Diagnostic and integrated work-up for the management of Acute Lymphoblastic Leukemia

Diagnostic and integrated work-up for the management of Acute Lymphoblastic Leukemia

Author: Robin Foà and Antonella Vitale, Division of Hematology, University “La Sapienza”, Rome, Italy (March, 2007)

Acute lymphoblastic leukemia (ALL) represents a biologically and clinically heterogenous group of diseases characterized by the proliferation of immature hematopoietic cells. The diagnosis and classification of ALL is currently a multistep procedure based on morphology, immunophenotype, cytogenetics, molecular genetics, immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements, genomic profiling and relies on the simultaneous application of multiple techniques.


ALL has been defined by the presence of more than 30% lymphoblasts in the bone marrow (BM) or peripheral blood (PB) according to the French-American-British (FAB) Co-operative group classification system or by the blast count above 20% according to the World Health Organization (WHO) classification scheme. Unlike acute myeloid leukemia (AML), no single cytochemical test is specific for ALL; by definition, however, ALL is negative for myeloperoxidase (MPO) and lacks staining with the anti-MPO monoclonal antibody (MoAb). The morphologic/cytochemical examination recognizes three morphologic types: L1, L2 and L3; hovewer, only the L3 type of ALL still holds as a distinct entity characterized because of its morphology and also in view of its unique immunophenotypic and genotypic features


Immunophenotype is an essential component of the initial diagnostic evaluation of ALL and is also a valuable tool for monitoring disease after therapy and for the detection of minimal residual disease (MRD). The immunophenotypic characterization of blast cells has several objectives: a) lineage assignment, b) evaluation of cell maturation, and c) assessment of phenotypic aberrations.

Necessary and sufficient antibodies for diagnosis are:

  • cyMPO, CD117, TdT, cyCD3, CD7, cyCD79a, CD19

For a more refined level of characterization two different steps can be considered:

First step:

  • cyMPO, CD117, TdT, cyCD3, CD7, cyCD79a, CD19, CD33, CD34

Second step, once a diagnosis of ALL is made:

  • T-lineage: CD1a, CD2, CD3, CD5, CD4, CD8, CD52
  • B-lineage: CD10, CD20, CD22, cyIgM, sIg, CD52

B-lineage ALL (70-80% of cases) can be classified into four groups according to the expression of B-cell differentiation antigens and cytoplasmic and surface immunoglobulins (Ig); also T-ALL (15-25% of cases) can be classified into four groups based on the level of thymocyte maturation and antigen expression; T-ALL can be further classified according to the subtypes of T-cell receptor (TCR) molecules. Other markers are used to identity the maturation level of the blast cells and eventually establish atypical or aberrant phenotypes; a variable proportion of ALL express apparently non-lineage associated markers, e.g. myeloid antigens and CD34. The reported incidence of adult ALL showing myeloid antigen expression (My+ ALL) ranges from 15% to 40% and the most frequently expressed are CD33 (~25%) and CD13 (~20%); CD15 and CD14 can be found in ~15% of ALL cases, while CD11c is rarely present on ALL blasts. The presence of myeloid antigens can be useful in the immunologic monitoring of minimal residual disease (MRD). CD34 is the most commonly used antigen to define immature hematopoietic progenitor cells; about 70% of ALL cases are CD34 positive. The incidence of CD34 expression is more frequent in B-lineage ALL (70-80%) than in T-lineage ALL (20-30%); its expression has also been recorded in a high proportion of Ph chromosome positive ALL. The quantification of the level of expression of given antigens on the leukemic population may have therapeutic implications; this applies, in particular, to antibodies directed against CD20, CD22 and CD52 (all three antigens may be expressed by ALL cells). Thus, the percent of positivity and the degree of expression by the leukemic population at diagnosis and at relapse is important when considering the potential clinical utilization of such antibodies for the management of ALL patients.

