haematology recent advances

1. INTRODUCTION
• Introduction of new drugs, combinations of
chemotherapeutic agents & novel biologic treatments
have caused dramatic responses in treatment of various
malignancies.
• However, many patients who achieve clinical and
morphological remission after induction chemotherapy
relapse sooner or later and die from recurrence of their
disease.
• MRD, a term referring to disease that is undetectable by
conventional morphologic methods, have therefore
attracted increasing attention in recent years.
• New and powerful laboratory tools such as PCR assays
have extraordinary sensitivity and provide exciting new
insights into the detection, nature, quantification, and
kinetics of MRD.

2. DEFINITION
• A stage in leukemia treatment when patient is in
remission, symptoms of disease are absent but small
no. of leukemic cells still remain in body.
• When in complete remission, absence of malignant
disease detectable by clinical imaging, standard
histologic or cytologic means & when malignant cells
can only be found by very sensitive immunologic or
molecular techniques.
• Lowest level of disease detectable in patients in
complete clinical remission by most sensitive
methods available.

3. CONTENTS
• Background: The problem of MRD
• Techniques for measuring MRD
• Use of & common targets in MRD detection in
different leukemias, lymphomas & solid tumors
• Significance of MRD
 level of MRD as a guide to prognosis or relapse risk
 monitoring people for early signs of recurring
leukemia
 individualization of treatment

4. CONTENTS contd…….
• Areas of current research & controversies:
Clinical usefulness of MRD tests
Method of testing, and when to test
Is there such a safe thing as a safe level of
MRD?
Is MRD testing useful for all patients?
• MRD testing by hospital & other labs

5. • Advances in the treatment of numerous malignant
hematologic disorders over the past 3 decades have
resulted in improved response rates and, in many
cases, long-term disease-free survival.
• Though a large no. of patients achieve complete
clinical remission after initial treatment, large
majority of these patients relapse because of the
persistence of low numbers of malignant cells that
have not been eradicated with induction therapies.
• A morphologically normal bone marrow & normal
blood counts are compatible with significant amounts
of residual disease.

6. • Despite having achieved a CR, a patient may
still harbor up to 10 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.
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7. • The last decade has therefore seen the emergence of
various laboratory tools for the detection and
assessment of residual disease, including flow
cytometry, immunologic studies, FISH, and
cytogenetic assays.
• PCR techniques in particular have provided a means
to analyze tumor-specific DNA sequences with
custom-built probes and added a level of sensitivity
of detection in the range of 1 malignant cell in 10,000
to 10,00,000 normal cells.
• Despite this progress, however, many incongruities
exist among clinical studies that try to establish
prognosis, outcome, and clinical decision making on
the basis of these assays.

8. TECHNIQUES FOR MEASURING MRD
• MORPHOLOGY
• CLONOGENIC ASSAYS
• IMMUNOPHENOTYPE
ANALYSIS
• KARYOTYPE ANALYSIS
• FISH
• PCR

9. MORPHOLOGY
• Limited by its low sensitivity.
• Generally, only 1 of 100 cells can be identified as
malignant.
• Inability to distinguish between immature cancer cells
and early regenerating cells further restricts the
usefulness of standard morphologic methods.
• Sensitivity and specificity of morphology can be
enhanced when combined with other tools such as
immunophenotyping or FISH.

10. CLONOGENIC ASSAYS
• In vitro culture techniques have been developed in which
bone marrow samples from patients are grown under
conditions favorable for stimulation of leukemic cells.
• Advantage of blast colony assays is that they identify
populations of occult malignant cells that can be
expanded, hence allowing their biologic characteristics
and growth requirements to be further studied.
• Also single colonies can be analyzed by immunologic,
cytogenetic, or molecular techniques. Disadvantages of
clonogenic assays include their dependence on growth
rates of leukemic progenitor cells and the cumbersome
nature of assays targeted at single colonies.

11. IMMUNOPHENOTYPIC ASSAYS
• Use of monoclonal antibodies by means of flow cytometry or
fluorescence microscopy to detect nuclear, cytoplasmic, and
surface antigens that are expressed by malignant cells can be
fast and reliable.
• Sensitivity of detection can range as high as 1 abnormal cell per
10,000 to 10,0000 normal cells using double- or triple-color
immunofluorescence techniques or fluorescence microscopy
screening multiple slides per sample.
• Drawbacks however, can reduce the sensitivity of detection to
the level of light microscopy and morphology. These include
• (1) the lack of antigen specificity for malignant cells as these
cells represent the counterparts of normal cells with, in many
cases, identical or similar antigen profiles;
• (2) the existence of several subpopulations, some of them as
minor clones, that are difficult to identify; and
• (3) the inability to identify phenotypic switch, a phenomenon
that may occur at relapse.

