Acute lymphoblastic
leukemia (ALL)

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Based on the current guidelines and the current state of research, there are different diagnostic recommendations for patients with acute lymphoblastic leukemia. We have summarized the most important information on classification and diagnostic methods at MLL. In addition, we provide further links and literature on prognosis and therapy in 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
Recommendation:

obligatory

Classification
B-ALL: Diagnostic methods
T-ALL: Diagnostic methods
Prognosis and therapy

ALL: Classification

According to the WHO classification, ALL is classified together with lymphoblastic lymphoma (LBL) as lymphoid precursor neoplasms of the B- or T-cell type.

The term ALL is conventionally used for cases in which the peripheral blood and bone marrow are primarily affected, and the term LBL for cases in which lymph nodes or extranodal sites are primarily affected. If both the bone marrow and extramedullary organs/sites are affected, the distinction becomes arbitrary - however, many treatment protocols define leukemia when >20-25% blasts are present in the bone marrow. Classification into subgroups (Fig. 1) is primarily based on cytogenetic and molecular genetic criteria (WHO 2022). The immunophenotypic classification according to EGIL, which is used to classify the risk groups of the GMALL study group, is also clinically relevant. In addition, the immunological subtypes are associated with specific clinical and cytogenetic aberrations.

Fig. 1: ALL classification according to WHO (WHO 2022) and GMALL/EGIL

Table 1: Clinical Presentation of ALL (Gökbuget et al., Blood 2024 [2]; Onkopedia 2022, WHO 2022)

Feature	B-ALL	T-ALL
Typical age of onset	At any age, more common in children	More frequent in adolescents and young males
Mediastinal mass	Uncommon	Common (involvement of the thymus)
CNS infiltration	5-10% at diagnosis	Higher risk in T-ALL
Lymphadenopathy	Frequent	Common
Leukostasis symptoms (e.g., shortness of breath, neurological signs)	Possible with hyperleukocytosis	Possible with hyperleukocytosis

For B-ALL (B-lymphoblastic leukemia/lymphoma) and T-ALL (T-lymphoblastic leukemia/lymphoma, NOS), different diagnostic recommendations apply in each case. Therefore, we have summarized them separately for you below:

B-ALL: Diagnostic methods and their relevance

Cytomorphology from the bone marrow, together with cytochemistry, is part of standard ALL diagnostics. It is used to confirm the diagnosis by assessing the degree of infiltration of blasts in the bone marrow (Gökbuget et al. 2024 [1]) and to monitor the hematologic response to therapy.

In ALL, morphologically undifferentiated blasts are found that show no relevant reaction with myeloperoxidase (<3%) or non-specific esterase in the cytochemistry.

Immunophenotyping allows the categorization of ALL into the B- or T-cell lineage and by degree of differentiation (Gökbuget et al. 2024 [1]), on which the EGIL classification is based. The diagnostic confirmation of the immunophenotypic subtype has a clinical relevance in risk stratification and therapy decision. In addition, this analysis is also used as a therapy control to determine the measurable residual disease (MRD), especially in the absence of molecular markers.

Table 2: Classification of B-precursor ALL by immunophenotype according to EGIL

	B-precursor-ALL
Antigen	Pro-B-ALL	c-ALL	Pre-B-ALL
cCD22*	+	+	+
CD79a	+	+	+
CD19	+	+	+
CD24	+/-	+	+
CD20	-/(+)	+/-	+/-
slg*	-	-	-
clgM*	-	-	+
cCD3*	-	-	-
sCD3*	-	-	-
CD7	-	-	-
CD5	-	-	-
CD2	-	-	-
CD1a	-	-	-
CD4	-	-	-
CD8	-	-	-
CD10	-	+	+/-
HLA-DR	+	+	+
CD34	+	+	+
TdT	+	+	+

*c: cytoplasmic; s: "surface", membrane-bound

The karyotype is necessary for classification according to WHO; however, the prognostic assessment of the cytogenetic findings is even more important.

