to retain 6 core antibodies while increasing ROR1 and CD45
.
The improved protocol can effectively reduce the error between the room and the room, and can reduce the deviation rate
compared to the ERIC solution.
With the development of treatment, the clinical depth of MRD detection has put forward higher requirements, second-generation flow cytometry (NGF) came into being, its prognostic value is higher than that of conventional protocols, the results of peripheral blood and bone marrow are highly consistent, but it requires more sample size, antibodies, detection time and cost, so clinical promotion is limited
.

During the discussion session, the participating experts had a heated discussion
on whether MRD can be used as a cure standard for patients in the future, whether retaliatory progression will occur after MRD negativeization, the frequency of MRD testing, how to select mutant genes when using NGS as a detection method, and whether CLL elderly patients still need to take MRD negative as the evaluation target.

Conference Summary

At the end of the meeting, Professor Paolo Prospero Ghia introduced the composition of ERIC, its organizational mission and ongoing projects, and sincerely invited the participating experts to join in the efforts
to standardize clinical MRD testing.
Professor Xu Wei concluded that in this meeting, cwCLL and ERIC had an in-depth exchange on the application value and detection method of MRD in clinical practice, and thanked ERIC for its help
in establishing the consensus in China.
Professor Yi Shuhua said that the conference benefited a lot and hoped that exchanges could be strengthened in the future, continue to be in line with international standards, and lay the foundation
for the future comparison of treatment of different CLL populations in the East and the West.

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