Minimal Residual Disease (MRD)-an overview

What is MRD?

The term minimal residual disease (MRD) is used to describe the low-level disease which is not detectable by conventional cytomorphology. A stage in leukemia treatment when patient is in remission, symptoms of disease are absent but small no. of leukemic cells still remains in body.

The Role of MRD Assessment in Patient Care

  • A morphologically normal bone marrow & normal blood counts can have significant amounts of residual disease.
  • After treating cancer, any remaining cancer cells in the body can become active and start to multiply, causing a relapse of the disease.
  • Despite having achieved a CR, a patient may still harbor up to 1010 leukemia cells that persist at levels undetectable by conventional cytomorphologic methods such as light microscopy.
  • Patients are subjected to either undertreatment with risk of relapse or overtreatment with exposure to therapy-related morbidity and mortality.
  • The aim therefore is to predict impending relapse in subsets of patients with specific clonal abnormalities.

Why MRD present even after Rx ?

  • The treatment was not completely effective or that the treatment was incomplete.
  • Not all of the cancer cells responded to the therapy.
  • Cancer cells became resistant to the medications used.

How does MRD testing helps?

  • Show how well the cancer has responded to treatment.
  • Confirm and monitor remissions.
  • Find cancer recurrence sooner than other tests.
  • Identify patients who may be at a higher risk of relapse; and hence, identify patients who may need to restart treatment.
  • Identify patients who may benefit from other treatments, such as stem cell transplantation or combination therapy.

When to Test for MRD?

Patients may be tested;

  •  After the final cycle of a planned combination therapy.
  • After bone marrow transplantation.
  • During treatment to confirm the depth of remission.
  • After one year on maintenance therapy.
  • At regular intervals after treatment is completed.
  • At other specific times.

Techniques to Detect MRD

  • FISH
  • Flow Cytometry
  • PCR
  • NGS

 

Flow Cytomtery

  • FCM has progressed from using 2 and 3 color flow cytometers for patient specific immunophenotyping to 4 and 6 color cytometers with MRD detection levels of 10-4 cells, meaning increased levels of sensitivity and detection.
  • Some labs even now use 8 and 12 color cytometers, with the former being able to visualize cells at the individual level.
  • Both have the ability to measure MRD up to 0.001% cells, which is 1 MRD cell in 100,000 cells.

Multi-Parametric Flow Cytometry Analysis

  • Antigens are differently expressed by B and T lymphoblasts, and their expression is assessed by quantification of the signal emitted by fluorochrome-conjugated-specific monoclonal antibodies (MoAb).
  • Leukemic cells express immunophenotypic cell markers in abnormal patterns.
  • MFC traces these leukemia - associated aberrant immunophenotype (LAIP).
  • The LAIP must be identified at diagnosis, before any therapy in each ALL case, by comparing the marker profile of leukemia cells to reference bone marrow samples, through various combinations of monoclonal antibodies against surface, cytoplasmic, or nuclear leukocyte antigens.

How does MFC detects LAIP

  1. Cross-lineage antigen expression, e.g. CD7 in AML.
  2. Maturational asynchrony, i.e. simultaneous presence of early and late antigens, e.g. CD21 on CD19/34 positive precursor B-cells.
  3. Under/overexpression (e.g. underexpression of CD38 in ALL and overexpression of CD11a, CD44 in ALL).
  4. Absence of antigen, ectopic antigens (TdT positive cells in CSF)
  5. Unique antigens, e.g. NG2 in ALL identified by antibody 7.1

 

Advantages of MFC

  • Accurate quantification of MRD and the capacity to examine the status of normal hematopoietic cell maturation simultaneously.
  • The detection of aberrant LAIP by MFC is less laborious and faster.
  • This allows prompt reporting of the results, which is particularly useful in making therapeutic decisions. (TAT = few hours).
  • It is widely applicable to > 95% cases of precursor B-ALL and T-ALL and > 90% AML cases

Disadvantages of MFC

  1. The samples must be analyzed shortly after collection to avoid cell death.
  2. Postinduction regeneration of normal lymphoid cells co-expressing some ALL-type antigens can lead to false positive results in B-ALL cases.
  3. The bone marrow sample hypocellularity and, in some patients, phenotypic shift can induce erroneous or difficult interpretations.

RQ-PCR-based Quantification of Leukemia-associated Fusion
Genes or Overexpressed Genes.

  • Another leukemia-associated feature that can be used to distinguish leukemic from normal cells is represented by chromosomal abnormalities.
  • Chromosomal translocations in leukemia result in fusion genes which are very good and stable disease-specific markers as they are directly linked to leukemogenesis.
  • The most frequent fusion transcripts detected by reverse-transcriptase (RT-PCR) in ALL are: t(1;19)(q23;p13) with the E2APBX1 fusion gene
  • t(4;11)(q21;q23) with the MLL-AF4 fusion gene,
  • The two main types of t(9;22)(q34;q11) with BCR-ABL fusion genes t(12;21)(p13;q22) with the TEL-AML1 fusion gene.
  • The intrachromosomal microdeletion on 1p32 with the SIL-TAL1 fusion gene.
  • Similarly, t (8;21) (RUNX1-RUNX1T1),     inv 16 (CBFB-MYH11) and t(15;17) (PML-RARA) in AML can be used as target for amplification.

Advantages of PCR

  • This approach is relatively easy, rapid (2-3 days), highly sensitive (104 - 106) and leukemia-specific.
  • An advantage of monitoring MRD by targeting fusion transcripts is the strong association between the molecular abnormality and the leukemic clone, irrespective of the presence of intraclonal differentiation and cellular changes caused by therapy.

