Acute lymphoblastic
leukemia (ALL)

  • Method:
  • Anticoagulant:
  • Recommendation:
  • Method:
    Cytomorphology
  • Anticoagulant:
    EDTA
  • Recommendation:
    obligatory
  • Method:
    Immunophenotyping
  • Anticoagulant:
    EDTA or Heparin
  • Recommendation:
    obligatory
  • Method:
    Chromosome analysis
  • Anticoagulant:
    Heparin
  • Recommendation:
    obligatory
  • Method:
    FISH
  • Anticoagulant:
    EDTA or Heparin
  • Recommendation:
    obligatory
  • Method:
    Molecular genetics
  • Anticoagulant:
    EDTA or Heparin
  • Recommendation:
    obligatory

Acute lymphoblastic leukemia is characterized by a proliferation and accumulation of malignant lymphoid precursor cells of the B or T cell series. The incidence is 1/100,000 inhabitants per year. It is the most common malignant neoplasm in childhood. In adults, however, it accounts for only about 20% of acute leukemias.

Classification of ALL

WHO classification of ALL

According to the current WHO classification in 2017, ALL is classified together with lymphoblastic lymphoma as lymphoid precursor neoplasms of the B- or T-cell type. The detection of more than 25% blasts differentiates ALL from lymphoblastic lymphoma. A division into subgroups is made according to cytogenetic, molecular genetic and immunophenotypic criteria. Mature B-ALL or Burkitt's leukemia/lymphoma is classified as mature B-cell neoplasm. The differentiation of the mature cell Burkitt B-ALL is of high relevance, since the therapy of this subgroup differs significantly from the usual therapy regimens for B-precursor ALL.


Classification of ALL according to WHO 2017 (Swerdlow et al. 2017)

Precursor lymphoid neoplasms

  • B-lymphoblastic leukemia/lymphoma, not further classified (NOS)

  • B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities

t(9;22)(q34.1;q11.2); BCR-ABL1
t(v;11q23.3); KMT2A rearranged
t(12;21)(p13.2;q22.1); ETV6-RUNX1
Hyperdiploidy (hyperdiploid ALL)
Hypodiploidy (hypodiploid ALL)
t(5;14)(q31.1;q32.3); IL3-IGH
t(1;19)(q23;p13.3); TCF3-PBX1
B-lymphoblastic leukemia, BCR-ABL1 like
lymphoblastic leukemia with iAMP21

  • T-lymphoblastic leukemia/lymphoma

Early T-cell precursor lymphoblastic leukemia (ETP)
Pre-T-ALL
Pro-T-ALL
cortical T-ALL
mature T-ALL

  • Natural killer (NK) cell lymphoblastic leukemia/lymphoma


Clinically relevant classification of lymphoblastic leukemia according to immunological subtypes

of adult ALL patients show cytogenetic abnormalities (Moorman et al. 2007, Graux et al. 2006)

The classification according to immunological subtypes is of great importance in practice. The German Multicenter ALL Study Group (GMALL), for example, uses the EGIL classification, which classifies ALL primarily according to the degree of maturity of the leukemic cells into Pro-B-, common-, and pre-B-ALL and into Pro-T-, Pre-T-, cortical/thymic and mature T-ALL. The immunological subtypes of ALL are associated with specific clinical and cytogenetic or molecular genetic abnormalities (see Table 1).

Table 1: Classification of ALL (according to GMALL/EGIL)

Subgroup

Immunophenotyping
(characteristic markers)

Cyto-/Molecular genetics

Incidence

Common abnormalities

Molecular markers

B-lineage-ALL

HLA-DR+, TdT+, CD19+ a/o CD79a+ a/o CD22+



76%
B-precursor-ALL

Pro-B

CD10-

t(4;11)

ALL1-AF4

11%

Common

CD10+

t(9;22)

BCR-ABL1

49%

Pre-B

cylgM+

t(1;19)
t(9;22)

