Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with different clinical and biological aspects, characterized by the proliferation and accumulation of immature cells of the lymphoid lineage in the bone marrow, peripheral blood, lymphoid tissues and other tissues.

 

 

EPIDEMIOLOGY

ALL is the most common tumor in children, while it is relatively rare in adulthood. Its incidence has a bimodal distribution with an early peak between 2 and 5 years of age, and a subsequent increase over 50 years (Sallan SE, 2006) (Figure I). There is a slight predominance in males, with a male-female ratio of ~2:1.

 

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Figure I: Incidence by age (from Sallan SE; Hematology 2006)

 

Although in the great majority of ALL it is not possible to recognize genetic or environmental causes, the leukemia probably develops for a combination of environmental causes, the presence of a genetic susceptibility and the action of tumor oncogenes (Kinlen LJ, 2004; Redaelli A et al, 2005; Gao F et al, 2005; Greaves M, 2006). The higher incidence of ALL in patients with genetic syndromes (Down, Klinefelter, Fanconi, Bloom syndromes) or primary immunodeficiencies, such as the Wiskott-Aldrich syndrome and ataxia-telangiectasia, suggests a relationship between genetic abnormalities and leukemia. The hypothesis of an influence of hereditary factors is also based on the demonstration of leukemia development in monochorionic twins; it is possible to assume a transition of the leukemic clone via the placental blood. Moreover, the presence of a clonal rearrangement of the T-cell receptor (TCR) or of the immunoglobulin (Ig) genes, or of the sequence of an aberrant fusion gene has also been documented at birth in the blood of children who later developed a leukemia (Jeffrey W, 2002). Even if some viruses, especially retroviruses, are capable of inducing cancers in animals, there is no such evidence in human ALL with the exception for the Epstein-Barr virus, that can be associated with mature B-cell ALL (also known as “Burkitt type”).

Among the environmental causes that are recognized as risk factors one must recall:

– the exposure to high doses of radiation (for example, the explosion of an atomic bomb, accidents in nuclear reactors or some forms of radiation therapy);
– living near industrial sites (ie exposure to substances such as benzene).

Young survivors of the atomic bomb in Japan and children exposed to radiation in utero have shown an increased incidence of leukemia. Exposure to external agents such as solvents, radiation, chemicals, pesticides, drugs has been related to the development of childhood leukemia. Also the exposure to radiations or chemical solvents in uterus may give rise to the development of leukemia, even if the latency of its onset suggests the requirement of the addition of other leukemogenic events. Smoking and exposure to electromagnetic fields or low doses of radiation are yet to be defined as risk factors.

One or more of these factors can cause an alteration of the lymphoid cell maturation towards a neoplastic cell. Thus, lymphoblasts lose their ability to give rise to differentiated cells; they expand and accumulate, initially in the bone marrow and subsequently in the peripheral blood and in other organs and tissues giving rise to the appearance of a leukemia.

 

PATHOGENESIS

The precise pathogenetic events leading to the development of ALL are unknown, although in recent decades the evidence that chromosomal defects and molecular abnormalities are consistently present in ALL patients has progressively increased (Armstrong SA, 2005; Pui CH, 2008). Even if the mechanisms that lead to these abnormalities are still unclear, it is certain that many are due to the loss of control by anti-oncogenes, the activation of oncogenes or the activation of new proteins with potential transcription activity. It is possible to assume that ALL originates from multiple important genetic lesions that occur in hematopoietic progenitors, already committed to differentiate into B or T cells, including mutations that confer the capacity of unlimited self-renewal and those leading to a developmental arrest in a stage-specific manner. The leukemic cells present clonal rearrangements in the genes of the Ig and/or TCR. The cloning and characterization of recurrent chromosomal translocations has allowed the identification of genes that are critical for the process of leukemogenesis (Rowley JD, 1998; Greaves M F, 2003). The use of gene expression analysis to characterize the differences between various types of leukemia with chromosomal aberrations has strengthened the notion that specific chromosomal abnormalities define specific leukemias (Haferlach T, 2003; Chiaretti S, 2005). These genetic lesions are important for the initial event of leukemia, but alone are not sufficient to generate a full leukemic phenotype; this means that other potential pathogenetic events are necessary (Mullighan CG, 2007).

The dominant theme of contemporary research in the pathobiology of ALL is to identify new genetic alterations and to understand the consequences of these genetic lesions, in terms of effects on cell proliferation, differentiation and survival. These genetic lesions and their protein products are also potential targets for the development of therapeutic strategies aimed at specifically targeting the transformed tumor cell.

 

SYMPTOMS

There are no specific symptoms or clinical signs of the disease; their onset is unexpected, different in each patient, variously associated, and they are largely similar between adult and pediatric patients. In general, patients report a brief history of fatigue, fever, night sweats, loss of weight and, sometimes, bone pain and hemorrhagic manifestations. These symptoms and signs are the result of the leukemic cell proliferation in the bone marrow and blood, and of the possible impairment by the same leukemic cells of other organs and systems such as liver, spleen, lymph nodes, mediastinum, central nervous system (CNS) and, less frequently, bones, joints, skin, kidneys and lungs. The reduced bone marrow reserve causes fatigue, pallor or tachycardia due to the consequent anemia, recurrent or prolonged infections due to the neutropenia, hemorrhagic manifestations (petechiae, bleeding gums, epistaxis, etc) because of the thrombocytopenia. There may be symptoms such as nausea, headache, or vomiting as a sign of an impairment of the CNS, whose involvement at diagnosis is found in 5-10% in both children and adults. A particular situation is linked to the testicular localization, often unilateral, present in a greater proportion of children than of adults. In this case, the work-up should be completed by a biopsy with histological examination performed on both testicles; this type of localization is much more common at the time of relapse rather than at the onset of the disease.

The complete blood count (CBC) may show parameters – such as anemia, leukopenia or leukocytosis, thrombocytopenia, etc – which can be shared with other hematological diseases. In children, more than in adults, there is often leukopenia. Also other laboratory analyses may show nonspecific abnormal results (Table I). An expert morphologic analysis of peripheral blood smears is necessary because they may show the presence of blasts. If the clinical picture and the CBC lead to a suspicion of acute leukemia, the bone marrow aspiration allows the execution of most of the investigations that lead to a correct diagnostic and prognostic work-up of ALL. Once the diagnosis of ALL is made, it is also important to perform a spinal tap, which consists of the analysis, via a very thin needle placed between two lumbar vertebrae, of the cerebrospinal fluid for the possible presence of leukemia cells, a sign of impairment of the CNS.

 

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 Table I:

 

 

LABORATORY DIAGNOSIS AND CLASSIFICATION

The diagnosis and classification of ALL are based on a “multistep” procedure which includes the analysis of the morphology, cytochemistry, immunophenotyping, classical cytogenetics and molecular genetics, and, when necessary, the rearrangement of the Ig genes and the TCR genes (Jabbour E, 2005; Vitale A, 2006; Faderl S, 2010). This approach allows a correct clinical and biological diagnosis of leukemia, but also helps in the choice of treatment (Figure II).

