Introduction:

• After induction therapy, even patients who achieve complete clinical remission, contain as many as 10¹⁰ malignant cells in marrow.  This is responsible for relapse in 15-20% of children & 50-60% of adults with ALL and varying proportion of patients of AM
• Goal of detecting MRD by sensitive techniques is to adjust patient’s therapy in order to reduce both the risk of relapse and of over-treatment

Technique used for MRD detection should be

• Specific- Discriminate malignant from normal cells
• Sensitive- Able to detect up to 1 leukemic cell in at least 10⁴ normal cells
• Reproducible- Widely applicable in different labs
• Quantitative- Provide a numerical estimate of positive cells

Techniques

• Immunophenotyping by flow cytometry.
• Leukemic cell DNA / RNA by PCR
• Cytogenetics
• FISH
• Southern blotting
• Next generation sequencing

MRD by flow cytometry

• Sensitivity of 1 in 10⁴ (Provides direct measurement of number of leukemic cells)
• Result is available in 2-3 hours
• Ex: In T-ALL patients – tumor cells have double expression of TdT & cCD3

MRD detection by PCR

• PCR relies on detection of one / two molecular targets that distinguish the leukemic cell from normal cells
• They include
  • Junctional regions that result from clonal immunoglobulin (B-cell) & T cell receptor gene (T-cell) rearrangement
  • Fusion transcripts due to specific chromosomal translocations
• Results available in 24 – 72 hours
• Procedure
  • BM samples are processed for DNA extraction and unique Ig or TCR gene rearrangement is identified by using combination of primers for the V, D & J segments of different Ig heavy of Ig light genes
  • To detect MRD the original clone of DNA (i.e. present during presentation) is identified based on its unique size and migration mobility.  Identification of residual leukemia is carried out by using the same combination of primers that yielded a signal in presentation sample (Ex – VH3 / JH etc)

Chromosomal Translocations as molecular targets for MRD analysis

• Detection of fusion transcript by PCR / FISH
  • ALL – t (1; 19): E2A – PBX, t (12 ;21) – ETV 6 AML1, t (9 ; 22) – BCR – ABL
  • AML  - t (8;21) AML 1 – ETO, t (15;17) PML – RARA, inv (16)