In particular, cytogenetic aberrations with t(8;22) or t(4;11), which are defined as unfavorable prognostic factors in the GMALL study group, have therapeutic relevance. Patients with hypodiploid karyotype also have an unfavorable prognosis, whereas hyperdiploid ALL is associated with a good prognosis.

Table 3: Recurrent chromosomal aberrations in adult ALL

Chromosomal aberration	Phenotype	Genes	Frequency	Prognosis
t(1;19)(q23;p13)	Pre-B-ALL	TCF3::PBX1	3%	unfavorable *
t(4;11)(q21;q23)	Pro-B-ALL	KMT2A::AFF1 (MLL-AF4)	6%	unfavorable
t(6;11)(q27;q23)	B-ALL	KMT2A::AFDN	< 1%	unfavorable
t(8;14)(q11;q32)	Pre-B-ALL	IGH::CEBPD	< 1%	intermediate/unfavorable
t(9;11)(p21;q23)	Pro-, Pre-B-ALL	KMT2A::MLLT3	< 1%	unfavorable
t(9;22)(q34;q11)	c-ALL	BCR::ABL1	25-30%	unfavorable
10p12/11q23	Pro-, Pre-B	KMT2A::MLLT10	< 1%	unfavorable
t(11;19)(q23;p13)	Pro-, Pre-B-ALL	KMT2A::MLLT1	1%	unfavorable
t(12;21)(p13;q22)	Pre-B-ALL	ETV6::RUNX1	< 1%	favorable
t(14;14)(q11;q32)	Pre-B-ALL	IGH::CEBPE	< 1%	intermediate/unfavorable
t(14;19)(q32;q13)	Pre-B-ALL	IGH::CEBPA	< 1%	intermediate/unfavorable
t(14;20)(q32;q13)	Pre-B-ALL	IGH::CEBPB	< 1%	intermediate/unfavorable
t(17;19)(q22;p13)	Pre-B-ALL	TCF3::HLF	< 1%	unfavorable
9p	Pre-B-ALL	CDKN2A	15%	-

* improved through more intensive chemotherapy

Table 4: Ploidy groups in ALL (according to WHO 2022)

Ploidy group	Chromosome number	Phenotype
near haploidy	24-31	c-, Pre-B-ALL
low hypodiploidy	32-39	c-, Pre-B-ALL
high hypodiploidy	40-43	B-ALL
high hyperdiploidy	51-65	c-, Pre-B-ALL

Highly hyperdiploid ALL show a characteristic pattern of gains of chromosomes 4, 6, 10, 14, 17, 18, and 21 and the X chromosome. Patients with low hypodiploid karyotype typically show losses of chromosomes 3, 7, 13, 15, 16, and 17.

FISH can detect the presence of a BCR::ABL1 or KMT2A rearrangement within 24 hours. Furthermore, FISH is often used in addition to classical chromosome analysis to confirm detected aberrations and to establish a baseline for follow-up to detect residual disease under therapy or to detect cytogenetically cryptic aberrations.

The so-called “chromosome painting” with 1-3 or 24 colors (24-color FISH) on metaphase chromosomes is performed in addition to classical chromosome analysis if the karyotype cannot be unambiguously clarified by chromosome analysis after Giemsa banding. This is often the case in complex aberrant karyotypes.

Molecular genetics allows, among other things, the identification of high-risk groups with t(9;22)(q34;q11) or t(4;11)(q21;q23) by detection of the corresponding fusion transcripts BCR::ABL1 or KMT2A::AFF1 (formerly MLL::AF4).

Table 5: Common recurrent fusion genes in B-precursor ALL

Cytogenetic	Fusion gene	Subtype	Frequency
Cytogenetic	Fusion gene	Subtype	Children	Adults
t(1;19)(q23;p13)	TCF3::PBX1	Pre-B-ALL	5-6%	3%
t(4;11)(q21;q23)	KMT2A::AFF1 (MLL::AF4)	Pro-B-ALL	2%	6%
t(11;19)(q23;p13)	KMT2A::MLLT1	Pro-B-ALL, Pre-T-ALL	< 1%	< 1%
t(9;22)(q34;q11)	BCR::ABL1	c-, Pre-B-ALL	2-5%	25-30%
t(12;21)(p13;q22)	ETV6::RUNX1	c-ALL	10-20%	< 1%

Furthermore, transcriptome analysis (RNA-Seq) can be used to characterize the gene expression profile, such as in the BCR::ABL1-like subgroup, thereby enabling the identification of therapeutically targetable structures.

