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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.
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.
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
CONTENTS contd…….
• Areas of current research & controversies:Clinical usefulness of MRD testsMethod of testing, and when to testIs there such a safe thing as a safe level of
MRD?Is MRD testing useful for all patients?• MRD testing by hospital & other labs
• 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.
• 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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• 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.
TECHNIQUES FOR MEASURING MRD
• MORPHOLOGY• CLONOGENIC ASSAYS• IMMUNOPHENOTYPE
ANALYSIS• KARYOTYPE ANALYSIS• FISH• PCR
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.
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.
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.
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.
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.
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.
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.
TECNIQUES USED FOR DETECTION OF MRD
• It is important to note that even with these very sensitive tests a negative result does not imply clearance of all residual disease
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
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.
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.
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.
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.
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.
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.
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.
Molecular Markers of Minimal Residual Disease in Hematologic Malignancies
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
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.
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.
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.
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
• 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.
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.
• 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.
• 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.
• 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
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.
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.
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.
Availability of MRD testing.
• MRD testing is available in some clinical trials in the UK, Europe, Australia and the US.
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.
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
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
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.