 

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Figure II: Diagnostic framework

 

Morphology and cytochemistry

The first “step” in the diagnosis of ALL is the observation, by an optical microscope, of peripheral blood and bone marrow smears stained according to the May-Grunwald-Giemsa method. ALL is characterized by the presence in the bone marrow of immature lymphoid cells in a proportion equal to or greater than 30%, according to the classification proposed originally by the “French-American-British (FAB) cooperative group” (distinguished morphologically in L1, L2 and L3), or to 20%, according to the most recent scheme proposed by the “World Health Organization (WHO)” (Figure III). The division between the morphological forms L1 and L2 is almost no longer used, because it does not bear prognostic implications; the exception is represented by the L3 form, that represents a separate entity with a distinct clinical-biological characterization, a different therapeutic approach and prognosis (see below) (Bennett JM, 1976; Harris Nl, 1999).

Even if there is not a specific cytochemical test for ALL, the lymphoid leukemic cell is, by definition, negative for myeloperoxidase (MPO). Other cytochemical reactions, such as periodic acid-Schiff (PAS) staining or the test for non-specific esterase, can be positive in some subgroups of ALL, but they are not specific, being also present in cases of acute myeloid leukemia (AML). Over 95% of ALL express the nuclear enzyme deoxynucleotydil-terminal transferase (TdT), which is normally negative in AML and in all chronic leukemias (Table II).

 

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Figure III: ALL morphology. EMBED PowerPoint.Show.8

 

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Table II: ALL cytochemistry.

 

Immunophenotype

The immunophenotypic analysis represents a crucial moment in the diagnostic work-up of ALL. The leukemic cells express surface and intracytoplasmic antigens, whose characterization allows the identification of the cell line of origin, its level of differentiation and maturation, and the presence of aberrations (Szczepanski T, 2006; Orfao A, 2006). In almost all cases, the immunophenotype of the lymphoid blasts does not change over time and allows the detection of leukemic cells even when they are present in very small percentages. Immunophenotyping is performed by flow cytometric techniques using monoclonal antibodies (mAbs) conjugated to fluorochromes that permit the simultaneous analysis of the different antigens expressed. The instrument used for the analysis is the flow cytometer; conventionally, to consider as positive a particular antigen on a leukemic cell, it is necessary that a surface antigen is expressed on at least 20% of the cells (10% for intracytoplasmic antigens). Another parameter to evaluate is the intensity of the fluorescence, capable of distinguishing not only the leukemic cell from its normal counterpart, but also to “quantify” the levels of antigenic expression important in the setting of minimal residual disease (MRD) monitoring during and after therapy, even when present in very small numbers (sensitivity equal to 10-4). It is possible to quantify the expression of an antigen by MIF (Mean Fluorescence Intensity) and by ABC (Antibodies Bound per Cell).

mAbs allow to identify and differentiate the lymphoid cells committed along the B-cell lineage (ie CD10, CD19, CD79a), from those of the T-cell lineage (ie CD1a, CD3, CD7, cCD3) and from those of the myeloid lineage (ie MPO, CD13, CD33). There are also non-lineage-specific mAbs (ie CD34). According to the different patterns of positivity of the various mAbs, it is possible to distinguish B-lineage ALL, which represent the majority of cases (75-80% of adult cases, 85-90% of pediatric cases), from the T-lineage cases (20-25% in adults, 10-15% in pediatric cases) and the more immature forms from the more mature ones (Table IIIa and b). Classification schemes allow a precise immunological definition of ALL (Bene MC, 1995; Bene MC, 2005). According to the presence of specific surface and intracytoplasmic antigens and to the presence or absence of surfaces/cytoplasmic Ig, B-lineage ALL can be classified into four groups. Similarly, T-lineage ALL can also be subdivided into four groups, depending on the level of maturation of thymocytes; T-ALL can be further classified based on the TCR profile (α/β and γ/δ).

 

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Table III: ALL immunological classification.

 

Although it is relatively easy to differentiate ALL into the B or T lineage, there is a small percentage of cases in which it is difficult to distinguish between lymphoid and myeloid forms; these cases can coexpress both lymphoid and myeloid antigens on the same cell (biphenotypic leukemia) or on different populations (hybrid leukemia); there is no firm consensus about the diagnostic criteria to use in these cases. The “European Group for the Immunological Characterization of leukemias (EGIL)” suggested a scoring system based on assigning a specific score for B, T or myeloid antigens and on their associations (Bene MC, 1995). According to this strict system, four groups can be identified; the most common group is the one that expresses both myeloid and B lymphoid antigens on the same blasts. Less commonly, cells coexpress myeloid and T lymphoid antigens. Rarely blast cells express both B- and T-lineage antigens, or myeloid, B- and T-lineage antigens together. A review of this classification made in 2008 by the WHO (Vardiman JW, 2009) brought some changes in the diagnosis and classification of leukemias with difficult characterization. Leukemias previously defined as “hybrid” and “biphenotypic” are now considered globally as “Mixed Phenotype Acute Leukemias (MPAL)” and cases without lineage markers as “Acute Undifferentiated Leukemias (AUL)”. The criteria that define MPAL were significantly modified. The clinical significance of biphenotypic leukemia or MPAL has still to be conclusively defined and there is a lack of uniformity in treatment; for example, it is not clear if it is better to use an induction therapy based on regimens for lymphoid or myeloid leukemias. It will be necessary to increase the number of cases through multicenter studies to gain useful information for optimal treatment.

Other markers are used to identify the level of maturation of the leukemic cells and, possibly, to establish atypical or aberrant phenotypes that may be indicative of a specific underlying genetic lesion. A variable proportion of ALL expresses markers apparently not lineage-associated, such as myeloid antigens and CD34. The incidence of adult ALL presenting myeloid antigens varies from 15% to 35%, while in pediatric ALL it varies from 4% to 15%. This wide range can be related to the number of antigens used, the sensitivity of the mAbs used, the established cut-off and technical factors (eg the sensitivity of the flow cytometry or the “gating” strategy). The presence of myeloid antigens on leukemic blasts does not seem to have an adverse prognostic factor (Vitale A, 2007), but it may be useful for the immunological monitoring of MRD.

CD34 is the antigen most commonly used to define the immature hematopoietic progenitor; in fact, it is present in only 1% of bone marrow cells, it is not lineage-restricted and can be expressed both in ALL and AML. About 70% of ALL cases express CD34 and it is much more frequent in B-lineage ALL (70-80%) than in T-ALL (20-30%); moreover, CD34 is present in a high percentage of Ph (Philadelphia)+ patients.

The quantification of the antigenic expression on the leukemic population may have therapeutic implications. mAbs have, in fact, clinical applications in lymphoproliferative diseases. This is true particularly for antibodies directed against CD19, CD20, CD22, CD33 and CD52; all these antigens may be expressed on lymphoid blasts. Thus, the positivity rate and the degree of expression by the leukemic population evaluated at diagnosis and at relapse are important when considering the potential clinical use of these antibodies in the management of patients with ALL. A recent work (Raponi S, 2011) analyzed the expression of specific antigens – CD19, CD20, CD22 and CD33 – for which mAbs are available for clinical use, in a wide series of ALL. It has been shown that lymphoid cells show a trend in the expression of these antigens during the differentiation or in association with the presence or absence of molecular abnormalities; these differences could be taken into account for a possible clinical use of mAbs.

