Research Article: Molecular Diagnostics, Targeted Therapy, and the Indication for Allogeneic Stem Cell Transplantation in Acute Lymphoblastic Leukemia

Date Published: November 10, 2011

Publisher: Hindawi Publishing Corporation

Author(s): Anthony Oyekunle, Torsten Haferlach, Nicolaus Kröger, Evgeny Klyuchnikov, Axel Rolf Zander, Susanne Schnittger, Ulrike Bacher.

http://doi.org/10.1155/2011/154745

Abstract

In recent years, the panel of known molecular mutations in acute lymphoblastic leukemia (ALL) has been continuously increased. In Philadelphia-positive ALL, deletions of the IKZF1 gene were identified as prognostically adverse factors. These improved insights in the molecular background and the clinical heterogeneity of distinct cytogenetic subgroups may allow most differentiated therapeutic decisions, for example, with respect to the indication to allogeneic HSCT within genetically defined ALL subtypes. Quantitative real-time PCR allows highly sensitive monitoring of the minimal residual disease (MRD) load, either based on reciprocal gene fusions or immune gene rearrangements. Molecular diagnostics provided the basis for targeted therapy concepts, for example, combining the tyrosine kinase inhibitor imatinib with chemotherapy in patients with Philadelphia-positive ALL. Screening for BCR-ABL1 mutations in Philadelphia-positive ALL allows to identify patients who may benefit from second-generation tyrosine kinase inhibitors or from novel compounds targeting the T315I mutation. Considering the central role of the molecular techniques for the management of patients with ALL, efforts should be made to facilitate and harmonize immunophenotyping, cytogenetics, and molecular mutation screening. Furthermore, the potential of high-throughput sequencing should be evaluated for diagnosis and follow-up of patients with B-lineage ALL.

Partial Text

Acute lymphoblastic leukemia (ALL) is a heterogeneous disorder, which consists of various clinical, morphological, and immunological phenotypes, underpinned by extreme genetic diversity [2–4]. Adaptation of treatment intensity to the probability of relapse in the individual patient requires a thorough understanding of the risks represented by the various stratified leukemia subtypes. This has been achieved, to a large extent, using a broad spectrum of diagnostic techniques including cytomorphology, immunophenotyping, cytogenetics, fluorescence in situ hybridization (FISH), and molecular techniques. The panel of known prognostically important molecular alterations is constantly increasing, as demonstrated by the recent detection of alterations of TGF-beta and PI3K-AKT pathway genes and prognostically adverse deletions at 6q15-16 in T-ALL [5]. In Philadelphia-positive (B-lineage) ALL, deletions of the IKZF1 gene confer a more adverse prognosis [6, 7]. Genetic alterations are now detectable in most ALL patients, when cytogenetic and molecular techniques are combined. These genetic alterations are linked to distinct clinical profiles and show specific interaction with other mutation types [8]. Following the success of the tyrosine kinase inhibitor (TKI) imatinib in chronic myeloid leukemia (CML), research focused on targeted therapy strategies for Ph-positive ALL and other ALL subtypes [9–13]. Imatinib has since become part of pre- and posttransplant treatment for patients with Ph-positive ALL [13, 14]. Rituximab was included in treatment of CD20-positive ALL [15–17]. This paper characterizes the most important molecular markers in patients with acute lymphoblastic leukemia, paying attention to their impact for treatment decisions, and discusses methods for their detection.

According to the WHO classification published in 2008 [1], different reciprocal rearrangements form the category “B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities” (Figure 1). Many of these genetic alterations provide useful markers to monitor the minimal residual disease (MRD) load [18].

