Introduction

Today, an important feature of treatment management especially in acute lymphoblastic leukemia (ALL) is proportionality of treatment rate to the risk of disease relapse, in order to adjust the intensity of treatment and avoid the risk of relapse and treatment-associated cytotoxicity in such patients. To this end, we need to pursue the methods and approaches to improve efficacy of treatment and avoid the risk of relapse [1]. To achieve this goal, the topic of minimal residual disease (MRD assay and measurement of residual cancer cells not detectable by morphological methods) has been addressed, which is independent of clinical predictors.

In view of recent progress in prediction of treatment and its outcome based on MRD assay results, the need for studies and accurate integrated presentation of MRD data has been raised to improve the treatment rate and quality. Multiparameter flow cytometric immunophenotyping (FCM) and quantitative polymerase chain reaction (PCR) are the most common MRD assay methods in ALL with their own advantages and disadvantages [2–4]. In this review article, we first explain the importance of MRD evaluation in ALL. Then, after a brief review of each method, the advantages and disadvantages, gold markers, standardization method and optimum time for MRD assay, the pitfalls of this method will be reviewed, and the best method will be discussed. Eventually, we will take a look at recent developments in the field of MRD assay methods.

Why should we detect MRD in ALL?

Nowadays, with progress in treatment of childhood ALL, over 80 % of children with ALL can be cured with a combination of modern chemotherapy and bone marrow (BM) transplantation (in case of high-risk patients) [5]. However, 20 % of ALL children and 30 % of patients undergoing allo-HSCT experience disease relapse after treatment. The relapse rate can reach up to 60 % in high-risk patients [6]. Furthermore, despite increased overall survival, ALL is the second cause of cancer-related mortality in US children [7]. Moreover, prediction of relapse by current indicators such as the number of blasts at diagnosis and the presence of specific chromosomal abnormalities [e.g., t(4,11) and t(9,22)] is not favorably adequate [8].

In recent years, MRD assay in the early stages of treatment provides more constructive prognostic information than classical prognostic factors. Moreover, maximum decline in MRD before transplantation is currently considered a prerequisite for achievement of allo-HSCT, and patients with a high pre-transplant level of MRD are at a high-risk of relapse [9, 10]. Therefore, MRD assay before allo-HSCT is an important parameter determining the appropriate time and outcome of transplant, which can contribute to post-transplant clinical management [10, 11].

Consequently, MRD is a valuable tool for faster detection of patients who are more likely to relapse. Moreover, the clinical impact of MRD before HSCT and during treatment has made it a valuable parameter for association with clinical markers [12]. However, a number of fundamental problems should be solved before routine use of MRD in clinical decisions, which will be explained below.

Methodological approaches

Multiparametric flow cytometry (FCM) immunophenotyping

FCM is able to detect leukemia-associated immunophenotypic patterns (LAIPs). LAIPs can be detected in over 95 % of ALL patients by FCM [13, 14]. In this method, a combination of fluorochrome-labeled monoclonal antibodies against specific cell surface markers is used to identify the immunophenotypic pattern of the cell surface. Application of this method in MRD assay is increasingly developing due to introduction of new specific markers.

Polymerase chain reaction

Since each lymphocyte has a specific V-D-J set encoding various domains of TCR and Ig, the junctional region of clonal Ig and TCR gene rearrangements provide fingerprint-like sequences for each lymphocyte. As ALL is usually derived from a lymphoid progenitor, all the leukemic cells derived from a lymphoid precursor are detected by the same rearrangement in Ig and TCR genes [15, 16], which can be identified using the primers standardized in collaboration with BIOMED-1 and -2 framework in over 95 % of patients. This method can detect most common rearrangements of Ig/TCR including IGH, IGK, TCRG, TCRD and SIL-TAL as well as light chain IGK (VK-JK) and IG lambda (IGL, VL-JL) rearrangements [17].

MRD can be assessed in lymphoblastic leukemia through the expression of chromosomal abnormalities and transcripts such as MLL-MFFI, TCF3-PBX1, ETV6-RUNX1, E2A-PBX and BCR-ABL as well as other fusion genes [18, 19]. Each of these methods has its own limitations and capabilities for MRD monitoring, which will be discussed below. Further, optimization of the markers used in each method can result in increased application of the method.