The immunophenotype is an essential step in the diagnostic of acute leukemias and is an important tool for the monitoring of MRD (see below).

 

Cytogenetics and molecular biology

Cytogenetic analysis is a further important step in the characterization of ALL. By conventional cytogenetics it is possible to identify alterations of the karyotype only in a proportion of ALL cases because of the difficulty of the lymphoid blast to enter mitosis; the more recently developed technologies can identify the presence of karyotypic abnormalities not previously detectable (Speicher MR, 2005). It is important to identify these alterations at the time of diagnosis because of their proven prognostic value (Mancini M, 2005; Moorman AV, 2007; Pullarkat V, 2008). The techniques for the detection of chromosomal abnormalities are nowadays:

– Conventional cytogenetics
– Fluorescence in situ hybridization (FISH)
– Comparative genomic hybridization (CGH)
– Spectral karyotyping (SKY) analysis.

Cytogenetic abnormalities may be structural, numerical or both. The majority are structural and they are generally translocations; less frequently, they are deletions. Although the mechanisms leading to these anomalies are still unclear, it is certain that many of them result in a loss of control by anti-oncogenes, activation of oncogenes or of new proteins with potential transcriptional activity. Numerical anomalies are less common; hyperdiploidy is present in 5-10% of adult patients and the association with a better prognosis is less obvious than in children (present in about 25% of cases); on the contrary, the presence of hypodiploidy (2-4% of ALL) is associated with a poor prognosis for both. Many of the most common translocations have been also studied by molecular biology, using the reverse-transcriptase polymerase chain reaction (RT-PCR) techniques and variants (Chomczynski P, 1987; Beillard E, 2003; Elia L, 2003). The lymphoid blasts are also characterized by the presence of specific and clonal rearrangements of the Ig and TCR genes (Van Dongen JJ, 2003) that differentiate them from reactive lymphoid proliferations. It is possible to identify such clonal rearrangements by molecular analysis based on DNA sequencing techniques and to design specific probes for each individual patient by sequencing the rearrangement of the leukemic cells at the onset of the disease. Chromosomal abnormalities are of two types: those in which the “breakpoint” occurs within the genes involved, leading to the production of a fusion transcript and to a chimeric protein (qualitative change), and those in which there are alterations of the Ig/TCR gene rearrangement (quantitative change). Qualitative abnormalities produce functional fusion genes. One of the most common is the translocation t(9; 22)(q34; q11), which gives rise to the fusion gene BCR-ABL1; another is the translocation t(1; 19)(q23; p13), wherein the gene E2A (TCF3) joins the PBX1 gene. The rearrangement involving the MLL gene (ALL1) on chromosome 11 in the region q23 results in a fusion gene with AF4 on chromosome 4 in the region q21; in addition to this, there are other different partners that can join the MLL gene. Quantitative abnormalities are related to unusual Ig and/or TCR gene rearrangements, with consequent alterations of the proto-oncogene which regulates the Ig/TCR gene sequences, leading to a protein deregulation (eg deletions of SIL-TAL1/tald on chromosome 1p32 in T-ALL). Qualitative fusion transcripts are predominant in B-lineage ALL, while the rearrangement aberrations are, instead, much more common in T-ALL. Other lesions recently identified in T-ALL include Notch1 and JAK1 mutations, the overexpression of TLX1 (HOX11), TLX3 (HOX11L2) and TAL1 LYL1, and the ABL1 rearrangement (Graux C, 2006; Grabher C, 2006). It should be noted that the role of mutations in the JAK genes (JAK1, JAK2 and JAK3) is emerging in ALL, since JAK2 mutations have been recently described in B-cell forms (Mullighan CG, 2009); this finding has obvious potential therapeutic implications, in view of the availability of JAK2 inhibitors. Recent studies (Mullighan CG, 2009; Martinelli G, 2009) have described the deletion of IKZF1, which encodes the transcription factor IKAROS, on chromosome 7 in the region p12, in many BCR-ABL+ cases, suggesting that the association of these two genetic alterations may cause an arrest in the B-lymphoid differentiation process which may lead to the leukemia onset. Furthermore, the deletion of IKZF1, within Ph+ cases, identifies a subgroup of patients with a more unfavorable prognosis (Martinelli G, 2009).

A list of the main genetic abnormalities in B- and T-ALL is shown in Table IV. At the moment, only some of these translocations are associated to defined biological, clinical and prognostic characteristics.

 

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Table IV: Principal ALL genetic abnormalities (modified from Vitale A et al. [15]).

 

The incidence of some transcripts is different between adults and children (Harrison CJ, 2009) (Figure IV); for example, the presence of BCR-ABL1 is observed in 25-30% of adults and increases progressively with age, while it is present in children only in 2-5% of cases. On the contrary, the TEL-AML1 (ETV6-RUNX1) transcript is present in less than 1% of adult patients and in 20-30% of pediatric cases, and SIL-TAL1, identifiable in T-ALL, is present in 10.5% of adults and in 10-20% of children. Other transcripts have a similar incidence in both populations, as MLL-AF4, which is present in 5-7% of both adult and pediatric patients, except for cases with less than one year of age (50%), and the E2A-PBX1 transcripts which is present in 3-5% of cases.

 

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Figure IV: Age distribution of the main cytogenetic/molecular anomalies (from Harrison CJ, 2009).

 

Genetic profile

The possibility of studying the gene expression profile by microarray technologies has opened new avenues in the characterization of leukemias (Haferlach T, 2003). The power of this analysis is broad: I) it can define the genetic profile of each neoplastic form; II) it may help to identify the normal counterpart of the neoplastic cell; III) it can identify a pattern of genes that characterize a group of patients with different responses to chemotherapy and therefore have prognostic value; IV) it may identify new therapeutic targets for future targeted therapies; V) it may help to give a more precise definition of drug sensitivity or resistance. In the context of adult ALL it is possible to identify two well-defined groups that are of B- and T-cell lineage; further analyses have identified a distinct genetic profile correlated to cases with E2A-PBX1 and MLL-AF4 transcripts. In addition, an integrated analysis of adult and pediatric cases has shown a close similarity between forms with the same molecular rearrangement regardless of age (Chiaretti S, 2005; Yeoh EJ, 2002; Kuchinskaya E, 2005) (Figure Va and Vb). With the improvement in knowledge and technologies, it is not unrealistic to suppose that, in the future, through genetic profiling we will be able to define the diagnostic, prognostic and therapeutic profile for most forms of leukemia

 

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Figure  V: ALL Microarrays. A) Adults (from Chiaretti S et al, 2005); B): Children (from Yeoh  EJ et al., 2002).

 

MicroRNA

MicroRNAs (miRNAs) are short non-coding RNA sequences (about 22 nucleotides) that modulate the expression of specific target genes, such as certain oncogenes or tumor suppressors. Every single microRNA regulates several genes and each gene can be modulated by more than one microRNA. The activated genes are transcribed into messenger RNA (mRNA), which lead to the production of proteins by ribosomes. During cell development and in different environmental conditions, the balance between activated and inactive genes is finely modulated, and alterations of this balance can lead to malformations or disease states. Therefore, increases or decreases in the expression of miRNAs may play a key role in the development of many cancers. So far, thousands of species of miRNAs have been identified and further information regarding their role is still being collected (Bartel DP, 2004).