Clonal cytogenetic anomalies are detectable in 50–70% of all cases of T-cell ALL. Reciprocal translocations usually involve the T-cell receptor (TCR) genetic loci; TCRA and TCRD (14q11.2), TCRB (7q35), or TCRG (7q14-15). The partner genetic loci reported are usually transcription factors particularly HOX11 (TLX1, 10q24), HOX11L2 (TLX3, 5q35); others include the MYC (8q24.1) or TAL1 (1p32) genes. Other fusion genes are, for example, CALM-AF10 or NUP214-ABL1. For molecular MRD measurement, suitable fusion transcripts are available for only 10–20% of T-ALL patients. If appropriate targets are available, quantitative real-time PCR can achieve sensitivity of 10−4 to 10−5. In the alternative, clone-specific TCR rearrangements of the leukemic T cells could equally serve for MRD monitoring in remission with comparable sensitivity. However, amplification of clone-specific TCR rearrangements is highly laborious as patient-tailored assays are required. Furthermore, molecular clonal evolution can lead to false-negative results [43].

After patients achieve complete remission following either chemotherapy or HSCT, the MRD load should be serially assessed [53]. It is thus desirable to identify a sufficiently specific leukemia-specific marker before-therapy, such as the BCR-ABL1 fusion. The preferred MRD technique depends on the desired level of sensitivity or the depth of remission. Cytogenetics has a sensitivity of 10−2 cells. Interphase fluorescence in situ hybridization (FISH) allows to evaluate 100–200 cells. Immunophenotyping using multi-parameter flow cytometry achieves sensitivity levels of 10−3 to 10−5 [54, 55]. Real-time PCR is particularly useful, as it can achieve a sensitivity of 10−4 to 10−6 [56]. Additionally, molecular techniques can be used to access MRD in ALL even in the absence of fusion genes, by assessing the levels of clone-specific rearrangements of the immunoglobulin or T-cell receptor [57] and have been introduced into treatment stratification already. In a study from the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL), a total of 196 patients with standard risk ALL were investigated at repeated time points in the first year by quantitative PCR monitoring of clonal immunoglobulin or TCR rearrangements. Three risk groups could be defined. Patients with a rapid decline of the MRD load to <10−4 or below detection limit in the early treatment period (days 11 and 24) were classified as low risk and had a three-year relapse rate of 0%. Patients with an MRD of ≥10−4 until week 16 formed the high-risk group with a 3-year relapse rate of 94%. The remaining patients had an intermediate risk [58]. In another study from the GMALL, postconsolidation samples of 105 patients with standard risk ALL were investigated by real-time quantitative PCR for clonal immune gene rearrangements. All patients were beyond the first year of chemotherapy, in hematological remission, and were MRD negative before study entry. The relapse rate was 61% in patients converting to MRD positivity thereafter, whereas only 6% of continuously MRD-negative patients relapsed [59]. In recent years, molecular diagnostics in the acute lymphoblastic leukemia have progressed rapidly. PCR-based analyses in combination with other approaches (cytogenetics, FISH, and immunophenotyping) have allowed us to define various distinct ALL subtypes, part of which already defines separate entities within the WHO classification of 2008, for example, the t(9;22)/BCR-ABL1 or the t(12;21)(p13;q22)/ETV6-RUNX1. Deeper insights into the networks of molecular markers have facilitated the understanding of the heterogeneity of the clinical courses within distinct genetic subgroups and improved therapeutic decisions, for example, regarding the indication to allogeneic HSCT within T-lineage ALL [49]. Screening for deletions of the IKZF1 gene might improve risk stratification in patients with Ph-positive ALL [6, 7]. Distinct levels of the MRD load as assessed by RQ-PCR have been defined as guidelines for therapeutic decisions [19, 62]. Molecular diagnostics and immunophenotyping have become the basis for targeted therapy in ALL, as demonstrated by the use of tyrosine kinase inhibitors for BCR-ABL1-positive ALL, and rituximab for CD20-positive B-cell precursor ALL [16] or mature B-ALL/Burkitt lymphoma [19], which improved the prognosis of these previously highly adverse subtypes. Screening for BCR-ABL1 mutations can be helpful to identify patients with Philadelphia-positive ALL who may have a benefit from second tyrosine kinase inhibitors or novel compounds targeting the T315I. Considering the recent introduction of high-throughput sequencing into hematological diagnostics [63], the potential of this novel technology should be explored for mutation screening, the definition of new therapeutic targets, and follow-up diagnostics in the acute lymphoblastic leukemias.   Source: http://doi.org/10.1155/2011/154745

 

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