Why and how do we standardize each method for MRD detection?

Given the importance of MRD, the entire process of PCR and FCM requires standardization. In fact, all the stages of PCR screening (including sequencing, primer design and results analysis) and flow cytometry (sample preparation and conditions as well as the choice of appropriate antibodies) require standardization in interlaboratory and international levels. ESG-MRD-ALL organization and the European Commission have presented methods and information for standardization between laboratories at the international level so as to obtain MRD assay with the same quality in different laboratories [20].

The following principles should be observed in PCR-based MRD:

  1. 1.

    The quantity of sample DNA should be sufficient.

  2. 2.

    At least two targets for Ig/TCR-PCR should be available.

  3. 3.

    Sensitivity of the selected targets should be at least 10−4.

  4. 4.

    An authenticated method should be used to assess MRD results [4].

  5. 5.

    Specific amplification should be effectively separated from any unspecific amplification (which is possible by using DNA of healthy subjects as control) [20].

  6. 6.

    Positive control should be used to confirm or rule out false positive results [4].

The following points should be considered in standardization of FCM-based MRD:

  1. 1.

    Standardization of sample preparation and staining.

  2. 2.

    Monoclonal antibodies should be coordinately used as a combination of 4 or 6 colors between the laboratories.

  3. 3.

    The use of different flow cytometry brands and statistical analysis software does not seem to have a negative impact on interpretation of results.

  4. 4.

    A few immunophenotyping compounds should be used to distinguish leukemic cells from healthy immature cells [21].

What are the golden markers for MRD detection by PCR and FCM?

Considering the fact that sensitivity and specificity of a method can significantly affect the interpretation of its results, different methods are used to determine MRD during patient monitoring. The main variable in determining sensitivity and specificity of a test is the marker used in the experiment, which should have high sensitivity, specificity and stability during the course of disease to avoid false negative results. The results of Ig/TCR-PCR rearrangements in patients with B-lineage ALL have indicated that almost all these patients have a rearranged immunoglobulin heavy chain (IGH) [22]. In most cases, rearrangements of IGH are in the form of complete recombination, with incomplete rearrangement of DH-JH detectable in 20 % of patients [23]. It is noteworthy that incomplete rearrangement of IGH is most common in infants with ALL [24]. In fact, IGH gene loci is the most sensitive marker of B-lineage ALL. However, due to ongoing and secondary rearrangement processes, it is prone to oligoclonality (presence of more than one clone of leukemic cells) in 30–40 % of cases [25]. However, if IGH is used in combination with the highly stable “endpoint rearrangements” (that is IGK-Kde rearrangements), it will be a good marker for monitoring MRD in these patients [26]. There is 60 % prevalence of Igκ rearrangements in ALL patients [27]. Incomplete TCRD rearrangements (especially Vδ2-Dδ3 and Dδ2-Dδ3) are also good markers for MRD monitoring due to good stability and high sensitivity [28], but their prevalence is low [29]. Rearrangements of TCRG are not good candidates in these patients due to low sensitivity [28]. As a result, IGH rearrangements are the most appropriate candidates for MRD monitoring in B-ALL. In patients with T-ALL, rearrangements of TCRG (especially VH3) [29] and IGH, with a respective prevalence of 84 [30] and 94 % [31], are the most common targets in these patients. However, TCRB rearrangements are less common (54 %) and are often used as a supplement for MRD assay in these patients, being added to screening protocols through BIOMED-2 multiplex TCRB PCR assay guidelines [32]. In general, the use of TCRA and TCRB as an independent marker has been limited due to the large number of gene segments [29].