A chromosomal region rich in miRNAs frequently lost in malignant T cells has been identified and this particular region encodes about 12% of all genomic miRNAs. A particular type of miRNA has also been identified, called miR-203, which is silenced by both genetic and epigenetic mechanisms in many forms of malignant human and murine blood, including chronic myelogenous leukemia and some forms of ALL. The silencing of miR-203 results in a defect of regulation of the oncogene ABL-1 and of the oncogenic fusion protein BCR-ABL1, while the re-establishment of miR-203 results in a subsequent reduction of ABL1 and BCR-ABL1, and, consequently, in a decrease in tumor cell proliferation (Bueno MJ, 2008). In a recent work (Chiaretti S, 2010) the expression levels of miR-223, that is involved in myeloid differentiation, were evaluated in a particular subgroup of T-ALL expressing a gene profile closer to the myeloid forms. These cases had significantly higher levels of miR-223 compared to the other T-ALL analyzed, with values comparable to those observed in AML.

 

Minimal residual disease (MRD)

The study of MRD allows a direct measurement of the “amount” of disease that chemotherapy can eliminate and of the residual one. This analysis has recently gained great importance because disease-free survival (DFS) depends on the ability to control residual disease (Campana D, 2009).

The conventional criterion to define a leukemic patient in complete remission (CR) is based on the presence of less than 5% of blast cells at the bone marrow examination by optical microscope. However, even if the patient is in morphological RC the “amount” of minimal disease can vary. Leukemic cells can be distinguished from the normal hematopoietic progenitors by morphological and cytochemical features, immunophenotypic analysis, presence of cytogenetic and/or molecular abnormalities and by the presence of a Ig and/or TCR gene rearrangements. However, not all cases carry known genetic alterations (eg BCR-ABL1, MLL-AF4, etc) suitable for molecular analysis and it is not possible to demonstrate the presence of Ig and TCR gene rearrangements in all patients. All the different biological characteristics of the lymphoid blasts have been used in an attempt to identify a small number of leukemic cells admixed within the normal ones; the most commonly used and reliable techniques are those based on immunophenotypic analysis and on molecular analysis by RT-PCR for given genetic abnormalities or for the Ig and TCR gene rearrangements.

The immunophenotypic analysis by flow cytometry is based on the expression of aberrant antigens by leukemic blasts and on the identification of markers that may be present on the leukemic cells and not on normal cells (Szczepanski T, 2006; Vidriales MB, 2003). Overall, flow cytometry can be used to monitor MRD in about 85-90% of cases. The use of this approach requires a deep knowledge of the immunophenotype expressed by normal hematopoietic cells, not only in the steady-state, but also during chemotherapy and active regeneration. PCR is a reliable and accurate method for monitoring MRD (Beillard E, 2003; van der Velden VH, 2007). As mentioned, two main methods of analysis may be used to distinguish the leukemic cells from normal cells. One is the molecular analysis that allows to detect fusion genes such as BCR-ABL1, MLL-AF4, E2A-PBX1 and TEL-AML1; the other is the molecular analysis that identifies the clonal rearrangement of the Ig and/or TCR genes (Gabert J, 2003; van der Velden VH, 2003). Rearrangements of the heavy chain Ig (IgH) genes are present in 95% of patients with B-lineage ALL. Rearrangements of the TCR genes occur in 95% of T-ALL (TCRd, TCRg and TCRb) and in 50-70% of B-ALL (TCRd and TCRg).

In order to have a prognostic value, MRD must be “reliable” and must meet the requirements of:

– Specificity: ability to recognize normal cells from leukemic cells.
– Sensitivity: ability to detect at least one leukemic cell in a context of ≥10,000 normal cells.
– Reproducibility: standardization of methods.
– Applicability: done at the right time in a recognized laboratory.

The reliability of MRD monitoring is also linked to the knowledge of the advantages and disadvantages of the methods used to determine it. Flow cytometry, fusion gene and Ig and/or TCR gene rearrangement analyses carry advantages and disadvantages (Table V).

 

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Table V: MRD advantages and disadvantages.

 

Flow cytometry is helpful when it is not possible to design specific probes by PCR and PCR can detect the presence of residual leukemic cells in cases not recognized by flow cytometry; so, when possible, the two techniques can be used tandem. The cut-off level commonly chosen to define the positivity of MRD is 0.01%. This limit of detection is routinely used by flow cytometry and molecular analysis, and has been shown to be able to discriminate between patients with different risks of relapse. However, to date, there are no universally accepted terms to define the presence or absence of MRD; a proposal has been recently made by the “European Working Group for Adult Acute lymphoblastic Leukemia (EWALL)” (Bruggemann M, 2009) (Table VI).

 

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Table  VI. Suggestion for the definition of minimal residual disease (MRD) (adapted from Bruggemann M et al, 2009).

 

In ALL, both for adults and children, a low risk of recurrence is associated with a good early response to chemotherapy, which can be documented by the level of reduction of MRD. In adults, this reduction appears to occur at a slower rate than in children and only a few patients reach a state of negativity. Prospective studies are ongoing to evaluate not only the modalities of “clearance” in adult patients, but also to establish the prognostic and therapeutic potential of minimal disease (Bruggemann M, 2006; Borowitz MJ, 2008; Basso G, 2009; Bassan R, 2009).

One of the most important challenges in the treatment of leukemia is the ability to distinguish patients who need less intensive, and therefore less toxic, treatment rather than those who need more aggressive treatments. The presence or absence of MRD represents a way to define these two categories of patients and consequently to treat them in the most appropriate manner.

 

STAGING AND PROGNOSIS

The continuous evolution of technologies allows increasingly sophisticated strategies for the characterization of leukemic cells and, thanks to an integrated clinical and biological approach, a prognostic stratification of patients with the opportunity of adapting the treatment according to the risk level is possible (Foà R, 2002; Schultz KR, 2007).

Adult ALL are conventionally divided into two groups: a) high risk and b) standard risk. This division is nowadays based on: a) different clinical features, such as age, white blood cells (WBC) count, the involvement of organs and/or systems (mediastinum, liver, spleen, central nervous system, and so on), the time to obtain a RC; and b) a number of biological characteristics, such as the presence of cytogenetic/molecular alterations associated to a poor prognosis (ie BCR-ABL1 or MLL-AF4) and the presence of cells with a more immature phenotype (pro-T/pro-B). Age and WBC count are the most important clinical risk factors; patients with more than 60 years of age are considered a group with a poor prognosis, while adolescents and young adults have a more favorable prognosis when treated with pediatric-like protocols (see below). Regarding the WBC count, there is a cut-off value to distinguish between high and low risk; in general, this is >30 x 109/L for B-lineage ALL and >100 x 109/L for T-ALL. Within the GIMEMA group, the cut-off is the same for B- and T-ALL (>50 x 109/L). A high WBC count is also representative of the contemporary presence of important tumor masses. From a biological point of view, the presence of traslocations such as t(9;22) and t(4;11), and their corresponding molecular rearrangements (BCR-ABL1 and MLL-AF4) represent per se a negative prognostic factor. Also reaching CR in a short time (3-5 weeks), ie within the first cycle of chemotherapy, is an important prognostic factor, because patients with an early CR have a better overall survival (OS). In recent years, the best parameter to assess response to therapy has become the MRD evaluation (see above) (Table VII).

