|Year : 2016 | Volume
| Issue : 2 | Page : 47-53
Assessment of minimal residual disease in childhood acute lymphoblastic leukemia
Sonia K Parikh, Urmila P Uparkar
Department of Medical and Pediatric Oncology, The Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India
|Date of Web Publication||14-Jul-2016|
Sonia K Parikh
Room No. 80, Medical OPD, The Gujarat Cancer and Research Institute, M.P. Shah Cancer Hospital, NCH Campus, Asarwa, Ahmedabad - 380 016, Gujarat
Source of Support: None, Conflict of Interest: None
The improving cure rate of childhood acute lymphoblastic leukemia (cALL) is considered as success story in the field of oncology. It has become possible due to progressive refinement of treatment over the period of years. Assessment of minimal residual disease (MRD) is one such tool to further refine and personalize treatment of cALL. Assessment of MRD is no longer a research tool; it has become an integral part of comprehensive management of cALL. Prognostic importance of MRD in cALL is well accepted, but translation of this new information in improving therapy has just begun especially in developing countries like ours. There is increasing understanding among clinicians regarding importance of MRD assessment in day to day clinical practice. Still, there are many issues and lack of clarity with respect to MRD assessment. We are making an attempt in this review to address such issues faced during practical implication of MRD assessment.
Keywords: Childhood acute lymphoblastic leukemia, minimal residual disease, review
|How to cite this article:|
Parikh SK, Uparkar UP. Assessment of minimal residual disease in childhood acute lymphoblastic leukemia. J Appl Hematol 2016;7:47-53
| Introduction|| |
Childhood acute lymphoblastic leukemia (cALL) is the most common childhood cancer; accounting for 25-30% of all childhood cancer cases.  It is wonderfully curable, with success rate of up to 90% in good risk cases treated with appropriate chemotherapy protocol. ,, In this era of personalized medicine and targeted therapy, "one size does not fit all" is very appropriate when it comes to management of cALL. Conventional prognostic criteria are based on pretreatment profile of the patient and tumors. It is based on clinical (age, gender), laboratory (total white cell count), immunophenotypic, and cytogenetic parameters.  Early response to treatment (clearance of blasts cells early during induction and induction failure) has been incorporated into the risk stratification of cALL.  Efficacy of treatment assessed at molecular level when disease in still under morphological remission is an evolving concept. Minimal residual disease (MRD) is the detection of residual leukemic cells not detectable by light microscopy. Assessment of MRD gauges the treatment response at molecular level and it is one such approach to further refine the therapy.
| Definition and concept of minimal residual disease|| |
Cancer chemotherapy destroys cancer cells by fractional cell kill, i.e., it destroys fixed proportion of cells and not the fixed number of cells. There is 99.9% cell kill (i.e., 3 log) with each dose of chemotherapy and there is regrowth of tumor in between the cycle (i.e., 1 log) which results in net 2 log reduction. Morphological remission is defined as <5% blasts in the bone marrow (BM). This corresponds to a level of 1 in 20 malignant cells. Induction of a morphologic remission may result from not more than a reduction from 10 12 to 10 10 leukemic cells.  Despite achieving morphological remission, there are significant numbers of cells present in the body that are responsible for subsequent relapse. There are sensitive techniques available to detect very low levels of leukemic cells in either peripheral blood (PB) or BM.  These assays can detect as low as 1 abnormal cell in 1 million cells which corresponds to MRD of 0.0001% (10−6 )  or as high as 1 abnormal cell in 10,000 normal cells which corresponds to MRD level of <0.1% (10−3 ). ,,, Most commonly MRD level presently acceptable is MRD level <0.01% (10−4 ).
Many different terminologies are coined by researchers to describe MRD. It is referred as low level disease, subclinical disease, no detectable disease with morphological assessment or incomplete remission. In simple words, MRD is persistence of leukemia in the BM, which is under morphological remission. Technically, the term MRD refers to residual leukemic cells that remain following the achievement of "complete" remission but are below the limits of detection using conventional morphologic assessment. ,, This morphologically undetectable leukemia is thought to be responsible for relapse in the future, and it is a major hurdle to cure of cALL. ,
| Minimal residual disease as a prognostic factor|| |
Several clinical studies evaluated MRD and its ability to predict clinical outcome. MRD is an evolving prognostic factor. MRD status is one of the most powerful predictors of disease-free and overall survival (OS) for cALL. ,, It is an important risk factor for de novo and relapse ALL,  as well as in ALL patients undergoing stem cell transplantation. 