Cytogenetic and molecular analyses

The study of cytogenetic abnormalities is the basis for unraveling molecular events that may be involved in the disease, such as the role of fusion transcripts that derive from translocations, tumor suppressor genes from deletions, or the control of cell cycle regulatory genes. The incidence of chromosome abnormalities in ALL can only be established approximately, because it depends on the techniques used and on the percentage of cytogenetic failures; chromosomes generally display a poor morphology (e.g. ALL cells do not always produce good metaphases) and important abnormalities can be missed. Chromosome abnormalities in ALL can be numerical, structural or both. New molecularly based technologies have been developed which enable the recognition in malignant cells of genomic rearrangements undetected by conventional cytogenetics:

  • Fluorescence in situ hybridization (FISH): the great advantage of FISH is the detection of known DNA sequences on metaphases or in interphase nuclei, combining the resolution of molecular analysis with cytogenetics.
  • Comparative Genomic Hybridization (CGH): the normal DNA and tumor DNA are labeled with different fluorescent colors; the chromosomal regions that are over-expressed (gains) or underrepresented (losses) in the test genome are seen with respect to the reference color.
  • Spectral Karyotyping (SKY) analysis: this technique allows to identify chromosome bands of unknown origin, including translocations, insertions, complex rearrangements marker chromosomes.

The chromosome abnormalities represent an independent and prognostic predictive factor for ALL.
Most of the more common karyotypic structural rearrangements have been studied at the molecular level; in molecular terms, chromosomal abnormalities or their submicroscopic equivalents are of two general types: those in which the breakpoint occurs within the involved genes, leading to the production of a fusion RNA transcript and a chimeric protein (qualitative change), and those which represent Ig/TCR rearrangement errors (quantitative change). Qualitative abnormalities can produce functional fusion genes; one of the most common is the t(9;22)q(34;q11) which forms the BCR-ABL fusion gene. Quantitative abnormalities result from Ig/TCR rearrangement errors which juxtapose the proto-oncogene to regulatory Ig/TCR sequences, leading to deregulated protein expression, for example the SIL-TAL1/tald deletions on chromosome 1p32 in T-ALL.
The detection of leukemia-associated clonal genetic changes at the karyotypic and genetic levels has been extensively tested by molecular biology techniques, based on reverse-transcriptase polymerase chain reaction (RT-PCR). Two types of PCR targets can be used: leukemia-specific breakpoint fusion regions of chromosome rearrangements (translocations, deletions or inversions) or junctional regions of leukemia clone-specific rearranged Ig/TCR genes. More recently, real-time quantitative PCR (RQ-PCR) has been used for MRD detection.

The main molecular genetic abnormalities identified in B-ALL are:

  • BCR-ABL / t(9;22)(q34;q11)
  • MLL-AF4 / t(4;11)(q21;q23)
  • TEL-AML1 / t(12;21)(p13;q22)
  • E2A-PBx 1/ t(1;19)(q23;p13)
  • E2A-HLF / t(17;19)(q22,p13)
  • MLL-v / t(11;v)(q23;v)
  • c-MYC-IgH / t(8;14)(q24;q32)
  • IL3-IgH / t(5;14)(q31;q32)

The main molecular genetic abnormalities identified in T-ALL are:

  • SIL-TAL1 / TAL1 deletion
  • C-MYC-TCRα /δ / t(8;14)(q24;q11)
  • HOX11-TCRα/δ / t(10;11)(q24;q11)
  • LMO1-TCRα/δ / t(11;14)(p15;q11)
  • LMO2-TCRα/δ / t(11;14)(p13;q11)
  • TAL1- TCRα/δ / t(1;14)(p32;q11)
  • TCL1- TCRα/δ / inv(14)(q11q32)

