Multiparameter flow cytometry can be used to

Minimal residual disease testing in Acute Leukemia Anjum Hassan MD Assistant Professor of Pathology and Immunology, Director FISH laboratory in Anatomic Pathology, Washington University in St Louis, School of Medicine, St Louis Missouri, USA, Email: ahassan@path.wustl.edu Acute leukemia (AL) is a complex disease with considerable phenotypic and morphologic heterogeneity. The phenotype is often assessed by multiparameter flow cytometry ideally on aspirated bone marrow. These leukemia associated phenotypes (LAPs) are crucial for disease monitoring. In addition, there are more than 100 recurring cytogenetic abnormalities encountered in acute myeloid leukemia (AML) and similarly, multiple acquired genetic abnormalities are responsible for aberrant proliferation and differentiation arrest seen in acute lymphoblastic leukemia (ALL). WHO requires a combination of both immunophenotypic and cytogenetic studies to definitively classify AL. Outgrowth of minimal residual disease (MRD) in AL is responsible for the occurrence of relapses. These are cells present in the bone marrow after treatment and these can be monitored by molecular, biologic and or immunophenotypic detection methods. Defining MRD is thought to be crucial in defining patient-tailored post remission therapy which might reduce the risk of relapse or diminish morbidity and mortality in AL. This may also allow detection of impending relapses for early intervention. Immunophenotyping by flow cytometry (FC) is a cost effective and fast tool for detection of MRD. Essentially all cases of AL possess a unique phenotype with multiple aberrancies which tend to remain stable during the course of relapses. By defining these LAPs on malignant cells at diagnosis, flow cytometry can be highly reliable in MRD parameter assessment at different stages AL, consequently predicting survival and forthcoming relapses. Multiparameter flow cytometry can be used to detect MRD in acute leukemia because the malignant cells have phenotypic differences that are reproducible and not present in normal cells of similar lineage. In acute leukemia there can be 10 12 malignant cells at the time of diagnosis. 1 Complete remission (CR) is defined as less than 5% blasts in the bone marrow, however, several studies have shown the presence of up to 10 10 neoplastic cells in patients with CR and from the point of complete remission to overt clinical relapse with 5% or more blasts. There are no well established guidelines to monitor the level of residual blasts if the patients are in clinical remission. Thus, patients with 1010 blasts are essentially treated with regimens not too different from those with much lower levels of blasts. 2 Several studies have already established the direct relationship between tumor load and prognostic outcome therefore, it is logical to assume that if more sensitive methods of detection of tumor cells are made available, this will increase the chances of early intervention before the clinically evident relapse and possibly reduce disease recurrence and reduce the likelihood of developing drug- resistant mutants. 3-7 Later is dependent on the ability of blasts to undergo cell division and as these malignant cells emerge by mutations, the larger volume of these cells in a CR setting is likely to lead to more drug-resistant blasts. 8,9 This highlights the importance of detecting minimal residual disease and subsequent section will address the general approach for this testing in acute myeloid and acute lymphoblastic leukemia. Several methods have been employed for detection of MRD in AL with different sensitivities and advantages. For a comparison of these different strategies please see Table 1. This review will focus on immunological mechanisms of MRD testing specifically, multicolor flow cytometry (MFC). MRD testing in acute myeloid leukemia For AML, previously described aberrations include asynchronous antigen expression such as expression of CD34, an early marker, and CD15 a late marker; lineage infidelity such as expression of lymphoid markers on myeloid blasts; antigen over expression and absence of lineage specific antigens, normally expected in a particular leukemic blast (Table 2). These features specify a certain leukemia associated phenotype (LAP) which, ideally, should either be absent or only infrequently present on normal bone marrow or peripheral blood cells. 10-12 MFC can be em-ployed to study the LAPs. In AML, LAPs are not well defined due to inherent heterogeneity of this disease. However, high levels of sensitivity can be reached by using multiple antibodies and MFC. 13-15 The ideal assay for detec- 306