Limitations

  • Applicable to only a minority of patients as these leukemia specific markers can only be identified in around 40-45% of B-ALL, 15-35% of T-ALL and around 20% of AML.
  • Cross contamination of RT-PCR products between patient samples is a major pitfall resulting in up to 20% of false-positive results.
  • This cross-contamination is difficult to recognize, as leukemia-specific fusion gene transcripts are not patient-specific markers.
  • The number of transcripts per leukemic cell may vary among patients with the same genetic abnormality and among different cells within the leukemic clone, and might be affected by therapy.
  • Therefore, precise quantitation of MRD with this technique can be difficult.
  • This cross-contamination is difficult to recognize, as leukemia-specific fusion gene transcripts are not patient-specific markers.

Next Generation Sequencing

  • The novel next generation flow (NGF)-MRD approach takes advantage of innovative tools and procedures.
  • Sample preparation, antibody combinations (including choice of type of antibody and fluorochrome), and identification of B-cell precursor (BCP) pathway in the BM, which allows to define the degree of immunophenotypic deviation of BCP-ALL cells from normal BCP (also in regenerating BM).
  • NGF-MRD is faster and reproducible, it has a greater applicability (>95%).
  • Moreover, the costs of reagents and assays are estimated to be lower than those of NGS.
  • However, it requires fresh material analyzed within 24 h after sampling.
  • Finally, NGF-MRD strategies provide a full insight into the composition of normal cells and aberrant cells, and can help to better characterize ALL cell population changes such as treatment-induce immunophenotypic shifts heterogeneity in the blast cell population with a de - differentiation to immature stem like-cells and aberrations in other lineages

 

MRD Testing in Specific Blood Cancers

  1. Acute Lymphoblastic Leukemia (ALL)
  • MRD is detected through flow cytometry, PCR and next-generation sequencing (NGS).
  • MRD is part of routine testing in the treatment of pediatric and adult ALL.
  1. Chronic Myeloid Leukemia (CML)
  • MRD is detected through PCR.
  • PCR can detect the Philadelphia (Ph) chromosome which is found in 95% of all CML patients.
  • PCR can detect one Ph+ CML cell among one million normal cells.
  • MRD monitoring helps predict treatment resistance and guide the course of treatment.
  • PCR is one factor used in deciding whether to discontinue or change tyrosine kinase inhibitor (TKI) therapy.
  1. Chronic Lymphocytic Leukemia (CLL)
  • MRD is detected by flow cytometry and PCR.
  • Patients who remain MRD negative after the end of therapy for CLL may have better treatment outcomes.
  • Patients who are MRD positive after the end of treatment may be candidates for treatment intensification, consolidation and maintenance strategies.
  1. Lymphoma
  • MRD is detected through flow cytometry and PCR.
  • MRD testing is used in follicular, mantle cell and diffuse large B-cell lymphoma (DLBCL).
  • MRD testing helps detect patients who are at risk of relapsing. These patients can then receive additional treatment.
  • Patients who are treated for mantle cell lymphoma and achieve an MRD-negative status have been shown to have longer remissions before their disease progresses.
  • Several studies have shown that DLBCL patients who achieved remission after treatment and were also MRD negative were more likely to remain in remission than MRD positive patients who had achieved remission.
  1. Myeloma
  • MRD testing in myeloma uses flow cytometry, next-generation sequencing and imaging tests.
  • Imaging techniques such as PET-CT scans, in addition to other tests, allow doctors to find disease outside the bone marrow.
  • Studies have shown that patients who achieve an MRD-negative status after treatment live longer without disease progression.

Practical issues: what must be known for adequate sampling and data interpretation?

  1. Source of material
  • MRD can be quantified in peripheral blood or in bone marrow however, MRD levels in BCP-ALL tend to be 1 to 3 logs lower in peripheral blood than in bone marrow.
  • Therefore, bone marrow assessments might be replaced by analysis of blood samples in T-ALL but not in BCP-ALL.
  1. Time point of MRD assessment
  • MRD is a time point–dependent variable. MRD levels at different time points have different prognostic value for relapse.
  • Early MRD assessment identifies patients with a rapid tumor clearance and a very low risk of relapse, whereas any persisting MRD at the end of consolidation therapy is associated with a particularly poor prognosis.
  1. Correct interpretation of MRD results
  • MRD quantification techniques have a lower limit of detection and a lower limit of quantification.
  • Therefore, MRD negativity is not synonymous with the absence of residual disease.
  • Current treatment protocols require a sensitivity of at least 10-4
  • NGS MRD detection claim to reach sensitivities down to 10-7.
  • Amount of input DNA is crucial for reaching a particular sensitivity.

 

Future of MRD Studies in Acute Leukemia

  • MRD detection is one of the successful examples where complicated basic research was transferred into high-technology laboratory diagnostics.
  • MRD diagnostics is very likely to be included in all acute leukemia treatment protocols as MRD data provides the most optimal evidence of the in vivo response to treatment, thus providing the clinician a better knowledge and control of the clinical course in individual patients.
  • Introduction of newer targeted therapies will create additional applications of MRD monitoring and may result in newer techniques for MRD detection like microarrays and immunobeads.
  • An EQA program is necessary to minimize interlaboratory variations and ensure uniformity of protocols being followed in all diagnostic MRD laboratories.

 

 

References

  1. Recent Advances in Hematology-3, 2011
  2. Della Starza et al. Frontiers in oncology;2019
  3. Monika Bruggemann and Michaela Kotrova, American Society of Hematology Educational Program;2017.
  4. Dongen et al. The American Society of Hematology;Blood first paper;2015
  5. Maggie L. Shaw. FCM, NGS, and PCR. Detecting MRD in Patients with ALL; 2020.
  6. Dario Campana. MRD in ALL, Semin Hematol.NIH; 2010