Multiple studies ,, have shown that patients with detectable MRD are at high risk (HR) for relapse in the future. Rapidity of clearance and reappearance of MRD affect its prognostic value. Faster the clearance of MRD, early on treatment may positively influence the outcome. Large, prospective studies of MRD in cALL have conclusively demonstrated that the probability of long-term relapse-free survival is directly related to the level of residual disease, both early in the course of treatment, and at later time points.  Another study of 240 children with cALL evaluated MRD by semi-quantitative polymerase chain reaction (PCR) on BM samples from several time points during treatment.  MRD status at day 33 (end of induction) and day 78 (start of consolidation) were used to identify three risk groups with significantly different rates of relapse at 3 years. Low-risk patients (those with no MRD detectable at either time point) had relapse rate of 2%. Intermediate-risk patients (those that were neither low-risk nor HR by MRD assays) had a relapse rate of 23% while HR (with ≥10−3 [0.1%] blasts detected at both time points) patients had a relapse rate of 75%. 
MRD is very sensitive and specific marker predicting relapse. ,, However; it is still not an independent marker for ALL prognosis. It is very well studied in B-cell ALL. Data on T-cell ALL is limited. T-cell immunophenotypes remain HR factor irrespective of MRD status. , So, the prognostic value of MRD should be interpreted in the context of other conventional prognostic factors.
| Minimal residual disease in risk stratification|| |
Till date, risk stratification of cALL is based on conventional prognostic criteria as mentioned previously. Currently, most cooperative groups have revised the risk stratification criteria on the basis of MRD status assessed during treatment at various time points. National Cancer Institute  has incorporated MRD of >0.01% in postinduction marrow as HR feature. Risk stratification on Berlin-Frankfurt-Münster (BFM) protocols includes treatment response evaluated via MRD measurements at two-time points, postinduction (week 5) and end of consolidation (week 12). The BFM risk groups include, standard risk patients having MRD-negative (<0.01% or <10−4 ) at both time points, intermediate risk patients with positive MRD at week 5 and low MRD (<10−3 i.e., >0.01-0.099%) at week 12 and HR patients with high MRD (≥10−3 i.e., 0.1%) at week 12. Patients with a poor response to the prednisone prophase are also considered HR, regardless of subsequent MRD. Phenotype, leukemic cell mass estimate and central nervous system status at diagnosis do not factor into the current risk classification schema. However, patients with either the t(9;22) or the t(4;11) are considered HR, regardless of early response measures. ,, At present ongoing, UKALL 2011 protocol, depending on MRD status, patients are categorized into three groups. Low-risk if MRD <0.005% at end of induction, intermediate risk if postconsolidation MRD <0.5% and HR if MRD ≥0.005% at end of induction and/or with postconsolidation MRD ≥0.5% MRD, in later scenario patients are taken off the protocol. In another ongoing Children's Oncology Group (COG) study AALL0932, PB MRD at D8 and BM MRD at D29 have been incorporated along with other risk factors. MRD-based risk assessment is more precise and may reflect the biology of cALL which in turn helps in the precise therapeutic decision and personalizing therapy based on the risk.
| Use of minimal residual disease in treatment decision|| |
Ultimate goal of MRD assays is to guide therapeutic decisions by recognizing patients who have responded very well to therapy and thus should be spared further therapy and distinguishing them from patients in whom therapy must be continued or intensified to minimize the likelihood of clinical relapse. As it is an evolving field, high level of evidence is still lacking. As of date, there is no meta-analysis available addressing this issue. Many randomized controlled trials are ongoing and results of few randomized studies are available. Modifying therapy decision based on MRD assessment at various time points during treatment has been shown to improve outcome in precursor B-cell cALL. UKALL 2003 , study is a large randomized study till date evaluated the role of augmentation of postinduction therapy based on MRD defined HR subgroup of children and young people with clinical standard-risk and intermediate-risk ALL. Five hundred and thirty-three patients were randomly assigned with end-induction MRD levels of >0.01% to receive standard or augmented therapy. Augmented therapy included additional dose of pegaparginase, vincristine, and methotrexate. Patients who received augmented therapy had more adverse events like asparaginase-associated hypersensitivity and pancreatitis, and methotrexate related mucositis and stomatitis. Importantly, this excess toxicity was not associated with increased treatment-related mortality. Patients treated with augmented therapy had a 5 years event free survival (EFS) that was significantly better than those who received standard therapy (89.6% vs. 82.8%, P = 0.04), although 5-year OS was not statistically significantly different. These results suggest that escalating care in patients with MRD at the end of induction therapy improves clinical outcome. , The UKALL 2003 study demonstrated that reduction of therapy (i.e., one rather than two courses of delayed intensification [DI]) did not adversely impact the outcome in non-HR patients with favorable end-induction MRD. ,
In another study, conducted by Conter et al. , during 2000-2006 in Ph-negative patients where BFM risk stratification was used. Among 80 patients who underwent allogeneic high dose stem cell therapy (HSCT) at a median time of 6 months from diagnosis, 68 had HR MRD or t(4;11) or no CR at day 33, their 5 years EFS was 51.7% compared with a 5 years EFS of 44.6% in patients with the same features were given chemotherapy only (P = 0.72). Intensive BFM therapy is effective for HR cALL if low MRD levels are achieved at the end of the induction/consolidation phase but cALL with high MRD levels at the end of induction/consolidation phase do poorly despite intensive BFM therapy or HSCT. 