60-80% of BCR::ABL1-positive B-precursor ALL or BCR::ABL1-like ALL additionally have an IKZF1 deletion, leading to an even less favorable prognosis (Mullighan et al. 2008, Martinelli et al. 2009, van der Veer et al. 2014, WHO 2022). Another prognostically unfavorable factor, especially when both alleles are affected, are mutations in the TP53 gene. These are most frequently observed in ALL with a low hypodiploid chromosome set or MYC rearrangements (Stengel et al. 2014).

T-ALL: Diagnostic methods and their relevance

Cytomorphology from bone marrow, together with cytochemistry, is part of the standard diagnostics of T-ALL. It is used to confirm the diagnosis by assessing the degree of infiltration of blasts in the bone marrow (Gökbuget et al. 2024 [1]) and to monitor the hematological response to therapy.

In T-ALL, morphologically undifferentiated blasts are found which do not show any relevant reaction with myeloperoxidase (<3%) or non-specific esterase in cytochemistry.

With the aid of immunophenotyping, ALL can be assigned to the B or T cell lineage and classified according to the degree of differentiation (Gökbuget et al. 2024 [1]). This is the basis for the EGIL classification (Table 5).

Confirmation of the diagnosis of the immunophenotypic subtype furthermore has clinical relevance with regard to risk stratification and therapy decisions.

This analysis is also used as a therapy control to determine the measurable residual disease (MRD), especially in the absence of molecular markers.

Table 6: Classification of T-precursor ALL by immunophenotype according to EGIL

T-precursor ALL
Pro-T-ALL	cCD3*, CD7, (CD10), (HLA-DR), (CD34), (TdT)
Pre-T-ALL	cCD3*, CD7, CD2 or CD5 (in >75% of blasts and highly expressed), CD4-CD8- or CD4+CD8+ (“double negative” or “double positive”), (CD10), (HLA-DR), (CD34), TdT
Cortical T-ALL	cCD3, (sCD3), CD7, CD5, CD2, CD1a, CD4+CD8+, (CD10), TdT
Mature T-ALL	sCD3*, CD7, CD5, CD2, CD4 oder CD8, (TdT)
ETP-ALL	CD7, CD8-, CD1a-, CD5 (<75% of blasts and weakly expressed), CD34, KIT, HLA-DR, CD13, CD33, CD11b, CD65**

*c: cytoplasmic, detectable inside the cell; s: "surface", detectable on the cell surface; ** often positive for one or more myeloid markers/stem cell markers

T-ALL occurs with a frequency of 15% in childhood ALL and 25% in adult ALL patients and characteristically affects males more often than females. Chromosomal aberrations can be detected in 50-70% of patients with T-ALL. The most common cytogenetic alterations represent translocations associated with aberrant expression of oncogenic transcription factors. Based on the oncogene involved, T-ALLs can be classified into molecular subgroups (Table 6). Furthermore, in addition to common copy number alterations such as deletions of 9p (65-70%) and 6q (20-30%), other non-subgroup-specific recurrent aberrations are observed (Tab. 7).

