Mrd

1. PRESENTOR : DR SADIYA
MODERATOR : DR PALLAVI
CHAIRPERSON : DR CHINTAMANI PATHAK​
MOLECULAR METHODS IN DETECTION
OF MINIMAL RESIDUAL DISEASE (MRD)
IN HEMATOLOGICAL MALIGNANCIES

2. CONTENTS
• What is MRD ???
• Role of MRD in patient care
• Remission critera
• Principle of MRD detection
• Techniques of MRD assessment
• FISH
• PCR
• NGS
• Summary

3. WHAT IS MRD???
The term minimal residual disease (MRD) is used to describe
the low-level disease which is not detectable by conventional
cytomorphology and detected by methods such as Flow
Cytometry (immunologic MRD) or Polymerase Chain Reaction
(PCR) (Molecular MRD).
A stage in leukemia treatment when patient is in remission,
symptoms of disease are absent but small number of leukemic
cells still remains in body.

5. Timeline for MRD Testing Development

6.  A morphologically normal bone marrow &
normal blood counts can have significant amounts
of residual disease.
 Any remaining cancer cells in the body can
become active and start to multiply, causing a
relapse of the disease.
6
THE ROLE OF MRD ASSESSMENT
IN PATIENT CARE

7.  After achieving Complete Response (CR), a patient may
still harbor up to 1010 leukemia cells that persist at
levels undetectable by conventional cytomorphologic
examination.
 Subjected to either undertreatment with risk of relapse
or overtreatment with exposure to therapy-related
morbidity and mortality.
7

8. Helps in antedating relapse and assess and quantify
residual disease
Therapy stratification
Risk stratify patients
Assess response to treatment
Surrogate marker for efficacy of treatment
Evaluation as a prognostic marker
WHY DETECT MRD???
8

9. Presentation title 9
 Normalization of neutrophil count to at
least 1.5x109/L & platelet count
>100x109/L
 BM aspirate and biopsy that demonstrates at
least 20% cellularity with<5% blasts
 No Auer rods
 Absence of extramedullary leukaemia
Partial remission: BM blasts 5-25%
Normocellular bone marrow with <5%
blasts
 No lymphoblasts in the peripheral blood
film
 Absolute neutrophil count of >500/cmm
 Platelets >100,000/cmm
 Absence of extramedullary disease
 No CNS disease

10. REMISSION IN CML 10
Hematological response:
 Normalization of peripheral blood values
& spleen size
Cytogenetic response:
 Proportion of Ph+ metaphases
 -100% : none
 -35-99% : minor
 -1-34%: major partial
 -0%: major complete
3. Molecular response:
Proportion of the residual BCR-ABL
transcript or protein detected through a
molecular method.
Major: ≥ 3 log reduction in BCR-
ABL/BCR level as compared to median
pretreatment level OR
BCR-ABL/ABL ratio =0.045
Complete: negative by RT-PCR

11. WHEN TO TEST???
Presentation title 11
Several prospective studies in childhood ALL
demostrated the most relevant time for detection of MRD
in bone marrow is at the end of induction treatment
(day 13/day 33).

12. WHY MRD PERSISTS EVEN
AFTER TREATMENT???
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.
12

13. Presentation title 13
PRINCIPLE OF MRD DETECTION
Molecular and cellular changes induced by
leukemogenic process are used to distinguish leukemic
cells from normal.
These leukemia associated features are identified using
various techniques at diagnosis or relapse , then used to
monitor MRD.

14. TECHNIQUES FOR MEASURING MRD14
MORPHOLOGY
CLONOGENIC ASSAYS
IMMUNOPHENOTYPE ANALYSIS
KARYOTYPE ANALYSIS
PCR
FISH
NGS

15. SAMPLES USED FOR MRD
DETECTION
• A bone marrow sample is required, and the preferred MRD
sample comes from the first or early pull of the bone marrow.3
• The second pull may have up to a 50% reduction in leukemic
cells
• he MRD sample should have a smaller volume (eg, 2 mL)
because a large sample volume (eg, 10 mL) may contain a
lower proportion of blasts
• Fresh peripheral blood samples
Presentation title 15