E2A-PBX1
BCR-ABL1

12%

T-lineage-ALL

TdT+,CD3+, CD7+

24%

Pro/Pre-T

sCD3-, CD1a-

6%

Pro-T

CD2-, CD5-, CD8-

Pre-T

CD2+ a/o CD5+ a/o CD8+

Cortical

sCD3+/-, CD1a+

12%

Mature T

sCD3+, CD1a-, TdT+/-

6%

Diagnostics of ALL

Prognosis of ALL

There are internationally accepted prognostic factors for adult ALL, whereby the various study groups use different criteria for risk stratification. The GMALL studies (see Table 7) define the high-risk group at initial diagnosis of ALL based on leukocyte count, immunophenotype and cytogenetics or molecular genetics (at least one unfavourable prognostic factor present, see Table 7). In addition, the response to therapy is monitored, especially by MRD control, and taken into account as an individual prognostic factor.

Table 7: Unfavourable prognostic factors in adult ALL

(GMALL study 08/2013)

High leucocyte count

> 30,000/µl in B-precursor-ALL

Subtype

Pro-B, ETP, mature T

Late CR

> 3 weeks (after induction II)

Cytogenetic / Molecular abnormalities

t(9;22) - BCR-ABL1
t(4;11) - KMT2A-AFF1

Minimal residual disease

Molecular failure after consolidation I (=MRD >10-4)
Molecular relapse (MRD >10-4 following CR)

Importance of the minimal/measurable residual disease

In several randomized studies, the detection of minimal residual disease beyond the cytomorphological detectability limit was shown to be a highly significant independent prognostic factor in both children and adults. MRD status during and after therapy influences both event-free survival and overall survival (Brüggemann et al. 2006, Berry et al. 2017, O'Connor et al. 2017). Therefore, MRD is now found in almost all clinical protocols for re-evaluation of individual risk assessment and optimal therapy control. In addition, continuous MRD monitoring of patients with negative MRD allows early detection of preclinical relapses and thus a rapid adaptation of the therapy strategy.

In ALL, MRD diagnostics can be performed during the course of the disease using both molecular genetic analyses and immunophenotyping. Molecular methods are based on the detection of leukemia-specific B-cell receptor and T-cell receptor rearrangements as well as leukemia-specific fusion transcripts. The sensitivity of MRD diagnostics using molecular genetics is 10-4 to 10-5. While the detection of typical fusion transcripts is very specific, clonal B-cell receptor and T-cell receptor rearrangements are not directly involved in oncogenesis. If these are altered by clonal evolution, false negative MRD results can occur. False positive results, however, are possible due to unspecific primer attachment in cases of high-grade bone marrow regeneration after therapy (Brüggemann & Kotrova 2017).

Immunophenotyping is used to define an individual leukemia-associated immunophenotype (LAIP) for each patient, which allows a quantification of MRD in the course of the disease. The sensitivity is about one log level below the sensitivity of molecular genetic methods. Like the specificity, it depends on the similarity of the leukemic blasts and the physiological precursor cells. In addition, phenotypic shifts are frequently observed both in MRD cells and in normal cells, which make the detection of leukemic cells more difficult, especially in antibody therapy (Brüggemann & Kotrova 2017).

Common entities of ALL of the B cell series

t(9;22)(q34;q11), BCR-ABL1 (WHO entity)

The translocation t(9;22)(q34;q11), which leads to a BCR-ABL1 rearrangement, represents the most frequent abnormalities in adult patients with ALL (25%). In children, the so-called Philadelphia+ ALL occurs in only 3% of patients (Pui et al. 2004).

In contrast to CML, the breakpoint in the BCR gene is found in 70% of BCR-ABL1+ ALL patients in the m-BCR region (minor) and only in 30% of patients in M-BCR (major). Additional cytogenetic abnormalities occur in 41-86% of BCR-ABL1+ ALL patients. The most frequent findings are gains of a derivative chromosome 22, a chromosome 8 and an X chromosome, the loss of a chromosome 7 and an isochromosome 8q, 9p deletions and hyperdiploidy (Moorman et al. 2007). Furthermore, in more than 60% of the BCR-ABL1+ B -ALL deletions of ICZF1 have been described, which are associated with an additionally unfavorable prognosis (Mullighan et al. 2009, van der Veer et al. 2014, Slayton et al. 2018).