In FCM, combined and optimized panels have been introduced in various studies. In more than 90 % of T-ALL patients, the blasts normally express nucleotide TdT/cytoplasmic CD3 (a combined marker limited to the thymus), but B-ALL blasts normally express the phenotypes of B cell precursors in BM [33]. In an extensive study by Borowitz et al. [34] on 2,143 children with B-ALL, the two panels of CD20/CD10/CD45/CD19 and CD19/CD34/CD45/CD9 were introduced as optimal for MRD assay in these patients. Other studies have also suggested the following common panels for MRD assay in B-ALL patients: CD20/CD10/CD19/CD34, CD58/CD10/CD19/CD34, CD10/CD34/CD19/CD45, CD10/CD11a/CD19/CD25 and CD10 ± CD20/CD38/CD19/CD34 [35–42].

Three combinations were introduced for T-ALL: CD99/CD7/CD5/surface(s), CD3, CD99/CD71/sCD3/c CD3 and TdT/CD7/s CD3/c CD3. In general, TdT/c CD3 (with a prevalence of 90–95 %) is the most common marker of T-ALL [43].

When should we monitor MRD to achieve a better diagnosis and treatment?

Children with undetectable MRD always have excellent prognosis at the end of induction, are good candidates for treatment attenuation and should not be subject to severe treatments (especially BM transplantation) [36]. Overall, clinical impact of MRD is highly dependent upon the therapeutic time point in which it is studied. This means that the initial time point is very important in terms of prognosis and increases the application of MRD in clinical stages. The children oncology group suggested MRD assay 8 days after induction in peripheral blood by FCM as an early time point method for identifying low-risk patients by studying 2,143 children with B-ALL. This period has been suggested to be 28–35 days for BM samples [44]. MRD assay at day 29 in BM by this method was suggested as the most important prognostic time point in multivariate analyses [34]. MRD assay by FCM in BM is recommended on day 15 for estimation of relapse [35]. Recently, MRD assay in BM has been enabled on day 15 in 94 % of ALL patients using 6-color flow cytometry. Moreover, MRD assay is recommended on day 78 to correct the risk estimate [36, 45]. In a study by Basso et al., day 33 was found to be the optimal time to perform PCR on BM sample [46]. Some studies have specified the patients with rapid decrease in the content of leukemic cells on day 19 (by PCR on BM sample) as candidates to reduce the toxicity of treatment [47]. On the other hand, the importance of MRD timing also depends on the type of leukemia. For example, MRD assay at end induction is valuable for B-ALL, but has a greater value for T-ALL in end consolidation, detecting extramedullary relapse in addition to general relapse [29] (Table 1).

Table 1 Golden time and sample based on clinical value in B- and T-ALL with PCR and FCM protocol
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What is the optimal sample for each method?

The choice of optimal sample for MRD assay depends on type of leukemia, patients’ conditions, the method used and the time of the survey. In patients with T-ALL, MRD assay is easier to perform on peripheral blood than on BM, enabling more accurate monitoring. On the contrary, MRD assay in patients with B-lineage ALL indicates that despite the presence of leukemic cells in BM, there may be no trace of leukemia in PB [51]. As a result, the sample preferred in patients with B-ALL and T-ALL is BM and PB, respectively. Furthermore, another feature in FCM is freshness of the samples, and delayed samples can adversely affect the results and cause false negative results, while this is not the case for PCR. In addition, MRD assay can be done by FCM on PB samples containing a few blasts for patients whose BM sample is not available [52, 53].

Can MRD be used as a surrogate marker for risk classification in clinical study?