16. MORPHOLOGY
• Low sensitivity
• 1 of 100 cell can be identified as malignant
• Inability to distinguish between immature cancer
cells and regenerating marrow cells
• Sensitivity and specificity can be enhanced when
combined with other methods.
16

17. CLONOGENIC ASSAYS
In vitro culture of
BM samples with
conditons
favourable for
growth of
leukemic cells
Formation of blast
colonies and
hence occult
malignant cells
can be expanded
Colonies can be
analysed using
immunologic
,cytogenetic or
molecular
techniques
17
DISADVANTAGE
 Sampling errors and dependence on growth
rates of leukemic progenitor cells
Cumbersome procedure

18. IMMUNOPHENOTYPING
 Use of monoclonal antibodies by means of flow cytometry or
fluorescence microscopy to detect nuclear, cytoplasmic and
surface antigens expressed by malignant cells
 Detection rates of 1of 10000 to 100000 normal cells using
double or triple color immunofluorescence techniques
 Fast and reliable technique.
18

19. Presentation title 19
Leukemia Cell lineage Marker combination Applicability
%
T lineage ALL (n=39) Tdt/CD5/CD3(CD19/CD3
3/HLA-Dr)
92
CD34/CD5/CD3 21
B lineage ALL (n=169) CD19/CD34/CD10/CD38 52
CD19/CD34/CD10/CD58 49
CD19/CD34/CD10/CD45 47
CD19/CD34/CD10/TdT 43
CD19/CD34/CD10/CD66c 31
CD19/CD34/TdT/IgM 17
CD19/CD34/CD10/CD22 11
CD19/CD34/CD10/CD13 10
CD19/CD34/CD10/CD15 10
CD19/CD34/CD10/CD21 6
CD19/CD34/CD10/CD33 6

20. LEUKEMIAASSOCIATED
IMMUNOPHENOTYPE (LAIP)
DEFINED BY LINEAGE INFIDELITY OR ASYNCHRONOUS
EXPRESSION OF DIFFERENTIATION MARKERS.
Asynchronous antigen
expression
Cross-lineage antigen expression
(myeloid)
Altered antigen expression
(over or under expression)

21. LAIP in AML
21
LAIP class Examples
Cross-lineage expression of
lymphoid antigens
CD33+,CD2+,CD34+:
CD34+,CD13+,CD19+
Overexpression HLA-DR++CD64++,CD33++,
CD34++:CD64++,CD4++,CD45++
Lack of expression of antigen HlA-DR -, CD33+, CD34+
Asuynchronous expression of
antigens
CD15+, CD33+, CD34+;
CD65+,CD33+, CD34+

22. 22
Lack of antigen specificity of malignant
cells
Existence of several sub-
populations or minor clones
Inability to detect phenotype switch
which is a common phenomenon after
relapse.
DISADVANTAGES
OF FCM

23. KARYOTYPING
 Important tool in risk stratification
 Unambiguously identify malignancy
specific markers and detect cytogenetic
signs of clonal evolution at relapse.
DISADVANTAGES:
1. Labour intensive (because it requires in
vitro cultures)
2. Dependency of karyotype identification on
dividing cells thereby missing populations
of residual cells with a low proliferative
index
23

24. FISH (FLUORESCENT IN SITU
HYBRIDISATION)
• Promising tool for detecting absence or presence of additional
chromosomes
• Can also detect or confirm gene or chromosomes changes
(detection of fusion of genes: BCR-ABL, TEL-AML, PML-
RARA etc.)
• Detects 1 cell in 1000 i.e it is below the desired range for
MRD detection.
24

25. 25

26. Presentation title 26

27. 27
• Large number of cells studied in a time efficient
manner
• No need for in-vitro cultures
• Quantifiability of results
• Applicable to archival materials such as blood smears
and histological sections
• Can be performed on peripheral blood samples
thereby avoiding the need of marrow aspiration
• Analysis of metaphase and non-dividing interphase
cells
ADVANTAGES OF FISH

28. PCR
Presentation title 28
PCR IN MRD

29. 29
PCR
Kary Mullis, the inventor of PCR, was awarded the Nobel Prize in
1993 Chemistry
It is quick, powerful, inexpensive DNA amplification technique
Standard RT PCR is useful in establishing the appropriate target
for MRD detection, RQ-PCR is the “gold standard” for MRD
detection and evaluation.