Table 7: Subgroup-defining recurrent chromosomal aberrations in T-ALL

Molecular subtype	Chromosomal aberration	Genes	Frequency
TLX1	t(10;14)(q24;q11)	TLX1::TRAD	5-10%
TLX1	t(7;10)(q34;q24)	TRB::TLX1	<1%
TLX3	t(5;14)(q35;q32)	TLX3::BCL11B	20% (children), rare in adults
TAL1	t(1;14)(p32;q11)	TRAD::TAL1	3%
TAL1	del(1p32)	STIL::TAL1	9-26%
HOXA	inv(7)(p15q34)	HOXA::TRB	3%
HOXA	del(9)(q34q34)/t(9;9)(q34;q34)	SET::NUP214	rare
HOXA	9q34/Episomal amplification	NUP214::ABL1	5%
HOXA	t(10;11)(p12;q14)	PICALM::MLLT10	5%
HOXA	t(X;10)(p11;p12)	DDX3X::MLLT10	3%
HOXA	t(4;11)(q23;p15)	NUP98::RAP1GDS1	rare
BCL11B*	BCL11B/14q32 rearrangements (frequent partners: 8q24 or 6q25)		<5%

*also in MPAL and undifferentiated AML

Table 8: Further recurrent balanced chromosomal aberrations in T-ALL

Chromosomal aberration	Genes	Frequency
KMT2A rearrangements	KMT2A::MLLT1 (most often), ELL, MLLT10, AFDN	8%
t(9;12)(q34;p13)	ETV6::ABL1	rare
t(11;14)(p15;q11)	TRAD::LMO1	rare
t(11;14)(p13;q11)	TRAD::LMO2	rare
t(7;9)(q34;q34)	TRB::NOTCH1	rare
t(4;14)(q25;q11)	TRAD::LEF1	rare
t(6;7)(q23;q34)	TCRB::MYB	rare

FISH can detect the presence of a BCR::ABL1 or KMT2A rearrangement within 24 hours. FISH is also frequently used in addition to classical chromosome analysis to confirm aberrations detected there.

Molecular genetic analyses can detect fusion genes that play a major role in the development of T-ALL (Table 8).

Table 9: Frequent recurrent fusion genes in T-ALL

Cytogenetic	Fusion gene	Frequency
1p32 deletion	STIL::TAL1	9%-26%
del(9)(q34q34)/t(9;9)(q34;q34)	SET::NUP214	rare
9q34/Episomal amplification	NUP214::ABL1	5%
t(10;11)(p12;q14)	PICALM::MLLT10	5%

In addition, mutations in various genes occur in T-ALL. These include activating mutations in the NOTCH1 gene in over 50% of T-ALL cases and FBXW7 mutations in about 20%. FBXW7 mutations contribute to NOTCH activation. The absence of a NOTCH1/FBXW7 mutation and/or the presence of a mutation in PTEN (6-10%) and N-/K-RAS genes have been described as high-risk features (Trinquand et al. 2013). TP53 mutations occur in approximately 9-15% of adult T-ALL patients and are associated with a significantly poorer prognosis (median overall survival: 1.6 years vs. 9.5 years in TP53 wild-type patients). The mutations often occur together with NOTCH1/FBXW7 mutations. Unlike in myeloid neoplasms, biallelic status has no additional adverse effect on prognosis (Harris et al. 2025).

ALL: Prognosis and Therapy

The GMALL study divides ALL into standard and high-risk groups based on the following factors: Leukocyte count, immunophenotype, late CR, cyto/molecular genetic aberrations, and MRD. For an overview, see the Onkopedia guideline ALL. The European Leukemia Net (ELN) also provides an overview of therapy management (Gökbuget et al. 2024 [2]).

MRD status during and after therapy influences event-free survival and overall survival (Gökbuget et al. 2024 [2]). MRD is a highly significant prognostic factor and enables early detection of relapse. It is used to rapidly adapt the treatment strategy. MRD can be quantified using molecular genetics or immunophenotyping.

NGS (next-generation sequencing)-based MRD assays show superior sensitivity compared to conventional multiparameter flow cytometry and can detect residual disease in patients who are MRD-negative by flow cytometry. Achieving NGS-based MRD negativity correlates strongly with improved event-free and overall survival and is highly predictive of the risk of relapse after stem cell transplantation and CAR-T cell therapy (Stefan et al. 2025).