 

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Table VII: Prognostic factors in ALL (≠: the prognostic value is still under discussion)

 

There is, however, no complete agreement on the most important prognostic factors for ALL; a new prognostic stratification will need to integrate many more elements: a) “conventional” clinical and biological parameters (ie features at diagnosis, time to obtain CR, BCR-ABL1+, and so on); b) the immunophenotype (B/T); c) the monitoring of MRD; d) the presence of additional cytogenetic and/or molecular abnormalities (eg Ikaros, etc). Some points need to be better clarified, such as: 1) the “time point” to define the prognostic stratification; 2) the combination of MRD with conventional risk factors; 3) the therapeutic decision based on the same stratification. Age remains an important parameter both for prognosis and for outcome. Over the years, there has been a marked improvement (Seibel NL, 2008; Pulte D, 2009) of DFS in children affected by ALL (Figure VI); however, while in children a positive outcome is achieved in over 80% of cases, this is true only for 30-40% of adult patients (Goldstone AH, 2008) (Figure VII). Various reasons may account for this difference. Pediatric protocols are more intense, while adults typically receive less intensive treatment because of their poorer tolerance to chemotherapy. It has been shown that increasing the intensity of chemotherapy, but not its duration, will improve survival rates. Although in recent years the intensification of chemotherapy has produced significant progresses, the current adult regimens allow to obtain CR rates in 85-95% of cases, but the DFS still remains largely unsatisfactory.

 

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Figure VI: Improvement of overall survival at 5 years in pediatric patients (<15 years) (adapted from Siebel NL et al. [58]).

 

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Figure VII: Overall survival at 5 years in adult patients (adapted from Goldstone AH et al. [60]).

 

Mature B-ALL

Mature B-cell ALL (FAB L3) or lymphoma “Burkitt type” is a rare entity that represents from 1% to 5% of ALL or non-Hodgkin lymphomas (NHL) in children and adults. The WHO classification (Harris Nl, 1999) recognizes the stages of lymphoma and leukemia as a single entity known as Burkitt lymphoma/Burkitt cell leukemia subtype. This subgroup shows defined clinical and biological features. Clinically, patients present important tumor masses and, often, CNS involvement. From the biological point of view, they have a characteristic morphological appearance, an immunophenotype of mature B cells (CD19, CD20, CD22 positive and TdT negative), with expression of surface Ig and in 80% of cases there are typical translocations involving chromosome 8; the most common is t(8;14)(q24,q32), but variants have been described (Burmeister T, 2005; Blum KA, 2004). These translocations lead to an inappropriate expression of the proto-oncogene c-myc on band 8q24; c-myc influences the transcription of numerous proteins involved in cell cycle regulation, apoptosis, cell growth, cell adhesion and differentiation (Kenkre VP, 2009; Patte C, 2002). Prognosis of mature B-cell ALL (FAB L3) or lymphoma “Burkitt type” has been largely unfavorable with the use of conventional therapies, with CR rates between 30% and 70%, and DFS between 0% and 30%. The current treatment regimens, focusing on the unique characteristics of these forms, are based on intensive chemotherapy and an important intrathecal prophylactic treatment, making this type of ALL a curable malignancy (Kenkre VP, 2009; Patte C, 2002). With this intensive approach 80-90% of CR rates are obtained and the survival, with the addition of the monoclonal antibody anti-CD20 (rituximab), approaches 80% (Thomas DA, 2006). Cranial radiotherapy as CNS prophylaxis and maintenance therapy have not proven beneficial and are not recommended. Future improvements for this group of patients may be obtained through the standardization of gene expression profiles (to ensure a more accurate diagnosis and prognosis of the disease and to understand the mechanisms of resistance to treatment) and the development of new forms of targeted therapy.

 

RELAPSE

Recurrence of the disease is frequent in ALL, especially in adulthood, in which, despite improved CR rates, the DFS still remains low. Relapses occur in about one-third of adult patients with standard-risk ALL and in two-thirds of high-risk patients. This is still the main problem in adult ALL; disease recurrence is often regarded as incurable. There can be several causes of a treatment failure: the emergence of resistance to the drugs used, or the selection of a resistant subpopulation within the sensitive subpopulation eliminated by the chemotherapy, or even the induction of somatic mutations that make the neoplastic cells resistant. Factors associated with the probability of recurrence can be various. Among these:

– A high WBC count at diagnosis
– Age at diagnosis (<2 yrs, >60 years)
– The extent of the disease
– The involvement of the CNS
– A more immature immunophenotype (pro-T/pro-B)
– The time needed to obtain a CR (>4 weeks)
– The first remission duration (less than one year)
– The presence of cytogenetic/molecular abnormalities associated to a poor prognosis (ie BCR-ABL1 or MLL/AF4).

A second CR is possible, but rarely post-relapse treatment provides good results in terms of long-term survival (Giona F, 1997; Thomas DA, 1999; Fielding AK, 2007; Tavernier E, 2007; Oriol A, 2010). In order to avoid the possibility of a new relapse, treatment of relapses should be more aggressive. There is no doubt that the best treatment option for relapsed (refractory) ALL patients who achieve a second remission, is represented by an allogeneic stem cell transplant (SCT), regardless of the risk factors at diagnosis, whenever the age of the patient, performance status and donor availability permits it (Popat U, 2003; Doney K, 2003).

In ALL, both in adults and children, a low risk of recurrence is associated with a good (and early) response to chemotherapy, which can be documented by the level of MRD reduction. MRD monitoring performed at well-defined time points during chemotherapy, based on an accurate biological characterization performed at diagnosis, allows an individual assessment of early response to treatment with the possibility of intervention when the tumor is still minimal, adapting the treatment and using, if necessary, specific therapies.

 