tion of small numbers of leukemic cells should fulfill the following criteria: a) applicable in most cases, b) specific for neoplastic cell type, c) sensitive, d) allows quantization of tumor burden for prognostic purposes. Al-Mawali et al demonstrated in their study of 54 AML samples that, using 5 color flow cytometry, LAPs could be identified in 94% of cases. The post induction MRD levels in that study influenced both relapse free survival and overall survival as independent prognostic variables. 16 Using five color flow cytometry, specific LAPs will allow detection of very low levels of blasts considering the criteria outlined above. However, compared to ALL, AML LAPs are more heterogeneous and co-existence of several blast populations with different LAPs can be observed. When different LAPs are present, those with the largest logarithmic difference to normal bone marrow cells should be chosen for MRD monitoring. This guarantees better specifity and sensitivity when compared to approaches using more general inclusion of all LAPs in to one assay. Usage of three, four, five and up to ten color flow cytometry strategies using a wide panel of antibodies (see Table 2) has improved the sensitivity of MRD in most AML cases to between 0.1% and 0.01% of all nucleated bone marrow cells. One large study evaluating the prognostic relevance of MRD load involved 126 AML patients, all in morphologic remission. They summarized their patients into four risk groups: 1) very low risk=<10-4 residual leukemic cells- no relapses. 2) Low risk=10-4 to 10-3 residual AML cells-14% relapse rate 3) intermediate risk= >10-3 to 10-2 residual blasts- 50% relapse rate 4) high risk=>10-2 blasts- 84% relapse rate. Thus, MFC allowed highly differentiated risk stratification among patients in morphologic remission. 17 The timing of MRD monitoring is a subject of ongoing debate. Various studies have used post-induction and post-consolidation MRD monitoring to establish their relative prognostic significance. In a study of 100 AML patients, a threshold of 3.5 10-4 leukemic cells was reportedly able to stratify poor and good risk groups both after induction and post consolidation. In this study, patients without detectable MRD post consolidation fared much better in terms of overall survival and relapse free survival (P<0.001) regardless of their MRD level post induction. Best treatment results were shown with post induction MRD load of <4.5 10-4 residual leukemic cells, however, relapse rates did not differ much in those with similar or higher levels of MRD. The impact on relapse rate was much stronger post consolidation with MRD cut off of greater than or equal to 3.5 10-4 varying from 77% to 17% (P<0.001). 18,19 In summary, MRD monitoring has proven to be highly reliable in predicting survival and forth coming relapses in AML. Monitoring of MRD cells by molecular, cytogenetic or immunophenotypic methods are of crucial value for defining individualized patient management. Based on MRD frequencies, patient-tailored post remission chemotherapy could be designed, which might reduce either the risk of relapse or, diminish morbidity and mortality by avoiding unnecessary intervention (Figure 2). In an event, when impending relapses are predictable, early therapeutic intervention may be less intense than otherwise required in an overt relapse. One, very important application of MRD testing could be to quantify leukemia cell contamination in autologous peripheral blood stem cell product in order to guide the decision to whether or not to purge, and, in the later case to establish the efficacy of leukemia cell eradication. 20 Table 1. Methods of detection of MRD in AL TECHNIQUE PROS CONS SENSITIVITY MORPHOLOGY FISH PCR FC >5% Blasts Fast ; no dividing cells required Limited to leukemias with available primers for common recurrent abnormalities Applicable in ~80% of cases; cost effective; accurate for normal and abnormal cell types; rapid turnaround (1-2 days) Low sensitivity; not suitable for subclinical disease Labor intensive; low level of sensitivity especially with low cell counts High likelihood of false-positive results, expensive; applicable in only a subset of AML and ALL Not as specific as PCR; phenotypic shifts post treatment or therapy; subpopulations of blasts 1% to 5% 0.3% to 5% 10-4 to 10-5 Comparison of morphology, Florescent-in-situ Hybridization (FISH), Polymerase chain reaction (PCR) and Flow cytometry (FC) in detection of MRD Adapted from A. Al-Mawali et al. Am J Clin Pathol 2009; 131: 16-26 10-4 307