30. Presentation title 30

31. Presentation title 31
RQ-PCR permits accurate quantitation of PCR
products during the exponential phase of the PCR
amplification process
Real-time detection of fluorescent signals during
and/or after each subsequent PCR cycle
Quantitative PCR data can be obtained in a short
period of time

32. 1) RQ-PCR ANALYSIS USING
SYBR GREEN DYES
32
Dye binds to
minor groove of
dsDNA and
fluorescence is
emitted
PCR amplifies
DNA therefore
more dye binds
to dsDNA
Fluorescense
signal gradually
increase in
exponential
phase and hence
suggests
amplification of
target DNA

33. Presentation title 33

34. 2) ANALYSIS
USING
HYDROLYSIS
PROBES
Presentation title
34

35. 3) ANALYSIS
USING
HYBRIDIZATION
PROBES
Presentation title 35

36. Presentation title 36
An amplification plot of several 10- fold dilutions of a diagnostic leukemia sample is
shown. The samples were diluted in normal mononuclear cell (MNC) DNA. Based on the
(background) fluorescence intensity detected during the first three to 15 PCR cycles, a
threshold is determined. The cycle threshold (CT) is defined as the PCR cycle at which the
fluorescence exceeds the threshold for the first time. The CT value will be directly
proportional to the amount of target sequence present in the sample. The increase
in fluorescence, on the y-axis, is indicated as DRn.

37. Detection of leukemia specific rearrangements
37
Detection of
fusion transcripts
Clonality of Ig &
TCR gene
rearrangements
Aberrant genes
and aberrantly
expressed genes

38. Translocation Fusion
t (1;19) (q23;p13) E2A-PBX1
t (4;11) (q21;q23) MLL-AF4
t(9;22) (q34;q11) BCR-ABL
t (12;21)
(p13;q22)
TEL-AML1
Presentation title 38
RQ-PCR BASED QUANTIFICATION OF LEUKEMIA –
ASSOCIATED FUSION GENES
Distinguish leukemic cell from normal – Chromosomal abnormalities.
Chromosomal translocations in leukaemia result in fusion genes which are disease
specific.
Translocation Fusion
t(8;21) RUNX1-
RUNX1T1
Inversion 16 CBFB-MYH11
t(15;17) PML-RARA

39.  Breakpoint fusion sites at the DNA level can be used as
MRD-PCR targets as they are related to the oncogenic process
and therefore are unchanged throughout the disease course.
 They concern PCR targets at the DNA level instead of at the
RNA level, implying that these MRD-PCR targets are less
sensitive to degradation.
39

40.  Only one PCR target is present per cell, which makes
quantification easier.
 Fusion sites in each person is unique therefore patient specific
RQ-PCR strategies are applied
 Less risk of cross-contamination between two different
patient samples
Presentation title 40

41. Chromosome aberrations can be employed as tumor-specific
MRD-PCR targets in which the PCR primers are chosen at
opposite sides of the breakpoint fusion region
Feasible when the breakpoints of different patients cluster in
relatively small breakpoint areas
41

42. Presentation title 42
In many chromosomal
aberrations, the breakpoints of
different patients are scattered
over large areas.
Long distance PCR can be used
for rapid and efficient screening
of these sites

43. If after assessment by PCR we get low positive results and
clinical history is unknown;
1) confirm if CML has been previously diagnosed or treated
2)inform the clinician or pathologist that low levels are not
keeping with diagnostic presentation.
Precautions need to be taken when only single samples of
patient are being used because very low levels of BCR-ABL1
mRNA have been reported in healthy individuals
Presentation title 43

44. BCR-ABL1 44
Reciprocal translocation between chromosomes
9 and 22[t(9;22)] that gives rise to
a BCR::ABL1 fusion gene that leads to increased
tyrosine kinase activity .
Used for
• Diagnostic workup of CML, ALL and AML
• Assess optimal response and detect MRD if
achieved in a timely manner