TREATMENT

Chemotherapy

Treatment of ALL is typically divided into several phases: induction, consolidation, maintenance and CNS prophylaxis (Pui CH, 2006; Fielding A, 2008; Rowe JM, 2009; Bassan R, 2011). Despite the heterogeneity of the various clinical protocols, which use different prognostic factors, the general principles are: A) Eradication of leukemic cells from the bone marrow while preserving normal progenitors. The use of active drugs, with different mechanisms of action, administered at the maximum dose, represents the intensive therapeutic approach that allows to reduce the tumor mass quickly and to avoid resistance. B) In order to eradicate the leukemic clone, an early intensive treatment with an induction phase, followed by a consolidation, is generally used. The induction phase has the purpose of achieving a morphological CR in a few weeks, eg a normalization of the bone marrow reserve. C) To establish a maintenance therapy, which allows to reduce the risk of recurrence; there is still no accordance among the various international study groups about the type and duration of maintenance. D) To perform a CNS prophylaxis during induction, consolidation and maintenance. E) To adapt the protocols to prognostic factors such as, for example, patients with ALL under the age of 1 year or with a high WBC count or high risk patients for cytogenetic and/or molecular characteristics (eg the presence of the BCR-ABL1 or MLL-AF4). In general, induction therapy is typically formed by the association of several drugs, including vincristine, prednisone and anthracyclines (daunorubicin or idarubicin or doxorubicin), which represent the “backbone” of treatment; it is common to associate these drugs with L-asparaginase and intrathecal methotrexate for CNS prophylaxis. The doses and the combination of the drugs are different according to the protocols used and if they are for pediatric or adult patients. For consolidation therapy, drugs such as cytosine arabinoside or high dose methotrexate, also exploiting their ability to pass the blood brain barrier for the prophylaxis of CNS, are generally used. The standard maintenance therapy involves the use of drugs such as 6-mercaptopurine and methotrexate associated with monthly reinductions with vincristine and prednisone for a total duration of 2-3 years. CNS prophylaxis begins already during the induction phase through medicated lumbar punctures with drugs such as methotrexate and/or cytosine arabinoside and/or prednisone in the spinal canal; it may continue after the consolidation with cranial radiation therapy and during maintenance by performing other medicated lumbar punctures. For high risk patients, for example due to the presence at diagnosis of a high WBC count (eg >50.000mm3) or of the BCR-ABL1 abnormality or to the non-response to induction therapy, it is necessary to implement a more intensive approach including a SCT from a HLA-matched donor, or if not possible, an autologous transplant (Larson RA, 1995; Gökbuget N, 2000; Annino L, 2002; Takeuchi J, 2002; Thomas X, 2004; Kantarjian H, 2004; Rowe JM, 2005).

 

Transplant

Allogeneic hematopoietic SCT is the most intensive therapy for ALL. Which patients are most likely to benefit from this procedure and which is the optimal time to perform it are important aspects that still need to be fully defined. An allogeneic transplant in first remission for high risk patients, such as Ph+ ALL, even when treated with a tyrosine kinase inhibitor (TKI), and those with a poor initial response to treatment, is widely accepted. Transplant can improve the outcome of t(4;11)+ adult ALL patients, but its benefit in children with this genetic profile is controversial. The evaluation of MRD may certainly help to better define which patients should be sent to an allogeneic transplant. However, due to the traditional debate about transplant compared to chemotherapy, opinions continue to be controversial (Popat U, 2003; Hahn T, 2006; Larson RA, 2008; Fielding AK, 2008; Goldston AH, 2009; Forman SJ, 2009).

Since about 60-70% of patients do not have a sibling donor, much has been invested in improving alternative stem cell sources: from an unrelated donor (matched unrelated donors, MUD), from umbilical cord blood, from haploidentical siblings. In recent years, the results of allogeneic transplants based on reduced intensity conditioning regimens (RIC) have been published; low rates of transplant-related mortality and good percentages of OS at 3 years have been reported, suggesting that RIC transplants are a promising alternative for selected patients in whom common conditioning regimens are not indicated (Stein AS,  2009; Forman SJ, 2009).

The role of autologous transplant in the treatment of ALL is still under discussion; despite the many practical benefits, this procedure has in fact failed to improve survival in both adults and children. Nowadays, the assessment of MRD can make this type of transplant possible for those categories of patients considered at high risk, but not suitable for an allogeneic transplant because of age, morbidity or lack of a suitable donor (Doubek M, 2009; Giebel S, 2010).

Considering the morbidity and mortality associated with these procedures and the increasing prospects of targeted therapies (see below), the need for allogeneic transplant needs to be reassessed.

 

Adolescents and young adults

Adolescents and young adults, aged between 15 and 20 years, are a particular group treated with both pediatric or adult protocols. Recent data (Boissel N, 2003; de Bont JM, 2004; Testi AM, 2004; Hallbook H, 2006; Ramanujachar R, 2007; Stock W, 2008), comparing the results obtained from pediatric protocols and those for adults fot this subgroup of patients, have shown a better survival benefit with pediatric protocols (Ribera JM, 2009) (Table VIII). If you compare the two subgroups treated with different protocols (adult-pediatric), there are no differences in design that can justify these results; the reasons that may explain the difference include: the therapeutic approach (more aggressive for children), the protocol scheme and a series of factors such as support systems, family support, much more present in the pediatric groups (Schiffer CA, 2003). Based on these results, protocols for the treatment of adolescents and young adults with “pediatric like” schemes have been carried out with positive results (Barry E, 2007; DeAngelo DJ, 2007; Ribera JM, 2008; Huguet F, 2009); the incidence of deaths related to chemotherapy, remission rates and DFS has documented a favorable trend in these studies compared to those reported from previous programs for adults. One of the main open questions is the age limit (40-50 years?) to which this more aggressive approach can be extended. A recent study (Haïat S, 2011) showed greater toxicity in patients older the age of 40 treated with “pediatric-like” protocols.

 

Foà_Acute_Lymphoblastic_Leukemia_EN_Tabella_8

Table VIII:  Retrospective comparison between adolescent and young adult treated with pediatric or adult protocols (adapted from Ribera JM et al, 2009).

 

Elderly

The management of elderly patients with ALL remains a therapeutic challenge. The presence of comorbidities, cytogenetic abnormalities and adverse biological characteristics creates significant difficulties for the success of treatment programs using conventional chemotherapy; elderly patients, commonly defined as older than 60 years, have a worse prognosis compared to younger patients when they are submitted to the same intensive care. Although the remission rates vary greatly, their chances of long-term survival is <20%; the intensification of chemotherapy in elderly patients reduces the incidence of resistance to leukemia, but also increases the incidence of death in RC for adverse events related to myelosuppression (Figure VIII) (Larson RA, 2005; Hoelzer D, 2005). In this subgroup of patients, therapy should be less intensive; palliative chemotherapy approaches are sometimes recommended for the poor performance status or the presence of comorbidities, such as diabetes, heart disease or kidney failure. There are only few published studies specifically dealing with this category of patients (Robak T, 2004; Sancho JM, 2006; O’Brien S, 2008).

In addition, Ph+ ALL (see below) occurs in more than 50% of elderly patients (Figure IX); the presence of the Philadelphia chromosome was considered until recently as a very poor prognostic factor. Paradoxically, now the presence of this translocation, with the advent of TKI, allows an improvement of prognosis with an increase in the percentage of RC and survival (Vignetti M, 2007; Ottmann OG, 2007). It is mandatory, in an elderly patient with ALL, to perform an immediate search of the BCR-ABL1 rearrangement.

 

Foà_Acute_Lymphoblastic_Leukemia_EN_Figura_8

Figure VIII: Comparison between outcomes in three different age ranges (from Larson RA [108]).

