Minimal residual disease (MRD) testing

Leukemia, malignant lymphoma, and multiple myeloma are common hematological tumors. Minimal residual disease (MRD) refers to the state where a small amount of leukemia cells remain in the body after treatment and remission of these hematological tumors, which is an important cause of leukemia recurrence. Accurate MRD detection can early predict leukemia recurrence, stratify patient risk levels, guide post remission treatment, and determine the optimal time for bone marrow or stem cell transplantation.

The importance of MRD detection

Leukemia, malignant lymphoma, and multiple myeloma are common hematologic tumors with minimal residual disease ( Minimal Residual Disease (MRD) refers to the state in which a small number of leukemia cells remain in the body after the above hematological tumors are relieved by treatment, which is an important cause of leukemia recurrence. Accurate MRD testing can predict leukemia recurrence early, stratify the patient's level of risk, guide post-remission treatment, and determine the optimal time for bone marrow or stem cell transplantation.

MRD detection method

The number of remaining hematological tumor cells after treatment remission is generally small and requires very sensitive methods to detect. The 2018 pediatric acute lymphoblastic leukemia protocol recommends the following four MRD evaluation methods:

1. Flow cytometry

The use of abnormal antigen expression between leukemia cells and normal cells to distinguish between leukemia cells and normal cells is the most widely used and fastest method. However, only antigens or combinations of antigens that do not overlap significantly from normal cells (including normal naïve cells) can be used for MRD monitoring; And the sensitivity of conventional flow cytometry detection is only 10-4; At the same time, the immune drift of leukocyte surface antigens can interfere with the detection of MRD.

2. Fusion gene quantitative RT-PCR

Surveillance sensitivity is high, but fusion genes are present in less than 50% of cases, resulting in very limited applicability.

3. IgH/TCR rearrangement quantitative PCR

Surveillance sensitivity is high and linear, and this regimen is available in more than 90-95% of cases. However, this method requires the design of patient-specific probes, which is cumbersome and time-consuming and will be missed due to mutations in the genes in the probe binding region brought about by the clonal evolution of leukemia.

4. Next-generation sequencing (NGS).

Based on BCR/TCR rearrangement for MRD surveillance, which can be applied in more than 95% of cases, overcomes some limitations of RT-PCR and enhances sensitivity when analyzing a sufficient number of cells (10-5-10-6)。 NGS provides prognostically relevant information on B cells and T cells during and after treatment and can be used to analyze immune system diversity, immune reconstitution, etc., and is of relatively stable quality, but guidelines for the use of calibrators, quality control, and correct interpretation of NGS data are still lacking.

Pan-cause medicine NEO-MRDTMInspection products

Pan-cause medicine MRD detection products are based on accurate and unbiased multiplex amplification technology for MRD detection, are easy to operate, have sensitivity up to 10-5-10-6, and are not affected by tumor cell surface antigen drift and clonal evolution. The test product first sequences and analyzes the TCR/BCR gene of the tumor sample before treatment to determine the tumor clone, and then detects the burden level of the tumor clone in the post-treatment sample, accurately and sensitively evaluates the MRD level, and can monitor whether new high-frequency clones appear, and evaluate the risk of recurrence due to the original tumor subcloning or evolutionary cloning.

1. NEO-MRDTMDetection principle

NEO-MRDTM first performed multiplex PCR amplification and sequencing analysis of TCR/BCR genes in pre-treatment tumor samples to determine tumor clones and then detected the burden level of tumor clones in post-treatment samples to accurately and sensitively assess MRD levels.


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2.    NEO-MRDTMTechnical characteristics and advantages

(1) Accurate and sensitive experimental system. The system is based on an accurate and unbiased Ig/TCR multiplex PCR assay system for tumor clonal identification and MRD detection. Through the continuous development of experimental techniques and data processing algorithms, we optimize the primer amplification efficiency to a very balanced one, and the balanced amplification system will bring high accuracy and sensitivity of MRD quantification.


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(2) Amplify the receptor chain to improve the detection rate of tumor cells. The experimental system includes IGH, IGK, IGL, TRB, TRG receptor chains, which can accurately identify the marker sequence of tumor cells, and each chain also includes primers in different positions (FR1, FR2, FR3), which can effectively avoid false negatives or inaccurate quantification caused by mutations of tumor cells.

(3) Original bioinformatics analysis tools. Due to the extremely low MRD levels of some samples, sequencing errors and sequence contamination need to be tightly controlled. Through our original data processing technology, we can effectively correct sequencing errors and remove trace contamination that may occur during the experimental process to ensure the accuracy of detection.