45. RUNX1-RUNX1(8:21)
• Quantification of RUNX1-RUNX1(8:21) is
considered a valuable tool
• > 3 log reduction rates in marrow = better outcome
• Low levels of transcripts may be detectable for
years after initial diagnosis as it has been detected
in mast cells
• Low level transcripts not always reflects residual
disease
• Compared and correlated with flow cytometry.
45

46. Presentation title 46
RQ-PCR BASED QUANTIFICATION OF
ABBERANT/OVEREXPRESSED GENES

47. WT1 EXPRESSION
 Wilms’ tumor gene WT1- potent transcriptional
repressor of several growth factors
 Overexpressed in all patients with AML and is thought
to play a role in maintaining the viability of leukemic
cells
47

48. Presentation title 48
 Regarded as a specific feature of the
malignant cells and consequently can be
used as an MRD-PCR target

49. HOX11L2
 A new recurrent translocation, t(5;14), has been
identified in a subset of T-ALL
 HOX11L2 gene: expressed in fetal thymus, fetal
spleen, and adult thymus
not expressed in adult spleen, adult peripheral blood,
or bone marrow
 Ectopic HOX11L2 expression can be used as an MRD-
PCR target
49

50. Presentation title 50
QUANTIFICATION OF JUNCTIONAL REGIONS OF
IG AND TCR GENE
REARRANGEMENT
 Mostly used in monitoring MRD in cases of ALL
 The immunoglobin and T cell receptor gene rearrangement
during normal B & T lymphocyte development generate
unique fusion of variable, diversity and joining (VDJ)
segments.

51. Presentation title 51
 These B & T clonal recombination generate
patient specific DNA length and sequences
which represent ideal markers for detection
and quantification of leukemic cells among
normal lymphocytes in remission phase.

52. ANTIGEN-RECEPTOR GENE
REARRANGEMENTS
Combined study of these 4 loci permits to identify one or more
rearrangement in virtually all cases of ALL.
Presentation title 52
B-lineage ALL T- lineage ALL
IgH >90 % 10-15%
IgKappa 50% 0 %
TcR delta 55 % 50 %
TcR gamma 55 % 90%

53. Presentation title 53
Disadvantages
 Most laborious, expensive and time
consuming.
 Limited applicability in AML
Advantages
- Good sensitivity.
- Applicable to most ALL patients
- Good reproducibility within same as
well as different laboratory.
Ig and TCR gene
rearrangement

54. 54

55. DROPLET DIGITAL PCR
Presentation title 55

58. Presentation title 58
NEXT GENERATION SEQUENCING

59. 59

60. 60

61. Presentation title 61

62. Presentation title 62
AVAILABLE NEXT GENERATION
SEQUENCING PLATFORMS
1. Illumina / Solexa’s GA
2. Life/APG’S SOLiD
3. Roche 454
4. Helicos Biosciences HeliScope
5. Pacific Biosciences SMRT
6. Oxford Nanopore

63. NGS IN AML 63
 AML related gene mutations can also be found as germline
mutations can be used for monitoring diseases
 VAF >50% is an indicator of germline origin
 NGS alone is inadequate for assessing molecular response but can
be used in conjunction with Flow cytometry
 Flow Cytometry cell sorting of suspicious progenitors with
subsequent NGS analysis can be helpful
 Recently there has been many developments and upcoming Error
correcting NGS can solve this problem

64. NGS IN B & T-CELL MALIGNANCIES
• Ig or TCR rearrangements represent unique genetic markers /
molecular signatures for B and T-cell malignancies
• NGS nowadays have become an attractive option for MRD
monitoring
• Can also detect new clonal evolution and emergence of sub-
clones
• Early time results are possible
• Classic Sanger sequencing can be employed to identify the
index clone at time of diagnosis
64

65. • A sensitivity of 1/1000000 can be achieved if input DNA
quality is adequate, this becomes concern because availability
of sufficient DNA is challenging in specimen with low
cellularity
• NGS can also be used for monitoring residual disease of Non-
Hodgkins Lymphoma and Plasma cell neoplasms
65