Minimal residual disease

Leukemia cells can be potentially distinguished from normal hematopoietic progenitors on the basis of morphologic and cytochemical properties, the immunophenotypic profile, karyotypic or genetic abnormalities and Ig/TCR gene rearrangements. These different characteristics have been exploited in an attempt to detect small numbers of blasts among normal cells and a variety of techniques have been developed for the detection of residual disease. The conventional criteria for remission in patients with acute leukemia are based on the morphologic examination of bone marrow (BM) samples and patients are considered to be in complete remission (CR) when BM aspirates contain less than 5% blasts. At the time of morphologic CR, however, the extent of minimal residual disease (MRD) varies considerably. The methods for MRD analysis include cytogenetics, FISH, Southern blotting, immunophenotype and PCR techniques. The applicability of these techniques for MRD detection depends on three parameters: a) specificity ability to discriminate between malignant and normal cells without false positive results), b) sensitivity detection limit of at least 10 3, and c) reproducibility and applicability (easy standardization and rapid collection of results for clinic application). Only a proportion of leukemias have specific markers such as chromosomal translocations, e.g. t(9;22), t(4;11) or t(1;19). Conventional karyotypic analysis may be used to monitor MRD if an abnormal karyotype is present at diagnosis; however, its low specificity and the risk of analyzing metaphases from normal cells represent major obstacles towards its routine use. The main advantage of FISH is that it provides interpretable information based on interphase cells with a low proliferative rate; nonetheless, the sensitivity of FISH analysis for MRD monitoring is limited. Immunophenotyping techniques using multicolor-gated flow cytometry are based on the aberrant expression of antigens by the leukemic cell population and on the identification of markers that may be found on malignant cells in combinations that are normally not observed in normal BM and PB cells; flow cytometry can be utilized to monitor MRD in about 85 90% of cases. The detection of leukemia-associated clonal genetic changes at the genetic level has been extensively tested by molecular biology techniques, based on PCR analysis.
Thus, in ALL patients MRD can be studies using three techniques that enable detection of leukemic cells with a sensitivity of 10 3-10 6:

  1. flow-cytometric immunophenotyping, using aberrant or leukemia associated phenotypes
  2. polymerase chain reaction (PCR) analysis of breakpoint fusion region chromosome aberrations
  3. detection of clone specific Ig and TCR gene rearrangement by PCR amplification.

One of the aims of MRD investigations is to estimate the amount of residual tumor rather than to establish its presence and, recently, real-time quantitative PCR (RQ-PCR) has been used for MRD detection in ALL. The greatest obstacle to the routine use of MRD studies in ALL therapy protocols is that none of the techniques currently available for MRD detection can be applied to all patients. Because PCR may detect residual leukemic cells in cases not amenable to flow cytometric investigation, and vice-versa, it is possible to apply the two techniques in tandem.

Gene expression profiling

Genomic profiling is becoming a reality that may profoundly modify the management of ALL patients. Hierarchical clustering of all adult ALL samples based on gene expression profile identified two well-defined groups which correlated precisely with the T- or B-cell immunophenotype of the leukemic cells. Further analysis identified gene expression profiles associated with the presence of either ALL1-AF4, BCR-ABL or E2A-PBX1 gene rearrangements. Furthermore, an integrated analysis of childhood and adult ALL has highlighted a strong similarityy between cases which harbor specific rearrangements regardless of the age of the patients. With the use of these technologies, it has been shown that genetically defined subgroups express different sets of genes. In individual cases, the genetic lesion could be classified by microarray analysis, while being negative by RT-PCR. It is now realistic to verify whether these innovative technologies will change our approach to the characterization of leukemias.


Following a diagnostic work-up, information obtained through karyotype, molecular genetics, immunophenotype, and, more recently, genomic profiling is progressively contributing to a better understanding of the biology of ALL. Only through such framework we can aim:

  • at unraveling further abnormalities
  • at adequately diagnosing all cases
  • at optimally evaluating the prognostic impact of each molecular markers
  • at monitoring minimal residual disease of individual subgroups of patients according to the genetic abnormalities identified at presentation
  • at using different therapeutic protocols based on prognostic indicators and, recently, also at designing innovative and specific treatment strategies.