ACRT Journal

Introduction

The field of immunophenotypic and molecular diagnostics has dramatically changed with several advancements and developments especially in the last two decades, where it became a key player in the diagnosis and even treatment of different diseases. The depth and quality of remission were always important in assessing the response to treatment in cancer patients and were based on imaging and laboratory tests compared to the baseline at diagnosis [1]. Minimal residual disease (MRD) assessment has gained clinical importance in hematologic malignancies as a measure for therapy response and became a cornerstone in risk stratification and guidance for management of some types. The initial use of MRD was in chronic myeloid leukemia via quantification of BCR-ABL transcripts to identify the level of remission [2]. This was followed by the extensive use of MRD in acute myeloid leukemia to guide prognosis, therapeutic decisions, need for stem cell transplantation or the addition of targeted therapies to the treatment regimen [3].

Chronic lymphoid leukemia/small cell lymphoma (CLL/SLL) is the most common leukemia of the adults in western countries with small monomorphic mature B-lymphocytes involving the bone marrow, peripheral blood and secondary lymphoid tissues such as the lymph nodes and the spleen. CLL currently accounts for around 7% of all non-Hodgkin’s lymphomas [4]. The MRD approach in chronic lymphoid leukemia (CLL) is different, where it was a debatable topic over years until the emergence of effective targeted therapies due to enhanced understanding of the pathophysiology and molecular biology of this disease [5]. The standard staging methods CLL were initially based on cytology and clinical assessment, making them less accurate than the current MRD, in determining the response to treatment and the depth of complete remission (CR). Given what is mentioned formerly, the need for an accurate definition of CR and the depth of remission based on MRD, in the era of precision and personalized medicine, has become the focus of clinical trials in CLL to guide treatment options in clinical practice [6].

The treatment of CLL had dramatically changed with the use of targeted therapies and chemoimmunotherapy leading to more prominent MRD negative disease. The importance of MRD status was highlighted in a number of clinical trials that assessed the relationship between MRD status and progression free survival (PFS) after treatment showing a longer PFS in patients with undetectable MRD disease even if they achieved only partial response than that in patients with complete response but detectable MRD disease [7].

The first method to be used for accurate detection of MRD in CLL was the flow cytometry that is dependent on the leukemiaassociated immunophenotype of the cells. The abilities of flow cytometry measurement of MRD in CLL were developed with time reaching very low thresholds of detection [3]. The molecular testing was then added to the tools of MRD assessment with polymerase chain reaction (PCR) and next generation sequencing (NGS) that lowered the threshold of detection and became technically validated assays [8].

The present review aims to provide an overview about the current techniques for measurement of MRD in CLL, their history and development, together with their role in clinical practice through guiding personalized treatment for patients.

MRD Techniques

Flow Cytometry

The use of flow cytometry as an approach to detect MRD goes back to 2007 in which an automated phenotyping of cells is performed through fluorescently labelled antibodies that target specific antigens on cell surface [4]. Compared to MRD by cytology that was limited to detect the presence of less than one CLL cell in a maximum of 100 leukocytes, flow cytometry was able to detect MRD of less than one CLL cell per 10000 cells in a sample of one to two million cells known as <10-4 or MRD4, which was developed later and became an accurate detection of one CLL cell per 100000 termed as <10-5 or MRD5 [1, 9]. Performing a flow cytometry requires the preparation of the sample through whole blood lysis with or without fixative agents before the addition of the fluorescent antibodies resulting in quantitative examination of the cells [10]. The initial standardized testing was dependent on four-colors set of antibodies and with evolution of flow cytometry, these sets reached six to eight colors [10]. Specific panels of fluorescent antibodies were established for the identification of CLL phenotype by flow cytometry for diagnosis and to check for MRD. These panels include different targeted cell surface antigens such as CD5, CD19, CD20, CD22, CD23, CD43, CD45, CD79b, CD81, CD200, and monoclonal surface immunoglobulin. The characteristic immunophenotype for CLL shows a positive expression of CD5, CD19, CD23, CD43, CD200 while a dim expression of CD20, CD22, CD45, CD81, monoclonal surface immunoglobulins and dim to negative expression of CD79b [4]. The flow cytometry approach to detect MRD5 in CLL with a standardized six-markers panel was validated in 2016 by the European Research Initiative on CLL and included the following core panel: CD19, CD20, CD5, CD43, CD79b and CD81 with a one-tube method that showed confirmed reliability [9]. Several studies showed that the novel combination of the NK-cell receptor and tumor specific antigen, CD160 with the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR1) has enhanced the sensitivity for MRD detection in CLL by flow cytometry [11]. CD160-ROR1FCA is being used in a single tube assay with a highly sensitive results, rapid application and simple gating strategy [12]. In addition, the enhanced ability of the flow to read more events in shorter period led to the reduction of the time and sample amount needed to perform a sensitive test that ca quantitatively detect residual disease in the 0.001–0.01% range [9]. The only exceptions for low sensitivity tests in CLL MRD using the flow cytometry are CLL patients with atypical phenotypes that are frequently uncommon and may lack CD5 and CD23 markers or normally express CD20, CD22 and CD79b [13-15]. It is essential in these cases to always compare to the pretreatment characteristic phenotype detected by flow cytometry at diagnosis [16]. Flow cytometry remains the most commonly used technique for diagnostic and MRD assessments due to its vast availability in laboratories, detections on peripheral blood samples, uncomplicated processing protocols and software with an acceptable cost. However, flow cytometry is still facing limitations such as the need for fresh blood to be processed within 48 hours