In ongoing COG protocol (AALL0932) for newly diagnosed standard-risk precursor B-cell ALL, the study design has Down's syndrome (DS) as separate entity. All non-DS are either average risk or low-risk (LR). LR being the patient with good cytogenetic and PB-MRD on D8 of therapy is <0.01%. These patients are randomized between study arm and standard arm. Study arm being consolidation of 19 weeks followed by maintenance therapy each cycle is 4 monthly with dexamethasone and vincristine intrathecally (IT) pulse, or standard arm of consolidation (4 weeks) followed by interim maintenance-I (IM-I) followed by DI (8 weeks) followed by IM-II (8 weeks) followed by maintenance therapy where pulse of dexamethasone, vincristine and IT is given every 3 monthly. The result of the study will clarify the importance of MRD assessment in therapeutic decision.
Various strategies have been tried to target MRD and improve the outcome of MRD positive HR cALL. These are intensive chemotherapy regimen, use of more number of drugs, use of stem cell transplant, immunotherapy, serial monitoring and detection of early relapse, cancer vaccines, and monoclonal antibodies. There is scarcity of evidence from randomized trials showing that intensified chemotherapy can overcome the poor prognosis of MRD positivity. Results are pending from large, ongoing trials (e.g., NCT01406756), i.e., intensifying therapy by adding agents such as clofarabine or using early transplant for MRD-positive patients.
| Serial minimal residual disease monitoring during surveillance in minimal residual disease negative patient|| |
Residual disease is a dynamic process and numbers of residual leukemic cells vary over time. It is unknown whether serial testing of MRD in patients who are MRD negative after the completion of therapy may detect relapses at earlier, more curable stage. The concept of molecular relapse has been well established in acute promyelocytic leukemia.  In a prospective study in adult population, MRD was detected in 77% of patients before clinical relapse. MRD was consistently negative in 6% of patients despite clinical relapse.  However, early detection of molecular relapse and starting therapy change the outlook is still investigational.
| Minimal Residual Disease-Based Response Criteria|| |
It is essential that clinicians and researchers use uniform terminology to report individual patient results and allow for the comparison of trial outcomes. European study group , on adult and cALL has proposed the definitions for assessing response based on MRD status. Complete MRD response: No MRD is detected with the assessment that complies with a set of minimal technical requirements for the method used. MRD persistence: Presence of a continuously quantifiable MRD positivity measurable at least two-time points with at least one relevant treatment element in between. MRD reappearance: Conversion from MRD negativity to quantifiable MRD positivity, ideally with confirmation from a second sample before a change in treatment. 
| Technical aspects of minimal residual disease assessment|| |
Method for Minimal Residual Disease Assessment
Different methods for MRD assessment are suggested; each method has different limits of detection and standardization. There are three types of techniques which are widely available and standardized to a great extent for MRD assessment. (i) Multicolor flow cytometry (FCM), (ii) reverse transcription-PCR of amplification of different fusion genes transcripts and (iii) real-time quantitative-PCR (RQ-PCR) of B-cell receptor gene immunoglobulin (Ig)/T-cell receptor (TCR) gene. 