In the past, risk assessment in ALL patients was based on clinical characteristics including age and WBC count as well as biological characteristics of leukemic cells such as immunophenotyping, karyotype and molecular genetic methods [54]. Over the past 15 years, MRD assay in BM of children with ALL has enabled identification of patients with different prognoses [55] and has become one of the most potent predictors of outcome in childhood ALL. The 4-year study by MRD-I-BFM 91 ALL showed that MRD-based classification of ALL patients is the most important criterion to classify approximately 80 % of patients in the multicenter international level [56]. The study conducted by this center as well that of Conter et al. on, respectively, 129 and 3,148 BM samples from patients with ALL in which PCR was done at two time points (day 33 and 78), and MRD completely replaced the common risk factors. In these studies, patients with negative MRD on day 33 were placed in standard risk group, and MRD-negative patients on day 78 were placed in high-risk group [48, 57]. Another study has shown that using MRD results (end induction, i.e., 5 weeks after TP1 diagnosis, and before consolidation treatment, i.e., 3 months after diagnosis of TP2), the patients can be divided into groups with different relapse risks. Accordingly, MRD-negative patients in both time points (with a sensitivity of at least 10−4) were placed in low-risk group, and MRD-positive patients at both time points (≥10−3) were placed in the high-risk group [57]. Godoy et al. [44] also studied Ig/TCR on 59 BM samples from ALL patients and found that a positive MRD result (with a sensitivity of 10−4) on day 28–35 in high-risk patients had a prevalence of 78 %. However, in another study, a positive MRD result in BM sample (with a sensitivity of 10−2) on day 15 of induction was classified in the high-risk group [58]. Flow cytometry-based classification is such that each patient having more than 10 % leukemic blasts on day 15 after induction is placed in high-risk group, and any B-ALL patient with MRD >10−3, which is still positive on day 78, is classified in the high-risk group [59]. In addition, the presence of MRD by FCM in peripheral blood on day eight and in BM on day 29 is associated with an adverse outcome [34].

What are the important pitfalls in MRD detection?

The change in applied targets due to instability of some rearrangements (clonal evolution) [60] and selection and expansion of subclones not identified upon diagnosis (oligoclonality) are important pitfalls of PCR. This problem may occur between diagnosis and relapse and may cause false negative results [61]. To overcome these pitfalls, detailed analysis of all molecular markers upon diagnosis and selecting two specific and sensitive targets of Ig/TCR rearrangement among them is recommended for each patient [62]. Thus, the origin of the dominant clone can be easily identified, and false negative results are avoided. After determining PCR targets upon diagnosis, clonality should be confirmed by homo-/heteroduplex analyses or by gene scanning to confirm their malignant origin and rule out contamination of normal cells with Ig/TCR gene rearrangements [63].

Changing expression of antigen during treatment, which is often the result of BM regeneration during this stage, is an important pitfall of FCM, which can lead to false positive results in 50–60 % of B-ALL cases [53, 64], and often occurs 78 days after BFM-type induction [36, 65]. Although checking the actual date of sampling is helpful in this regard, delayed treatment may cause untimely the expression of markers. In these cases, the use of two specific immunophenotypic compounds is helpful to reliably distinguish normal immature cells from malignant cells [2, 66]. In addition, 11b has been recently proposed as a good candidate marker to solve this problem, so that its expression is increased in the early stages of childhood ALL and remains high during treatment induction and consolidation (day 78). In more than 30 % of MRD-positive cases, expression of 11b is higher in blast cells than in memory cells, improving separation of remaining leukemic cells from rearranging BM cells [67].

CD49f is another good candidate to overcome this pitfall in B-ALL, such that detection of MRD using this marker is consistent with results of a standard panel of antibodies (with a sensitivity of 0.02 %), and its expression is indistinguishable in normal BM samples [68]. In addition, decreased expression of CD81 has been considered a determining factor for MRD, and fourfold reduction of it has been reported as an indicator of relapse after treatment induction with 87 and 99 % sensitivity and specificity to detect MRD in B-ALL, respectively [69]. To avoid false negative results in FCM-based studies, assessment of two specific immunophenotypes is recommended. Recently, the sensitivity of this method has been improved to some extent (≤10−6) with the advent of 6-color flow cytometry.

What is the optimal method for MRD detection?

Since MRD assay is very important especially for predicting disease prognosis, we should seek the most appropriate method to monitor it. This method should be specific for patient characteristics or leukemia type and should have such parameters as satisfactory sensitivity (at least 10−4, i.e., one malignant cell per 10,000 normal cells), extensive use, easy implementation and standardization as well as internal and between-laboratory reproducibility. In the largest study conducted to date, Neil et al. suggested 97 % consistency between the results of both methods by examining 1,300 PB and BM samples by PCR and FCM by regarding 0.001 % sensitivity [70]. However, each method has its own advantages and disadvantages (Table 2).