12. KARYOTYPIC ANALYSIS
• Cytogenetic analysis has become an important tool in
risk stratification .
• Cytogenetic studies provide specificity because they
can unambiguously identify malignancy-specific
markers and detect cytogenetic signs of clonal
evolution at relapse.
• The pitfalls of conventional cytogenetic analysis are
its labor intensity (because it requires in vitro
cultures) and the dependency of karyotype
identification on dividing cells, thereby missing
populations of residual cells with a low proliferative
index.
• Despite its specificity, the sensitivity of the method is
low and does not exceed that of morphologic
assessment of marrow smears.

13. FISH
• FISH has emerged in recent years as a promising new tool for
the identification on a molecular level of both chromosomal
aberrations and malignancy-specific DNA sequences.
• Although 100 times more sensitive than standard cytogenetics,
the sensitivity level achieved by FISH( one cell in 10³) is
markedly below that desired for MRD detection (one cell in
10,000)
• The main advantages of FISH, besides its specificity, are the
larger number of cells that can be studied in a time-efficient
manner and without the need for in vitro cultures, the
quantifiability of results, and the applicability to archival
material such as blood smears and histologic sections.
• In addition, FISH allows analysis of both metaphase and
nondividing interphase cells.
• Interphase FISH can even be performed on peripheral blood
samples, thus avoiding the need for marrow aspiration.

14. PCR
• Molecular techniques allow the detection of leukemia-specific
gene rearrangements by identifying either leukemia-specific
translocations or clone-specific immunoglobulin heavy chain
(IgH) gene and T-cell receptor (TCR) gene rearrangements.
• PCR assays are characterized by unparalleled sensitivity. A
single malignant cell can be identified among 10,000 to
10,00000 normal cells.
• Both PCR and its variants such as reverse transcriptase (RT)-
PCR and nested PCR have become the preferred tools in
studies addressing the detection and role of MRD.
• The drawback of PCR lies in its extraordinary sensitivity
-sample contamination and false-positive results can occur. In
addition, since RT-PCR measures transcript expression, a
malignant clone that transiently does not express its diagnostic
transcript would evade detection by this technique.

15. Patient specific testing
• Patient specific MRD detection using
immunoglobulin (IG) or T cell receptors (TCR) is
gaining popularity as a way of measuring MRD in
leukemias that do not contain a chromosomal
translocation or other leukemic specific marker. In
this case the leukemic specific IG or TCR clone is
amplified using PCR and the variable region of the
IG or TCR is sequenced. From this sequence PCR
primers are designed that will only amplify the
specific leukemic clone from the patient.

16. TECNIQUES USED FOR DETECTION
OF MRD

17. • It is important to note that even with these
very sensitive tests a negative result does not
imply clearance of all residual disease

18. Use of & common targets in MRD
detection in different leukemias,
lymphomas & solid tumors
• Acute lymphoblastic leukaemia (ALL)
• Targets: t(9;22) BCR-ABL, t(12;21) ETV6-
RUNX1 (TEL-AML1), Patient specific assays
for immunoglobulin and T cell receptor genes
• Uses: Chromosomal translocation MRD
detection is used as a standard clinical
practice. Patient specific assays are gaining
acceptance

19. Acute myeloid leukaemia (AML)
• Targets: t(15;17) PML-RARA, t(8;21)
AML1-RUNX1T1 (AML-ETO), inv(16)
• Uses: Chromosomal translocation MRD
detection used as a standard clinical practice.

20. Chronic lymphocytic leukaemia
• Targets: Cell surface proteins, Patient specific
assays for immunoglobulin and T cell receptor
genes
• Uses: Immunological methods are gaining
wider use as more advanced flow cytometers
are utilized for clinical testing. Patient specific
assays are still generally only used in research
protocols.

21. Chronic myelogenous leukemia
• Target: t(9;22) BCR-ABL
• Uses: MRD detection of the t(9;22) is
considered standard of care for all patients
with CML and is extremely valuable for
patients being treated with imatinib mesilate.