MRD testing in acute lymphoblastic leukemia Childhood ALL is highly sensitive to chemotherapy. Nearly all patients receive complete remission following therapy and majority achieve complete cure. About a third of patients subsequently relapse and most of these ultimately die of their disease. Detection of minimal residual disease has been established as an independent prognostic variable of high clinical relevance in de-novo and relapsed cases of pediatric and adult ALL. 21 It is already incorporated as a valuable tool in most American and European protocols as a stratification tool. Leukemia lymphoblasts differ from physiologic counterparts in qualitative and quantitative antigen expression patterns. In addition, immature phenotypes outside the context of normal tissues can also serve as unique identifiers of blasts. Such LAPs are present in vast majority of ALL cases if a panel of 6 to 8 antibodies is employed in strategic combinations. This approach currently reaches sensitivities of 10-3 to 10-4. Use of MFC has greatly improved the sensitivity/ specificity of this assay and allowed for simultaneous determination of multiple phenotypic patterns in leukemic cells. The stability of LAP is of major interest for purposes of targeted follow up. Comparing the patterns of immunophenotypic aberrancy to those of normal hematogones, Chen Weina et al found stable and reliable phenotypic differences which were retained in 80% of cases at relapse. However, there is increasing evidence documenting immunophenotypic changes in ALL during the course of disease with 30% to 70% of cases commonly reporting loss of CD10, HLA-DR, TdT and CD20 in addition to gain and/ or loss of myeloid antigens. Hence initial phenotypes only serve as orientation and guidelines and should not be used for planning strict gating strategies in follow ups. 22,23 Increased numbers of hematogones may also cause problems in MRD assessments especially in pediatric ALL setting. Hematogones are found in small numbers in most marrow specimens; however, they can occur in large numbers in healthy children 308

and in some disease states such as patients with autoimmune and congenital cytopenias, neoplasms and AIDS. They also commonly share overlapping morphologic features with blasts (Figure 2). In a study of immunophenotypic analysis of hemtogones involving 662 consecutive bone marrow specimens, McKena R W et al showed that hematogones exhibited a complex spectrum of antigen expression that followed the normal evolution of B- cells and completely lacked aberrancies such as asynchronous co-expression of earliest and latest antigens such as concurrent CD34 and CD20, and aberrant over- or under-expression of antigens normally present at a particular B-cell stage (Figure 2). In contrast, lymphoblasts in 49 cases of precursor B- ALLs exhibited at least one and often multiple immunophenotypic aberrancies. 24,25 Several other studies have suggested reliable methods of differentiating hematogones from neoplastic cells. Rimza et al found an increase in number of more mature precursor cells relative to least mature (CD34+ and TdT+) in bone marrow samples rich in hematogones. They also reported heterogeneous expression of adhesion molecules such as CD44 and CD54 on hematogones. 25,26 One should be mindful that an increase in immature cells including left shifted hematogones can be seen in the setting of early bone marrow recovery 309

such as post chemotherapy or transplant 27,28 In these setting there is even greater potential for mistaking hematogones with neoplastic cells, however, the maturational sequence is the rule among hematogones even in these cases and aberrant expression patterns are not noted. Other methods of detecting MRD which are in current use include sequencing of rearranged TCR or Immunoglobulin genes in ALL and PCR based detection of gene fusions resulting from recurrent cytogenetic abnormalities. Assays based on PCR or flow cytometry can detect one ALL cells among 10,000 to 100,000 normal cells in clinical samples. In most cases, MRD positivity is defined as the presence of 0.01% or more ALL cells; the risk of relapse generally is proportional to the level of MRD especially in the post chemotherapy induction phase. MRD is currently being used in several clinical protocols of risk adapted therapy. There is, however, considerable variability in time points selected for MRD testing and in the levels of MRD used to define risk of relapse. Nevertheless, its utility for treatment stratification and definition varies with the different protocols. The current COG protocol for instance recommends MRD testing at the end of remission induction (day 29) and the cut off levels used to assign risk are 0.1% and 1%. 29 Summary FC based MRD diagnostics has the distinct advantage of instant availability with regards to testing for blasts and normal marrow constituents at various time points in the natural history of acute leukemias. FC is widely used as a useful adjunct to diagnose remission status, overt relapse and MRD. Recent technical advances in routine flow cytometry with three lasers and up to eight colors and the new developments in software for data analysis make it a very reliable tool for MRD assessment provided the limitations of sensitivity, and treatment induced phenotypic shifts and difficulties in discriminating ALL cells from hematogones are carefully considered and standardized. This can only be achieved if a high degree of standardization can be reached for technical aspects and data interpretation and quality assurance programs are established to ensure reliable results. Intensive networking and open collaboration between clinical and diagnostic research groups will be the key to high level standardization and quality control for further improvements in MRD diagnostics. 310

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