Although at least one cluster containing at least ten leukemic cells is required for a positive MRD result, positive result of PCR needs a single copy of target sequence in a background of normal cells [56]. As a result, both methods may be similar in detection of MRD in 10−4 level. In fact, the difference between PCR and FCM is observed more frequently in samples with low levels of MRD (lower than 10−4), but high-risk patients are not affected, and both methods give the same results [48]. However, MRD monitoring by PCR sometimes indicates a positive result in PCR and a negative result with FCM. This inconsistency can be due to the fact that during PCR, dead cells are evaluated, but FCM cannot detect these cells. In general, we face less false negative results in PCR, and it is a sensitive method after MRD treatment to classify childhood ALL especially in the first 3 months after treatment [77]. However, among MRD assay methods, FCM is the one most affected with BM remission conditions [78].

The choice of method is also dependent upon patients’ conditions. For example, MRD assay by PCR is not suitable for CD10-negative B-ALL and ALL with t(4,11) (both of which show pro-B-ALL phenotype), as a PCR target with high prevalence is not found in these patients. However, FCM interestingly enables easy detection of MRD in all these patients. Therefore, FCM is a proper substitute for Ig/TCR-PCR in this group of ALL patients [79]. Moreover, despite high consistency between FCM and PCR results, FCM is used more frequently in some subsets of ALL, including childhood ALL and immature B- and T-ALL phenotypes [79]. In recurrent BFM-based protocols, FMC and PCR cannot be easily substituted in single time point, and percent consistency between them is mainly dependent on the time of their use [80] (Table 1). On the other hand, eliminating the false negative results using Ig/TCR rearrangement methods due to clonal expansion is inevitable [81], and only 40 % of ALL cases have specific chromosomal abnormalities detectable by PCR [82]. As a result, FCM can be used as a complementary method for PCR. Most studies emphasize the role of these two methods to optimize risk stratification in clinical procedures for reduction of treatment failure risk in children with ALL.

Table 2 Advantages and disadvantages of PCR versus FCM
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Future perspectives

MRD is one of the most interesting research topics in which sophisticated basic research is transmitted into advanced methods of laboratory diagnosis. Today, given the crucial role of Ig/TCR rearrangements in MRD assay, advanced methods including allele-specific oligonucleotides (ASOs), ASF-sequencing PCR-gene scan sequencing and high-throughput sequencing are used to identify these rearrangements. In ASO, the primers are designed in such a way that they complement the unique short binding regions of VJ (IGK & TCRG) or VDJ (IGH) rearrangements (as most common targets in ALL) and are used in combination with common primers and complementary probes to join J segments to quantitatively determine MRD by RQ-PCR. Sensitivity of this method is reproducibly ≤10−4 using a single primer and is increased five times by using the second primer [56, 83].

Generating sequencing technology can now give valuable information about clonality of ALL, changing our viewpoint to MRD. Recently, the use of PCR-gene scan sequencing analysis in Ig/TCR gene rearrangement has improved relapse detection on BM samples from B-ALL patients [84]. Next generation sequencing based on high-throughput sequencing globally amplifies and detects antigen receptors, and considers all clonal gene rearrangements in diagnosis [85, 86].

ASF sequencing is a highly sensitive tumor-specific sequencing-based method, with a sensitivity level enough to identify patients with at least one detectable genomic amplification. In PCR-based methods, it is not known whether the signal arises from specific amplification of DNA from leukemic cells or non-specific reproduction of DNA from normal cells, but ASF sequencing is a highly tumor-specific method and can provide useful information for MRD assay.

The sensitivity and reliability of flow cytometry is increasing by application of FCM 6-color. Due to the fact that this method is cheaper than other methods, with improvements that have occurred in this method [87], it can become a rapid and efficient method, which can be a good candidate to evaluate MRD according to patients’ conditions [75].

By far, MRD has been highly reflective of treatment response in vivo, providing more information to doctors to control clinical symptoms in the patients. As a result, it is fair to assume that such individualized medicine approaches will ultimately lead to improved outcome of patients with ALL.