Overall, there is a correlation of the translocation t(9;22)(q34;q11) with a higher age of the patients, with higher leukocyte values and with an unfavorable prognosis (Moorman et al. 2007). The treatment of BCR-ABL1+ ALL patients with a combination of chemotherapy and tyrosine kinase inhibitors currently leads to a significant improvement in the prognosis, but long-term observations indicate problems with the development of drug resistance (Ravandi 2017). Mutations that lead to resistance to tyrosine kinase inhibitors can be detected using next generation sequencing. Furthermore, the response to therapy can be quantitatively determined by MRD measurement.

1q23 (KMT2A, formerly MLL) arrangements (WHO entity)

Chromosomal abnormalities involving 11q23 are observed in about 10% of ALL patients in adulthood. In infants KMT2A rearrangements occur in 80% of ALL (Pui et al. 2004). Typically, a Pro-B-ALL phenotype is detected.

The most frequent translocations involving 11q23 are the t(4;11)(q21;q23) (KMT2A-MLLT2), t(6;11)(q27;q23) (KMT2A-MLLT4), t(9;11)(p22; q23) (KMT2A-MLLT3), t(10;11)(p12;q23) (KMT2A-MLLT10) and t(11;19)(q23;p13) (KMT2A-MLLT1), wherein more than 100 partner genes of the KMT2A gene are known. KMT2A rearrangements are associated with an unfavorable prognosis in B-precursor ALL; in particular, the translocation t(4;11)(q21;q23) (KMT2A-MLLT2) is considered a high-risk abnormality (Moorman et al. 2007, Moorman et al. 2010, Pullarkat et al. 2008).

t(12;21)(p13;q22), ETV6-RUNX1 (WHO entity)

Approximately 25% of children and 2% of adults with B-precursor ALL have a translocation t(12;21)(p13;q22) that leads to an ETV6-RUNX1 rearrangement and is considered prognostically favorable (Pui et al. 2004, Bhojwani et al. 2012).

t(1;19)(q23;p13), TCF3-PBX1 (WHO entity)

The translocation t(1;19)(q23;p13) occurs in about 5% of children and adults with ALL (Pui et al. 2004); often an unbalanced rearrangement with two cytogenetically inconspicuous chromosomes 1 and a derivative chromosome of(19)t(1;19)(q23;p13) is present. TCF3-PBX1 rearrangements were associated with an unfavorable prognosis, which could be improved by the application of more intensive chemotherapy regimens according to the ALL-BFM protocol, so that the t(1;19)(q23;p13) in children can currently be classified as prognostically favorable (Kager et al. 2007). This subtype is also no longer associated with an unfavourable prognosis in adults (Moorman et al. 2007, Burmeister et al. 2010, Lafage-Pochitaloff et al. 2017). The translocation t(17;19)(q22;p13) is a variant of t(1;19)(q23;p13) and leads to a TCF3-HLF rearrangement at the molecular level. According to the data available so far, it is associated with an unfavorable prognosis in children and adults (Moorman et al. 2012). ALL with t(17;19)(q21;p13) are not further classified as B-lymphoblastic leukemia/lymphoma according to the WHO classification 2017 (NOS).

High Hyperdiploidy (WHO entity)

ALL with a highly hyperdiploid set of chromosomes are observed in 25% of child ALL and 7% of adult ALL patients (Pui et al. 2004).