22. Follicular lymphoma
• Targets: t(14;18) IgH/BCL2, Patient specific
assays for immunoglobulin and T cell receptor
genes.
• Uses: The t(14;18) is regularly used for MRD
detection. Patient specific assays are still
generally only used in research protocols.

23. Mantle Cell Lymphoma
• Targets: t(11;14) IgH/CCND1 (IgH/BCL1),
Patient specific assays for immunoglobulin
and T cell receptor genes
• Uses: The t(11;14) is regularly used for MRD
detection, but the assay can only reliably
detect 40-60% of the t(11;14) translocations.
Patient specific assays are still generally only
used in research protocols.

24. MULTIPLE MYELOMA
• Targets: M-protein levels in blood, Patient
specific assays for immunoglobulin and T cell
receptor genes .
• Uses: M-protein level in the blood is standard
of care and is used for almost all patients with
multiple myeloma. Patient specific assays are
still generally only used in research protocols.

25. SOLID TUMORS
• Research into MRD detection of several solid
tumors such as breast cancer and
neuroblastoma has been performed. These
assays have been used to sample lymph nodes
and blood for residual or metastatic tumor
cells. Applicable targets for MRD detection
have been more difficult to determine in solid
tumors and the use of MRD in solid tumors is
much less advanced than the use in leukemia
and lymphoma.

26. Molecular Markers of Minimal Residual
Disease in Hematologic Malignancies

27. SIGNIFICANCE OF MRD TESTING
• MRD evaluation serves to identify and quantify
occult leukemic cells and then using this information
to determine prognosis & modify therapeutic
protocols with long term intention to individualize
and improve treatment outcome.
• The ultimate goal of MRD assays is to guide
therapeutic decisions by recognizing patients who
responded well to therapy & thus should be spared
further therapy distinguishing them from patients in
whom therapy must be continued or intensified to
minimize likelihood of clinical relapse

28. SIGNIFICANCE OF MRD TESTING
Level of MRD is a guide to prognosis or relapse risk
• The level of MRD at a certain time in treatment, is a
useful guide, to the patient's prognosis.
• MRD testing could predict outcome, and this has
now been shown.
• MRD is a powerful & independent prognostic
indicator.

29. SIGNIFICANCE OF MRD TESTING
• Monitoring people for early signs of recurring leukaemia
Another possible use is to identify if or when someone
starts to relapse, early, before symptoms come back.
• This means regular blood or marrow samples. This is
being explored mainly in CML, where one can study the
leukaemia in blood, which is easier to sample regularly
than bone marrow.
• The molecular tests can show tumour levels starting to
rise, very early, possibly months before symptoms recur.
Starting treatment early , might be useful: the patient will
be healthier; fewer leukaemic cells to deal with; the cells
may be amenable to treatment, since at clinical relapse
they have often become more resistant to drugs used.

30. SIGNIFICANCE OF MRD TESTING
• INDIVIDUALISATION OF TREATMENT
It identifies patients individual risk of relapse,
and can theoretically allow them to receive
just enough treatment to prevent it.
• Treatment intensification for patients with
slow clearance of leukemia & persistent or
resurgent MRD is well supported now
• Conversely patients with profound
cytoreductions at early stages of therapy are
clear candidates for less invasive (hence less
toxic) treatment regimens.

31. SIGNIFICANCE OF MRD TESTING
• It has also been used to determine quality of
grafts ,the efficacy of procedures to remove
malignant cells from autografts & to determine
its clinical significance if any
• It has been shown that the detection of high
level of MRD ( one in 10³ ) before
transplantation was universally associated with
relapse

32. • Treatment for MRD
• Generally the approach is to bring a cancer into remission first
(absence of symptoms) and then try to eradicate the remaining
cells (MRD) Often the treatments needed to eradicate MRD,
differ from those used initially. This is an area of much
research.
• It seems a sensible idea to aim to reduce or eradicate MRD.
What is needed, is evidence on which is the best method, and
how well it works. Treatments which specifically target MRD
can include.
• (a) intensive conventional treatment with more drugs, or a
different combination of drugs (b) stem cell transplant, e.g
marrow transplant. This allows more intensive chemotherapy
to be given, and in addition the transplanted bone marrow may
help eradicate the minimal residual disease (c) immunotherapy
(d) monitor the patient carefully for early signs of relapse.
This is an area of active research in several countries. (e)
treatment with monoclonal antibodies which target cancer
cells (f) cancer vaccines.