Multicolor FCM is an immunologic method which is based on detection of leukemia-associated phenotypes. Aberrant immunophenotypes are detected in majority of cALL and that allows detection of MRD via FCM. Various markers that can be used to monitor MRD in B lineage cALL are broadly categorized into (a) backbone markers such as CD10, CD19, and CD34. These markers denote immature B-lymphoid cells but cannot distinguish between normal and leukemic cells. (b) First and second generation markers such as CD45, CD38, CD72, CD13, CD33 and CD86, CD58, CD123, CD73, CD44, CD72, CD24, CD123, CD200, CD79, respectively. These markers can be used to define leukemia-associated immunophenotypes. , To perform multicolor FCM, it is helpful to know the baseline antigen expression of the leukemic cells. In postinduction or in remission marrow these residual leukemic cells are detected based on their antigen characteristics.  PCR-based methods detect patient specific Ig/TCR gene rearrangements and gene fusion transcript. Recurrent cytogenetic abnormalities suitable for routine MRD studies in clinical samples are present in approximately 40% of cALL.  Certain gene fusion transcripts are tumor-specific and it remains stable during the disease course. They can be a good target for PCR. Common gene fusions frequently used for MRD assessment are BCR-ABL1, MLL-AFF1, TCF3-PBX1, and ETV6-RUNX1 resulting in the expression of aberrant mRNA transcripts. ,, Patient-specific Ig/TCR rearrangements are highly specific, but the methods for detection is time-consuming, laborious and requires >3 weeks.
Choice of Method
Which method to use to measure MRD is largely determined by resources, expertise and how soon the results need to be available, as PCR-based detection will take 2-3 weeks as oppose few hours with FCM-based method. Most international studies have used PCR-based methods. PCR-based methods have longer and larger experience. Various studies have tried to compare the results of multicolor FCM-based versus PCR-based assessment of MRD. There is good concordance to both these methods ranging from 85% to 97%. ,,,,,, Certain studies have also confirmed the fact that when it comes to measuring MRD <0.001% PCR-based methods are more superior.  A good correlation between RQ-PCR and FCM was established in a few single institution studies, although discrepant results occurred in individual cases. , PCR-based detection of antigen rearrangement takes at least of 2-3 weeks; so treatment protocol which requires MRD status for early response to therapy, FCM is preferred. FCM is relatively faster, less expensive and easily available for day to day clinical use. Diagnostic FCM is widely available in most of the centers and so people are well trained. In resource-limited country like ours, by implementing standardization of setting up instruments, staining protocols and data analysis we can make FCM-based MRD detection more accessible.
It is important that residual disease is studied in one tissue type, either BM or PB. Studies have shown that levels of MRD are higher in BM than PB, at least in AML and B-lineage ALL. , This is not the case in T-lineage ALL where MRD levels in PB are similar to those in BM.  BM mononuclear cells (MNCs) are preferred over PB samples for MRD assessment. At least 10 6 BM MNCs is required for analysis. It requires 2 ml of BM sample and or 10 ml of PB sample. 
Timing of Minimal Residual Disease Assessment
Assessment of MRD has been studied during various time periods during induction, end-induction and postconsolidation. MRD levels earlier in induction, e.g., days 8 and 15, at end induction time points and postconsolidation, e.g., week 12 after starting therapy have also been shown to have prognostic significance. ,,
Limitations of Methods
MRD assessment is a laboratory-based test and each test has its own limitations. These can be biological limitation or technical limitations. Biological limitation is the immunophenotypic or genetic markers identified at the time of diagnosis which may not be identical to those expressed by the "leukemia stem cells." Thus, recurrent disease may arise from occult leukemia cells that lack the markers assayed on their more populous progeny. Alternatively, some ALL cells with abnormal markers may persist for some time after treatment but lack the malignant stem cell's ability to recapitulate the disease.  There are few issues related to immunologic monitoring like recovering BM cells may sometimes be misinterpreted as abnormal cells, because normal precursor cells have common antigens expressed by leukemic blast cells in a given patient. MRD is assessed in posttherapy marrow and so leukemic immunologic fingerprints can change with therapy.  Technical limitations are due to problem while performing the tests, which could be operator dependent, methodology and technology dependent and it requires standardization and validation as well as expertise. To overcome technical limitation the operator should be well trained with expertise in MRD detection and techniques need to be well standardized as well as validated.
Most studies ,, use cutoff value of >0.01% in BM MNCs as MRD HR and <0.01% as MRD low-risk. There are recent studies have increased the bar to higher notch. ,
| Conclusion and unanswered questions|| |
Testing for MRD has become a common integral part of the management of cALL. MRD status at various time points during treatment has prognostic value and is incorporated into the risk stratification criteria. PCR-based method is more widely used but immunologic method is equally useful with high concordance rate. There are certain unanswered questions like; is it must that the leukemic clone be completely eliminated to achieve long-term survival? If so, what should be the cutoff level? Does serial monitoring needed for detection of early relapse? Does personalizing therapy change the outlook? 
Financial Support and Sponsorship
Conflicts of Interest
There are no conflicts of interest.
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