 

Central nervous system (SNC)

CNS involvement at the time of diagnosis of ALL has an incidence ranging from 5 to 8% (Fiere D, 1993; Cortes J, 1995; Kantarjian HM, 2000; Lazarus HM, 2006; Jabbour E, 2010). Most ALL protocols include the assessment of the impairment of the CNS, confirmed by the presence of lymphoid blasts in the CSF, with or without neurological dysfunction of the cranial nerves, via a diagnostic lumbar puncture. The prophylaxis and treatment of CNS involvement may consist of intrathecal therapy (medicated lumbar punctures) with methotrexate alone or in combination with cytarabine and prednisone, of a systemic treatment with high-dose cytarabine and/or methotrexate, and of cranial radiotherapy. Once the patients have achieved a rapid “clearance” of the CNS involvement, there is no evidence that there are regimens superior to the standard therapy of ALL (Lazarus HM, 2006; Thomas X, 2004); these patients are considered at high-risk and often recommended for transplant procedures. Patients with CNS involvement have a worse outcome than those without CNS involvement. Recurrence in the CNS continues to be a major complication in the management of patients with ALL; preventing new episodes of CNS involvement is a major therapeutic challenge (Sancho JM, 2006). Therapies in adults are based on the model of pediatric therapies, which use multiple treatments including radiotherapy, systemic therapy, intrathecal therapy and their combinations. Cranial irradiation is effective, but is marked by a significant toxicity and also neurological sequelae. Systemic chemotherapy, especially with cytarabine and methotrexate, has been shown capable of reducing relapses in the CNS, but therapeutic levels of drugs in the cerebrospinal fluid are not reached. Intrathecal chemotherapy with or without high-dose systemic therapy is the most common approach for CNS prophylaxis. Liposomal cytarabine (see below), recently made available for clinical use, allows to reach prolonged levels of cytarabine in the CSF, a key requirement for CNS prophylaxis (Jabbour E, 2007).

 

Ph+ ALL   

Ph+ ALL is a particular subtype of acute leukemia, historically associated with an unfavorable prognosis and a different treatment. The frequency of the Ph chromosome increases with age: about 2-5% in children/adolescents, 22% among younger adult patients (21-50 years old) and over 40% in patients older than 50, making it the most common genetic abnormality in adult ALL (Figure IX) (Secker-Walker LM, 1991; Uckun FM, 1998; Burmeister T, 2008).

 

Foà_Acute_Lymphoblastic_Leukemia_EN_Figura_9

Figure IX: Frequency by age of Ph+ ALL (from Burmeister T et al, 2008).

 

Ph+ ALL patients have high rates of recurrence and rapidly develop drug resistance; only about 10% have a favorable long-term survival with standard chemotherapy alone (Dombret H, 2002; Gleissner B, 2002; Kantarjian H, 2004). Over the years, the only potentially curative treatment for these patients has been allogeneic hematopoietic SCT, for those considered eligible. Although the transplant-related mortality rate is in the range of 30-40% and the post-transplant morbidity is significant, this approach permits good results (Error! Post not found for word:avivi i &amp; goldstone ah, 2003; Laport GG, 2008), especially in the pre-TKI era (Fielding AK, 2009).

Recently, after the development of targeted therapies, treatment in Ph+ ALL patients has evolved rapidly and has impacted on prognosis. TKI represent the most important advancement in the management of ALL throughout the last decade (Gruber F, 2009; Ottmann OG, 2009; Fielding AK, 2010). The first inhibitor used in clinic has been imatinib (Glivec®), which inhibits the TK associated with the BCR-ABL1 fusion gene and not only (it also inhibits the c-Kit and the PDGF-R); second generation inhibitors, such as dasatinib (Sprycel®) or nilotinib (Tasigna®), are currently used in therapeutic protocols for cases resistant to imatinib, but also as induction therapy.

Different cooperative groups use TKI (first or second generation) in combination with chemotherapy as induction treatment (Wassmann B, 2006; Yanada M, 2006; de Labarthe A, 2007; Bassan R, 2010). The GIMEMA group, using imatinib in combination with steroids alone as first-line treatment for elderly patients (>60 years), could show that virtually all patients obtained a CR (Vignetti M, 2007); this has also been confirmed by other groups (Ottmann OG, 2007).  Dasatinib, a second generation inhibitor, has been used in different cooperative studies with good results, although the association with chemotherapy has increased the toxicity and side effects (Rousselot P, 2008; Ravandi F, 2010). In our experience (GIMEMA), dasatinib alone with steroids as first-line treatment of adult patients (including the elderly) has enabled to reach a CR in virtually 100% of evaluable patients, with a good tolerability (part of the treatment could be delivered home) and no deaths in induction (Foà R, 2013).

Despite the high percentage of CR and good OS, the level of long-term success of the TKI treatment, with or without chemotherapy, remains yet to be defined; however, this approach currently represents the most manageable and successful first-line treatment for Ph+ ALL patients and can be a “bridge” to transplant for eligible patients (Ottmann OG, 2009; Fielding AK, 2010). The optimal post-induction treatment is being actively investigated. Some issues are still open such as: the onset of a primary resistance to inhibitors, the appearance of mutations, how and whether to perform the CNS prophylaxis. In addition, when a relapse occurs, the prognosis is definitely unfavorable; even if a second CR can be obtained, there is no consensus on what is the most appropriate treatment for patients resistant to TKI.
New therapies

New therapeutic strategies and the evolution of those commonly used are necessary, in order to improve the long-term prognosis of ALL, especially in the adult population (Thomas DA, 2002; Pui CH and Jeha S, 2007). A new generation of “targeted therapies”, directed to the signal pathways that control the cell cycle, transcription genes, cell mobility, apoptosis and cellular metabolism, is emerging (Figure X).

 

Foà_Acute_Lymphoblastic_Leukemia_EN_Figura_10

Figure X: Potential “targets” for new therapeutic agents (from Pui CH et al, 2007).

 

In addition, there are new formulations of drugs already known which can improve efficacy and reduced toxicity. Among these:

  1. A) The so-called “standard” drugs in new formulations with increased tolerability and pharmacological activity. Examples of such drugs are represented by:

– Pegylated asparaginase (PEG-L-asparaginase) is a new form of asparaginase generated by E. Coli, which has a reduced immunogenicity and a longer half-life, and allows its use as a single dose during the induction chemotherapy.
– Liposomal agents: liposomal preparations of chemotherapeutic compounds alter the pharmacological properties and toxicity of the active part of the compound, increasing the efficiency and reducing toxicity. Examples of such drugs are represented by liposomal vincristine, liposomal daunorubicin and liposomal cytarabine; these drugs are already used in several clinical trials.

 

  1. B) New drugs with different mechanisms of action from those in use; there are several compounds under investigation. Some of the most promising from the therapeutic point of view are:

– Nelarabine (2-amino-9-BD-arabinosyl-6-methoxy-9H-guanione, GW Compound 506U78): an inhibitor of PNP (purine nucleoside phosphorylase-), metabolized to ara-G (9-BD-arabinofuranosylguanine), after phosphorylation by deoxycytidine kinase, induces apoptosis through the accumulation of dGPT in T lymphoblasts (DeAngelo DJ, 2007; Cooper TM, 2007).
– Clofarabine (2-chloro-2’fluoro-deoxy-9-beta-D-arabinofuranosyladenine): the mechanism of action includes the inhibition of ribonucleotide reductase (RnR) and of DNA polymerase, with a significant anti-tumor effects; it has been proven to be effective either alone or in combination with other drugs such as cytarabine or cyclophosphamide (Faderl S, 2005; Jeha S, 2006; Vitale A, 2009).