and the sensitivity of MDR5 only [17]. Efforts and research are currently in process to develop flow cytometry machines into next generation ones, aiming towards recording millions of events per sample and reaching the sensitivity of MDR6 [18].

(Figure 1) summarizes the steps of MRD detection by flow cytometry in conventional assay and single tube assay.

Figure 1: MRD detection using the conventional assay or the single tube assay

Polymerase Chain Reaction (PCR)

Detection by PCR and real time PCR is based on identification of specific DNA sequences to be targeted with labeled probes and amplified through fluorescent intercalation, hydrolysis or hybridization leading to quantitative results via measuring the increased fluorescence during the elongation of DNA strands [19]. This technique, specifically real time PCR, was used initially in quantification of viral load in infections such as hepatitis and human immunodeficiency virus, then its use was extended to measure MRD in some hematologic malignancies as BCR-ABL in chronic myeloid leukemia and Philadelphia positive acute lymphoid leukemia [20, 21]. In CLL unique DNA sequences were identified on the immunoglobulin gene (Ig) and specific primers were generated to target Ig heavy chain, Ig Kappa and Ig Lambda. These were followed by targeting the variable (V), diversity (D) and joining (J) segments of the Ig what is known as the VDJ fingerprint [22]. Currently, the MRD detection by PCR depends on the production of patient specific primers. This requires the sequencing of IGHV gene at diagnosis and the generation of allele specific oligonucleotide probes to target the IGHV gene and quantify the mutation [19]. Unlike the low sensitivity of consensus PCR using prepared known primers, the sensitivity of real time PCR MRD is confirmed to be 10-5 with the result being quantitative [23]. The real time PCR permits the presentation of a relative gene expression through equation and calculations. The efficiency correction method calculates the relative expression ratio from the real-time PCR efficiencies and the threshold cycle (CT). The analysis is done using the sigmoidal curve fitting methods that fit the experimental data to an empirical equation and results in the prediction of the PCR efficiency and an estimate of the initial copy number of the amplicon. The lower the CT, the greater the amount of amplicon [24]. Additionally, droplet digital PCR is a highly sensitive and reproducible technique that is still mostly used for research purposes and was recently used for the detection of MRD in CLL. The most studied gene in CLL using the digital PCR was TP53 gene were specific exons that encode for the 17p deletion were identified and tested using specific probes. The droplet digital PCR for quantitation of TP53 deletions or point mutations might replace the FISH procedure with accurate results and shorter duration of time [25]. Despite its advantages that include quantitative sensitive results and unnecessity of fresh samples, where testing can be done on frozen samples, real time PCR for MRD is still not widely used due to its requirements such as prolonged time, the need for expertise, complicated procedure  and elevated cost [23]. Multiple clinical trials have been studying the increase of real time PCR sensitivity for MRD through the production of advanced targeted probes and the combination to other modalities as flow cytometry and genetic sequencing [9].

(Figure 2) summarizes the steps in Real-time PCR of IGH gene variable.