3.    NEO-MRDTMadvantages

(1) Easy operation, standardized and automated process, reduce human operation error. Full automation from DNA to test reports is possible. Good repeatability.

(2) Built on an unbiased amplification system, the accuracy is high and the sensitivity can be up to 10-5-10-6, and is not affected by tumor cell surface antigen drift and clonal evolution.

(3) Among the 58 real clinical samples, the consistency between the two methods of 25 samples with positive NGS and flow cytometry was good, 14 samples were negative for flow cytometry, positive for NGS, and 19 samples were negative for both.


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(4) It can monitor whether new high-frequency clones appear, and assess the risk of recurrence due to subcloning or evolutionary cloning of the original tumor.

(5) The reconstruction of the immune system can be evaluated.

4. NEO-MRD indications:

Acute lymphatic leukemia (ALL)

Bone marrow, peripheral blood

Chronic lymphoid leukemia (CLL)

Bone marrow, peripheral blood

Multiple myeloma (MM)

marrow

lymphoma

Bone marrow, peripheral blood, lymph nodes

5. Sample delivery requirements:

Both bone marrow and peripheral blood require EDTA anticoagulation tubes, and samples should be noted before or after treatment.


Sample type

Sample volume

Delivery conditions

Before treatment

Bone marrow (fresh)

>0.5 ml

oC (Delivered within 48 hours)

Bone marrow (frozen)

>0.5 ml

dry ice

Peripheral blood (fresh)

> 2 ml

oC (Delivered within 48 hours)

Peripheral blood (frozen)

> 2 ml

dry ice

DNA

> 0.5 µg

dry ice

After treatment

Bone marrow (fresh)

>1 ml

oC (Delivered within 48 hours)

Bone marrow (frozen)

>1 ml

dry ice

Peripheral blood (fresh)

> 2 ml

oC (Delivered within 48 hours)

Peripheral blood (frozen)

> 2 ml

dry ice

DNA

> 1 µg

dry ice

6. The relationship between MRD detection sensitivity and sample volume


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7. Notes on detection process: Sensitivity refers to the proportion of tumor cells in the total nucleated cells


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(1) Specimen collection: EDTA blood collection is used, and the detection caused by blood dilution is inaccurate or false-negative during bone marrow aspiration.

(2) DNA extraction: compatible with most DNA extraction methods on the market, extracted DNA-20oSave.

(3) Library construction, based on our accurate and unbiased Ig/TCR multiplex PCR experimental system, adding internal reference evaluation and correcting deviations in the experimental process. To improve applicability and detection accuracy, B-ALL amplifies fully rearranged IGH (primer combinations include: FR1-J and RF3-J), IGH (D-J primer combination) without complete rearrangement, IGK rearrangement (FR1-J, FR3-J, FR1-kde, FR3-kde, intro-kde), IGL rearrangement (FR1-J and FR3-J) during detection; T-ALL amplifies TRB and TRG receptor chains during detection.

(4) NGS sequencing requires calculating the number of cells according to the amount of DNA at the time of library construction, and determining the amount of sequencing data according to the number of cells.

(5) Data analysis: Through original data processing technology, effectively correct sequencing errors and remove trace contamination that may occur during the experimental process to ensure the accuracy of detection.

(6) Automatic report generation: The test report can be directly generated by importing the quality control data into the analysis software.

(7) Report determination: Manually check the key indicators of the analysis data and report to ensure the correctness of the test report.

8. Inspection cycle

15 calendar days from the date of receipt of samples.


References

1.  Wu, J., et al., Minimal Residual Disease Detection and Evolved IGH Clones Analysis in Acute B Lymphoblastic Leukemia Using IGH Deep Sequencing. Front Immunol, 2016. 7: p. 403.

2.  Faham, M., et al., Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia. Blood, 2012. 120(26): p. 5173-80.

3.  Wu, D., et al., High-throughput sequencing detects minimal residual disease in acute T lymphoblastic leukemia. Sci Transl Med, 2012. 4(134): p. 134ra63.

4.  Wu, D., et al., Detection of minimal residual disease in B lymphoblastic leukemia by high-throughput sequencing of IGH. Clin Cancer Res, 2014. 20(17): p. 4540-8.

5.  van Dongen, J.J., et al., Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies. Blood, 2015. 125(26): p. 3996-4009.


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