The chromosome sets have more than 50 chromosomes and usually less than 66 chromosomes (Heim et al. 2015). Characteristic for this ALL subgroup is a pattern of gains on chromosomes 4, 6, 10, 14, 17, 18 and 21 and the X chromosome. More rarely, gains of chromosomes 5 and 8 can be detected. The chromosomes are usually present as trisomies, with chromosome 21 being the most frequently gained; in 90% of cases it is present in three or more copies. In addition, 50% of the highly hyperdiploid chromosome sets show structural changes, mainly partial gains of 1q, deletions of 6q and the isochromosomes i(7q) and i(17q) (Moorman et al. 2003, Paulsson & Johansson 2009). Children with a highly hyperdiploid set of chromosomes often have mutations and deletions in the PAX5 gene (Mullighan et al. 2007). In addition, mutations in FLT3, NRAS, KRAS and PTPN11 have been detected (Case et al. 2008).

In children, a highly hyperdiploid set of chromosomes is associated with a good prognosis, especially in the presence of the so-called "triple trisomy" with gains of one chromosome 4, 10 and 17 each (Paulsson et al. 2013). This subtype can also be considered prognostically favourable in adults (Moorman et al. 2007). However, if one of the recurrent translocations t(9;22)(q34;q11), t(1;19)(q23;p13) or an 11q23 translocation occurs, its prognostically unfavourable effect is valid.

Hypodiploidy (WHO entity)

Hypodiploid chromosome sets have less than 46 chromosomes. In both children and adults they occur in 1 - 5% of ALL (Pui et al. 2004, Harrison et al. 2004).

According to the WHO 2017 classification, a further subdivision is made into nearly haploid (23 - 29 chromosomes), low hypodiploid (33 - 39 chromosomes) and high hypodiploid (40 - 43 chromosomes) (Swerdlow et al. 2017), whereby high hypodiploid chromosome sets represent a very heterogeneous group and differ prognostically from karyotypes with <40 chromosomes (Harrison et al. 2004).

Patients with a low hypodiploid karyotype typically show losses of chromosomes 3, 7 and 17 from a diploid chromosome set. In addition, monosomes of chromosomes 13, 15 and 16 are usually present, somewhat less frequently losses of chromosomes 4, 9, 12 and 20. As a rule, both chromosomes 21 are retained. The set of chromosomes is often doubled (so-called hypotriploid chromosome set, biologically very low tetraploid chromosome set). This results in a typical pattern of two or four chromosomes, which allows a distinction between hypodiploid ALL with doubled chromosome set and highly hyperdiploid ALL (Charrin et al. 2004, Mandahl et al. 2012). In more than 90% of patients with low hypodiploid chromosome set a mutation in the TP53 gene was detected (Moorman et al. 2007, Holmfelt et al. 2013, Mühlbacher et al. 2014, Stengel et al. 2014). Furthermore, alterations of the RB1 gene as well as deletions of the ICZF2 gene are frequently found, whereby the latter are biallelic due to aneuploidy (Holmfelt et al. 2013).

The pattern of chromosomal losses of the nearly haploid ALL is very similar to that of the low hypodiploid ALL. Especially the sex chromosomes as well as chromosomes 14 and 18 are mostly preserved. Chromosome 21 is also typically present in two copies. While about 95% of patients with a high hypodiploid karyotype have a complex aberrant chromosome set, structural abnormalities are rare in karyotypes with <40 chromosomes, especially in nearly haploid chromosome sets (Pui et al. 1990, Charrin et al. 2004, Harrison et al. 2004). In contrast, a doubling of the nearly haploid chromosome set is frequently observed. In contrast to the low hypodiploid ALL, TP53 mutations are rarely detected in the nearly haploid ALL. This subtype is characterized by deletions, amplifications or sequence mutations in genes of the RTK and Ras signaling pathway and the ICZF3 gene (Holmfelt et al. 2013).

Overall, the prognosis for patients with hypodiploidy is unfavourable, although it has been described as particularly unfavourable in the presence of less than 40 chromosomes in both children and adults (Harrison et al. 2004, Moorman et al. 2007, Moorman et al. 2012).

Philadelphia-like ALL (provisional WHO entity)

The Philadelphia-like ALL comprises a group of the B-precursor ALL, which shows a similar gene expression profile to the Philadelphia+ ALL, but does not have a BCR-ABL1 rearrangement. Similar to BCR-ABL1+ ALL, the incidence increases with age. It occurs in less than 10% of children and about 25% of adults with B-precursor ALL (Roberts et al. 2014, Herold & Gökbuget 2017).