  1. C) “Targeted” therapies capable of inhibiting a specific molecular or cellular element important for a defined subset of patients (Thomas DA, 2002; Pui CH and Jeha S, 2007). There are multiple agents aimed at targeting different mechanisms of action involved in the pathogenesis of leukemia. Among these:

– TKI: the best known example is imatinib-mesylate (Glevec®), which inhibits TK associated with the fusion gene BCR-ABL1 and others (c-Kit and the PDGF-R); as discussed, its use in Ph+ ALL patients has changed the therapeutic approach and the prognosis of these forms. Second generation TKI, such as nilotinib (Tasigna®) and especially dasatinib (Sprycel®), are used in clinical protocols for the treatment of Ph+ ALL (see above). Ponatinib a multi-targeted TKI appears to be active also in cases carrying the T315I mutation.
– Flt3 (FMS-like receptor TK) inhibitors, such as CEP-701 and PKC412 which act selectively on leukemic cells that have high levels of Flt3, normally expressed on the surface of hematopoietic cells and overexpressed on both myeloid and lymphoid neoplastic cells (in this last case, often associated with another fusion gene MLL-AF4).
– Inhibitors of farnesyl-transferase (FTIs), an enzyme involved in multiple signals necessary to proliferation, angiogenesis and reduction of apoptosis; the inhibition of this enzyme results in an anti-leukemic activity. Pre-clinical studies have demonstrated the efficacy of these inhibitors, such as SCH66336 (Sarasar) in Ph+ ALL cases.
– Inhibitors of angiogenesis, the neovascularization system that promotes the survival and proliferation of the leukemic clone. There are studies showing that angiogenesis plays a role in the pathogenesis of ALL; modulators of angiogenesis, such as vascular endothelial growth factor (VEGF) or fibroblastic growth factor (FGF), have been isolated in the supernatant of ALL cell cultures and stromal cells from bone biopsies of ALL patients. Molecules, such as SU5416, which are capable of inhibiting the process of angiogenesis, especially at the level of the bone marrow, are now being studied.

  1. D) Monoclonal antibodies (mAbs): the leukemic cell surface expresses a variety of antigens, such as CD20, CD19, CD22, CD33 and CD52, which are potential “targets” for the treatment with mAbs; an essential requirement for mAb therapy is generally the presence of the target antigen on at least 20% of the leukemic blasts, but this cut-off point is not used by all investigators. mAbs can be used as single agents or in combination with other chemotherapies, as pre-transplant “purging”, post-transplant therapy and may be particularly effective in cases with persistent MRD (Gokbuget N, 2003; Jorge Castillo, 2008; Holzer D, 2011).

Several mAbs are available for clinical use. Within these:

– Anti-CD20 (rituximab)
– Anti-CD19 (antiB4-bR; genistein)
– Anti-CD33 (gentuzumab-ozogamicin; CMA676; mylotarg)
– Anti-CD52 (campath-1H; alentuzumab)

Other mAbs are under very active investigation, such as:

– The bispecific CD19-CD3 mAb (blinatumomab) which has shown very encouraging results in patients with non-Hodgkin’s B-cell lymphoma in relapse (D’Argouges S, 2009); these data make this mAb a particularly promising candidate for the management of other B-cell diseases. Indeed, a phase II study for B-lineage ALL patients in CR with persistence or reappearance of MRD after consolidation therapy, has shown the ability of this mAb to lead to a complete MRD clearance (Topp M, 2001; Topp M, 2012a). Moreover, blinatumomab is currently being tested also in a phase II study for relapsed/refractory B-precursor ALL adults patients, in which high rates of complete hematological and molecular remission, associated with a good tolerance, have been reported (Topp M, 2012b).
– Anti-CD22: Inotuzumab ozogamicin (IO) is an anti-CD22 monoclonal antibody attached to calicheamycin which targets B lymphocytes in early stages of development, causing DNA damage and apoptosis. Its use in indolent and aggressive lymphomas has shown promising results with transient liver toxicity and thrombocytopenia. IO has allowed to obtain a response in more than 50% of refractory/relapsed B-ALL patients. Many of these patients were then eligible for an allogeneic transplant. The response obtained do not seem durable, so this drug appears suitable as a “bridge” towards a subsequent allogeneic transplant (O’Brien SM, 2012; Kebriaei P, 2013).

 

New therapeutic strategies

New therapeutic options are emerging thanks to pharmacodynamic studies, that establish the correlation between the dose of drug administered and the real active one, and pharmacogenomic studies, that are helping to highlight the differences between the metabolism of the drug and the patient’s response to it (Pui CH, 2003; Cheok MH, 2009). These studies aim at identifying therapies with a greater efficacy and reduced toxicity.

Pharmacodynamics is the study of the absorption, distribution, metabolism and excretion of drugs. These studies describe the correlations between the pharmacokinetic properties of drugs and their pharmacological effects, either positive or negative. Although age, organ functions, concomitant therapies and the type and severity of the tumor influence the effects of drugs, most of the variability among patients are due to genetic differences in the enzymes that metabolize drugs, carrying drugs and are drug targets.

Pharmacogenomics is the science that deals with hereditary genetic factors that cause a different drug activity between different people. Polymorphisms in genes that encode enzymes that metabolize, transport and are drug targets can influence a person’s response to therapy. In particular, pharmacogenomics focuses on the genetic variability of these enzymes and how these variations interact with the effect of drugs. Ultimately, the intent is to develop models that provide precisely the response and toxicity of the drug for the individual patients, and to use these models to customize prospectively treatment regimens, improving the effectiveness and security through a better understanding of pharmacogenomics characteristics of the patient.

 

Supportive care

This term indicates all types of therapy different from that administered with the intent to treat the disease; these therapies are equally important in patient management. Supportive care includes transfusions of packed red blood cells (to improve anemia), platelets (to prevent or treat bleeding), antibiotics, antifungal and antiviral drugs administered to prevent or treat active infections, drugs administered to reduce leukopenia and parenteral nutrition in case of impossibility to eat spontaneously (often because of chemotherapy-associated complications), pain medication, psychological support, etc. Supportive therapy allows a better use of antineoplastic drugs and to attenuate or prevent some of the more severe side effects, allowing to continue treatment as much as possible.

 

FINAL CONSIDERATIONS

Today, a broad and integrated diagnostic approach that combines cytomorphology to multiparameter flow cytometry, to cytogenetic and genetic analyses, is essential for the work-up of a patient with ALL. In fact, a correct diagnosis is essential not only for the classification of this heterogeneous group of disorders, but it also plays a central role in the stratification risk and individual treatment decisions.

In the last decades the intensification of consolidation treatment, the use of myeloablative therapy associated to the infusion of hematopoietic precursors and the enhanced supportive care has improved the results. New therapeutic possibilities are emerging, not only through the use of targeted therapies and mAbs, but also for a better stratification of patients into risk groups based on either clinical and biological characteristics of the disease or the early assessment of response to treatment with MRD studies.

Over the years, we have acquired a deep knowledge of the genetic events that lead to malignant transformation and of the particular metabolic pathways activated in cancer cells compared to their normal counterpart. These genes and their protein products are now potential targets for the development of therapeutic strategies aimed at targeting specifically the transformed cell. Therefore, future therapeutic strategies should include the development of protocols for the treatment of ALL that include the use of new drugs, of biological agents and of targeted therapies, all aimed at increasing the cure rate of this disease. Certainly, the increasingly sophisticated techniques of next generation sequencing today available, and in continuous improvement, will allow a better understanding of the pathogenesis of the disease and will open the way to the identification of new genetic alterations with prognostic and therapeutic implications.

 

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