Figure 2: MRD detection of the IGH variable using Real-time PCR

Next Generation Sequencing (NGS)

Since the description of DNA structure in 1953, genetic sequencing started to be introduced and incorporated in clinical research with the use of electrophoresis followed by radioactive and fluorescent techniques [26]. Automated sequencing was established in 1990s as an efficient tool for genetic mutations identification in various diseases. Moreover, next generation sequencing, known as secondgeneration sequencing or high-throughput sequencing (HTS) was introduced in 2005 for clinical use with the ability to perform millions to billions of reads in a single run [27]. NGS has gained in the last decade an important role in accurate identification of tumor burden and MRD in different hematologic malignancies. Similarly, NGS use in CLL showed that some patients with undetectable MRD by flow cytometry and PCR at a sensitivity of MRD4, which is required by guidelines, have positive MRD by NGS at a deeper sensitivity [28]. This finding was considered as a potential explanation for the delayed relapse in patients with labeled MRD negative disease especially those with highrisk features as unmutated IGHV CLL [29]. In contrast to PCR, NGS has more sensitivity and does not require identification of primers upfront or the use of specific primers for each patient [30].

New NGS assays in CLL can reliably detect and quantify MRD beyond the level of MRD 10−5 and up to MRD 10−6 especially those IGHV based assays developed for the detection of MRD [30]. NGS depends mainly on an adequate quantity of DNA and can provide both quantitative and qualitative data that can be compared before and after treatment [31]. Recently, there is a clear evidence for the survival benefit of negative MRD in CLL patients after first line treatment over patients with positive MRD post chemoimmunotherapy or venetoclax-rituximab treatment. NGS is currently considered the most reliable tool to identify the most accurate MRD status in CLL [32]. NGS has the capability to identify clonal diversity and intraclonal dynamics through revealing the baseline rearrangements present at diagnosis, to which the results are compared post treatment to check the presence of these clones [33]. NGS role in prognostication and treatment guidance is progressing very fast. However, it is still limited to specific centers due to its high cost and the need for complicated software tools and expertise for the analysis and interpretation of the results. NGS for MRD in CLL is mainly restricted to clinical trials and needs more standardization to become available for clinical practice [34].

(Figure 3) summarizes the MRD modalities, the sensitivity and the advantages and disadvantages of each modality.

Figure 3: MRD detection modalities with advantages and disadvantages of each modality

PCR: polymerase chain reaction, NGS: next generation sequencing

Precision Medicine in CLL

As in the diagnostic field of CLL, the therapeutic field in CLL has developed in parallel in the last two decades leading to improvement in the outcome of CLL patients. The spectrum of studied and applied treatments ranged from chemoimmunotherapy to targeted therapy to immunomodulatory agents, until reaching combinations of these agents [35]. Treatment of CLL started initially with chemotherapy, mainly chlorambucil, then it was based combinations of chemotherapeutic agents until the addition of immunotherapy with the introduction of rituximab carrying high toxicity. The treatment paradigm was completely shifted in 2014 with the emergence of the novel drugs the Bruton Tyrosine Kinase inhibitors (BTKi), which switched the pattern from chemoimmunotherapy into the era of targeted therapies that are effective with less secondary adverse events [36]. Ibrutinib is a first generation BTKi that was approved initially by FDA in 2014 for refractory/relapsed (R/R) CLL and and for patients with CLL harboring the deletion of chromosome 17p (del17p) based on the result of the phase 3 RESONATE trial [37]. Ibrutinib then received the approval to be used in the frontline in the treatment of CLL after the results of phase 3 RESONATE 2 trial that compared it to chlorambucil [38]. Moreover, ibrutinib showed superiority in PFS and OS when compared to fludarabine, cyclophosphamide, and rituximab (FCR) regimen in phase 3 study E1912 [39]. Furthermore, targeting Bcl2 via the use of the Bcl2 inhibitor venetoclax, was another milestone in the era of targeted therapies. All these advancements led to survival benefit in CLL, which in turn incorporated the use of MRD to accurately assess the depth of response to treatment [40]. Moreover, the current decision for first line treatment in CLL depends on the physical fitness of the patients, molecular features of the disease, IGHV status and FISH cytogenetics including 17p deletion, and TP53 mutational status before starting treatment [34]. TP53 alterations that include both TP53 mutation and 17p deletion, indicates chemo-refractoriness and requires the use of biologics such as Bcl2 inhibitor to overcome the refractoriness [41]. In addition, the presence of  IGVH mutations in the absence of TP53 disruption indicates a durable response to chemoimmunotherapy making the survival close to general population [42].