In about 90% of Philadelphia-like ALL rearrangements have been shown to lead to activation of tyrosine kinase and cytokine receptor-mediated signaling pathways (Roberts et al. 2014). These include fusions of genes of the ABL class (ABL1, ABL2, CSF1R, PDGFRA, PDGFRB), rearrangements of the genes CRLF2, EPOR and JAK2, respectively, as well as other fusions and mutations that activate the JAK-STAT signaling pathway (TSLP, TYK2, FLT3, IL7R and SH2B3) (Den Boer et al. 2009, Roberts et al. 2014).

Gene expression analyses are not yet part of the standard diagnostics of ALL. However, since the definition of this subtype is based on the typical gene expression profile and the underlying genetic changes are very heterogeneous, the diagnosis of Philadelphia-like ALL is difficult. However, immunophenotyping can detect CRLF2 overexpression in about half of the cases (Iacobucci & Mullighan 2017); the mostly underlying and cytogenetically cryptic CRLF2 rearrangements can be confirmed by fluorescence in situ hybridization. In addition, the karyotype and subsequent FISH analysis can be used to identify a Philadelphia-like ALL by detecting rearrangements of the JAK2 and EPOR genes and ABL class genes. A further genetic characterization is possible by molecular genetic analysis, e.g. RNA sequencing (Harvey & Tasian 2020).

The Philadelphia-like ALL is associated with an unfavourable prognosis (Roberts et al. 2014, Harvey et al. 2020). In vitro and in vivo studies could show a sensitivity of ALL blasts to tyrosine kinase inhibitors (Tasian et al. 2017, Roberts et al. 2017). Several clinical studies are currently evaluating the therapeutic benefit of tyrosine kinase inhibitors in Philadelphia-like ALL.

iAMP21 (provisional WHO entity)

Intrachromosomal amplification of chromosome 21 is detected in about 3% of children with B-precursor ALL, in adults this subtype is very rare (<1%) (Gu et al. 2019). The prognostic significance of this abnormalitiy remains controversial. On the one hand, there are indications that iAMP21 should be assigned to high-risk abnormalities. On the other hand, the MRD status of these patients is possibly a stronger marker on which therapeutic strategies should be based (Moorman et al. 2016).

B-lymphoblastic leukemia/lymphoma, not further classified (NOS)

This group of B-precursors-ALL shows none of the genetic abnormalities defined by WHO 2017 that allow an assignment to an entity. However, rare recurrent genetic abnormalities are also found here.

Translocations of the CEBP family

In about 1% of B- ALL patients, transcription factors of the CEBP family are involved in a rearrangement with the IGH locus. These include the translocations t(8;14)(q11;q32) (IGH-CEBPD), t(14;14)(q11;q32) (IGH-CEBPE), t(14;19)(q32;q13) (IGH-CEBPA) and t(14;20)(q32;q13) (IGH-CEBPB). IGH translocations are associated with an unfavourable prognosis (Moorman et al. 2016, Lafage-Pochitaloff et al. 2017).

ZNF384-rearrangements

ZNF384 rearrangements are detected in about 3% of pediatric and 7% of adult B-precursor ALL patients, with different translocation partners described. Typically, a pro-B-ALL with expression of myeloid antigens or a mixed phenotype acute leukemia (MPAL) is diagnosed. According to the current state of knowledge, the prognosis in children and adults can be classified as intermediate (Iacobucci & Mullighan 2017, Gu et al. 2019).