66. MRD DETECTED NOW
WHAT????
66

67. Generally the approach is to bring a cancer into remission
first (absence of symptoms ) and then try to eradicate the
remaining cell (MRD) often the treatments needed to
eradicate MRD differ from those used initially.
Presentation title 67
TREATMENT OF MRD

68. DRUGS
STEM CELL
TRANSPLA
NT
IMMUNO
THERAPY
68
MONOCLONAL
ANTIBODIES

69. INTERLABORATORY STANDARDIZATION
Presentation title 69
 The need for standardization of MRD studies is essential.
 Standardization of RNA based studies has proved to be complex
because mRNA is labile
 It is less difficult to standardize genomic DNA values and therefore
much progress has been made in Ig and TCR rearrangement studies
 Methods to standardize the lab results to enable interlaboratory
comparsions like International Standardized Ratio (ISR) used for
calculating the standard BCR-ABL1 ratio, may be extended to other
MRD assays in the future.

70. Areas of current research and controversies ?
70
 Clinical usefulness of MRD tests
 In simple test like complete blood count , doctors have large
number of evidence to interpret results but MRD test is new &
there is little evidence so problem occurs in interpreting
results.
 Method for testing, and when to test
 There are controversies about best time to test and best method
to use.

71. Is there such a thing as a safe level of MRD?
There is also controversy about whether MRD is always bad,
inevitably causing relapse, or whether sometimes low levels
are 'safe' and do not regrow. It is usually assumed that cancer
cells inevitably grow and that if they are present disease
usually develops.
Is MRD testing useful for all patients?
Presentation title 71

72. Multiparameter Flow
Cytometry
RQ-PCR Next Generation
Sequencing
Availability Widely applicable Widely applicable One platform /Company
available
Sensitivity ~104-105 ~105 ~105
Applicability Nearly 100% Only in cases with well
defined translocation or
mutation (BCR-ABL1)
FDA-cleared for use in
ALL, MM & CLL
Sampling Requires fresh sample
(viable cells), analysed
within 24-48hr
Both fresh and stored
samples can be used
Both fresh and stored
samples can be used
Result available Within a few hours Within 1 week Usually 2 weeks
Limitations -Lack of standardization
-Significant technical
expertise required
-Risk of
immunophenotypic shift
can lead to false negative.
-Limited to patients with
well defined genetic
aberrations
-Expensive
Presentation title 72

73. Presentation title 73

74. 74

75. SUMMARY 75
 Measurements of MRD provide a powerful and independent
prognostic indicator of treatment outcome in children with ALL.
 Flow- cytometry & PCR have emerged as most promising
methods for detecting sub-microscopic levels of leukemia.
 Early detection of relapse and subsiquant changes in therapeutic
strategies will improve cure rates.
 Timely detection of MRD whould identify patients who need
more intensive therapy to remain in remission.

76. REFERENCES
Vonk, Christian M et al. “Molecular Minimal Residual Disease Detection in Acute Myeloid
Leukemia.” Cancers vol. 13,21 5431. 29 Oct. 2021, doi:10.3390/cancers13215431
Herrera, Alex F, and Philippe Armand. “Minimal Residual Disease Assessment in Lymphoma: Methods and
Applications.” Journal of clinical oncology : official journal of the American Society of Clinical Oncology vol.
35,34 (2017): 3877-3887. doi:10.1200/JCO.2017.74.5281
Lori A. Ramkissoon, Nathan D. Montgomery, Applications of next-generation sequencing in hematologic
malignancies, Human Immunology, Volume 82, Issue 11,2021, Pages 859-870.
Starza ID, Eckert C, Drandi D, et al. Minimal Residual Disease Analysis by Monitoring Immunoglobulin and T-Cell
Receptor Gene Rearrangements by Quantitative PCR and Droplet Digital PCR
Farhad Ravandi, Roland B. Walter, Sylvie D. Freeman; Evaluating measurable residual disease in acute myeloid
leukemia. Blood Adv 2018; 2 (11): 1356–1366.
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