33. Areas of current research & Controversies
• clinical usefulness of MRD tests
• MRD tests are new. The tests have been done
on relatively few people. Consequently there is
less evidence available, to guide doctors, in
interpreting the tests, or basing treatment
decisions on them.

34. • There are controversies about the best times to test,
and the best test method to use. There are national
and international approaches to standardize these. In
childhood leukaemia and chronic myeloid leukaemia,
there appears to be consensus emerging.
• Whereas some investigators claim that detection of
residual disease immediately following induction or
during the first 6 months predicts likelihood of
relapse, others have found that, in many patients,
MRD can persist for 24 months and even longer and
that time points farther away from diagnosis and
induction may be more useful as a prognosticator for
disease recurrence.
• Other data indicate the importance of serial
assessments and quantification of residual disease
measurements, rather than focusing on timing in
relation to induction or maintenance therapy.

35. • 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, cancer cells inevitably grow
and that if they are present disease usually develops.
• But there is evidence from some studies, that
leukaemic cells can lie dormant for years in the body
and do not regrow.
• For this reason, it may be that the goal of treating
MRD may be to reduce it to a "safe" level - not to
eradicate it completely.

36. • Current standards for evaluating response to
induction chemotherapy only document that disease
was sufficiently reduced to allow for normalization of
bone marrow function.
• However some consensus is emerging regarding the
most predictive prognostic levels of postinduction
disease as detected by flow cytometry which, in both
ALL and AML, appear to lie between 0.01% and
0.035% leukaemic cell involvement of a
morphologically normal appearing bone marrow. We
must keep in mind, however, that to reliably detect
0.01% of leukaemic cells, flow cytometry is pushed
to its extreme limits which may not be reached in
every clinical routine laboratory due to variability in
technical expertise

37. Is MRD testing useful for all patients?
• Some types of leukaemia are difficult to treat.
In these, it is not clear how MRD testing
would help. The patients may not do well on
current treatment, but sometimes it is not clear
what other treatment, if anything, might be
better. There is thus an argument that as the
test is not necessary: it might involve an
additional procedure for the patient; it will
contribute no useful information on treatment,
it is not necessary.

38. MRD testing by hospital & other labs
• Where done?
• MRD testing is not yet a routine test, nor is it carried
out in all places.
• Currently most MRD testing - in leukaemia research -
is done during clinical trials, and would be funded as
part of that trial, for patients enrolled on the trial. The
tests are specialised, so samples are usually sent to a
central reference laboratory in each region or country.
The tests are not done in most routine diagnostic labs,
as they tend to be complex, and also would be used
relatively infrequently.

39. COST
• MRD testing is technically demanding and
time consuming; the tests are expensive, so are
usually available only through specialist
centres, as part of clinical trials.

40. Availability of MRD testing.
• MRD testing is available in some clinical
trials in the UK, Europe, Australia and the US.

41. Interpretation of MRD test results.
• MRD tests are new and have been carried out
on relatively few people (a few thousand at
most). Researchers and doctors are still
building the extensive database of knowledge
needed, to show what MRD tests mean.This is
likely to change in future, as tests become
more routine.

42. Outstanding questions and future
perspective
• Each of the methodologies for studying MRD
in patients with acute leukemia has relative
advantages & disadvantages.
• Use of multiple approaches is desirable as it
can increase the no. of patients that can be
studied & offset the limitations of individual
methods as well as the accuracy of individual
measurements

43. Conclusion
• In practice ,it is impossible for every neoplastic cell to be
eliminated, therefore MRD detection & subsequent treatment
aims to decrease disease load to levels where risk of relapse is
least.
• The efficacy of MRD has been rigorously proven by
statistically sound studies .
• MRD determination is an evolving field in which technology
& understanding of results are continually being refined.
• However ,there are still a no. of unanswered questions
regarding the modalities & means of attaining the optimal
approach to MRD analysis with regards to specific leukemias
• There is no doubt that in future, therapeutic measures in
leukemia management will be closely interlinked to close
monitoring of leukaemic cell load in the patients

44. Conclusion
• MRD studies will become an integral part of
modern management of patients with leukemia
• The main challenge is to simplify methods
while maintaining or increasing their
reliability, thus disseminating the potential
benefits of MRD monitoring to all patients.