Rare genetically defined subgroups

In various research projects, RNA sequencing was used to identify further subtypes that differ from each other in their genetic characteristics. These include ALL with DUX4 rearrangements associated with a favourable prognosis, MEF2D rearrangements and PAX5 alterations (Iacobucci & Mullighan 2017, Gu et al. 2019, Mullighan et al. 2019). With the PAX5 P80R mutation, a subtype was identified for the first time that is defined by a point mutation. In addition, groups were detected that exhibit an ICZF1 N159Y mutation or rearrangement involving the genes BCL2/MYC, HLF (usually TCF3/TCF4-HLF) or NUTM1 and that show distinct gene expression profiles. For further cases that have not been classified so far, it could be shown that they correspond to already existing phenotypes without showing the respective characteristic genetic change (ETV6-RUNX1-like, KMT2A-like, ZNF384-like ALL) (Iacobucci & Mullighan 2017, Gu et al. 2019, Mullighan et al. 2019). These results show the heterogeneity of acute lymphoblastic ALL.

Subgroups of ALL of the T cell series

Early T-cell precursor lymphoblastic leukemia (ETP) (according to WHO 2017)

In the WHO 2017 classification, ETP is listed as a separate entity.
ETP-ALL occurs in about 11% of children and 7% of adults and is formed
from thymus cells in the early differentiation stage of T-cell
precursors (ETP). Due to their low differentiation potential and their
similarity to haematopoietic stem cells and myeloid progenitor cells,
their immunophenotype, besides the absence of CD1a, CD8 and the weak
expression of CD5, shows positivity for one or more stem cell markers or
myeloid antigens. The gene expression profile is also more similar to
myeloid leukemias than T-cell leukemias. A lower incidence of NOTCH1 mutations and a frequent occurrence of FLT3, DNMT3A, IDH1 and IDH2
mutations as well as mutations of the RAS gene family have been
described (Zhang et al., 2012). Furthermore, ETP-ALL is associated with a
significantly worse prognosis in children and young adults compared to
other T-ALL/LBL subtypes (Coustan-Smith et al. 2009).


Molecular subgroups of the T-ALL

Gene expression studies, in addition to the classification of T-ALLs by their immunophenotype, have also identified molecular subgroups with unique gene expression signatures. The four subgroups are based on the overexpression of the transcription factors TAL1, TLX1, TLX3 and the genes of the HOXA gene cluster. About 3% of pediatric patients with T-ALL show aberrant expression of TAL1 due to translocation t(1;14)(p32;q11). Furthermore, the cytogenetic cryptic intrachromosomal deletion in the short arm of chromosome 1 leads to the fusion gene STIL-TAL1. Both mechanisms lead to an overexpression of TAL1. Another recently discovered mechanism shows changes in the chromatin structure near the TAL1 gene, indicating the presence of a new enhancer region. Detailed analysis of this region led to the identification of mutations that result in a de novo binding site for MYB, which leads to the recruitment of additional transcriptional regulators and activation of TAL1 expression (Navarro et al. 2015, Mansour et al. 2014).      

In contrast, 8% of infantile T-ALLs and 20% of adult T-ALLs exhibit translocations involving the TRA/D locus (14q11) or the TRB locus (7q34) with the TLX1 oncogene (t(10;14)(q24;q11),t(7;10)(q34;q24)), resulting in the overexpression of TLX1. The overexpression of TLX1 seems to be associated with a more favourable prognosis and a low risk of recurrence (Ferrando et al. 2004).        

Another subgroup is defined by the overexpression of the TLX3 transcription factor, which is caused by the translocation t(5;14)(q35;q11) or t(5;14)(q35;q32), where TLX3 is under the control of the TRA/D locus(14q11) or the BCL11B gene (14q32). In contrast to TLX1, the prognosis seems to be less favourable and recurrences have been described more frequently in patients with TLX3 overexpression (Baak et al. 2008).  

The fourth molecular subgroup shows an aberrant expression of the HOXA gene cluster (7p15). Cytogenetically recurrent alterations resulting in overexpression of genes of the HOXA cluster were described in 5% of infantile T-ALLs and 8% of adult T-ALLs. Inversion 7 (inv(7)(p15q34)) predominantly leads to overexpression of HOXA9 and HOXA10. Cryptic deletions in 9q34 leading to the fusion gene SET-NUP214 and the translocation t(10;11)(p13;q10) leading to the PICALM-MLLT10 fusion gene have also been associated with the overexpression of HOXA genes.

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