The prognostic significance of MRD is influenced by disease-related factors (cytomic molecular risk, immunophenotype) and assay-related parameters (sensitivity, sample material, measurement time). Current expert panels therefore recommend integrating MRD information into clinical decision algorithms for therapy management in the context of these factors (Short et al. 2025).

Due to the complexity and longevity, therapy should be performed in a hematological center.

Biobanking of blood and bone marrow samples is recommended (Gökbuget et al. 2024 [1]) – preferably within a clinical trial or registry. These samples can support future molecular or cytogenetic testing, especially in cases of treatment resistance, relapse, or uncertain therapeutic response. Biobanked material may help identify eligibility for targeted therapies at later stages of treatment.

Table 10: Treatment Response Gökbuget et al., Blood 2024

Feature	B-ALL	T-ALL
Response to standard chemotherapy	Good in standard-risk patients	Similar to B-ALL; subtype-dependent (favorable for cortical T-ALL, poor for ETP/early T-ALL)
Role of stem cell transplantation	Indicated in high-risk cases (poor MRD response, high-risk genetics)	Indicated in high-risk cases (poor MRD response, ETP/immature T-ALL)
Targeted therapy options	e.g., TKIs for BCR::ABL1-positive cases	e.g., NOTCH1 inhibitors , BCL-2 inhibitors (in development)

Status: December 2025

Gökbuget N et al. Diagnosis, prognostic factors, and assessment of ALL in adults: 2024 ELN recommendations from a European expert panel. Blood 2024 [1];143(19):1891-1902.

Gökbuget N et al. Management of ALL in adults: 2024 ELN recommendations from a European expert panel. Blood 2024 [2];143(19):1903-1930.

Harris, EJ, et al. Clinical and molecular characterization of TP53-mutant acute lymphoblastic leukemia in adults. Blood Cancer J 2025;15(1):138.

Iacobucci I et al. Single-cell analysis of acute lymphoblastic and lineage-ambiguous leukemia: approaches and molecular insights. Blood 2023;141(4):356-368.

Martinelli G et al. IKZF1 (Ikaros) deletions in BCR-ABL1-positive acute lymphoblastic leukemia are associated with short disease-free survival and high rate of cumulative incidence of relapse: a GIMEMA AL WP report. J Clin Oncol 2009;27(31):5202-5207.

Mullighan CG et al. BCR-ABL1 lymphoblastic leukemia is characterized by the deletion of Ikaros. Nature 2008;453(7191):110-114.

Onkopedia guideline "Acute Lymphoblastic Leukemia" 2022; DGHO 2022.

Short NJ et al. Clinical use of measurable residual disease in adult ALL: recommendations from a panel of US experts. Blood Adv 2025;9(6):1442-1451.

Ștefan AI et al. The emerging role of next-generation sequencing in minimal residual disease assessment in acute lymphoblastic leukemia: a systematic review of current literature. Front Med (Lausanne) 2025;12(1570041).

Stengel A et al. TP53 mutations occur in 15.7% of ALL and are associated with MYC-rearrangement, low hypodiploidy, and a poor prognosis. Blood 2014;124(2):251-258.

Trinquand A et al. Toward a NOTCH1/FBXW7/RAS/PTEN-Based Oncogenetic Risk Classification of Adult T-Cell Acute Lymphoblastic Leukemia: A Group for Research in Adult Acute Lymphoblastic Leukemia Study. J Clin Oncol 2013;31(34):4333-4342.

van der Veer A et al. IKZF1 status as a prognostic feature in BCR-ABL1-positive childhood ALL. Blood 2014;123(11):1691-1698.

WHO Classification of Tumours Editorial Board. Haematolymphoid tumors [Internet]. Lyon (France): International Agency for Research on Cancer; 2022. (WHO classification of tumors series, 5th ed.; vol. 11). Available from: https://tumourclassification.iarc.who.int/chapters/63.

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Acute lymphoblastic leukemia (ALL)

ALL: Classification

B-ALL: Diagnostic methods and their relevance

Cytomorphology in B-ALL

Immunophenotyping in B-ALL

Table 2: Classification of B-precursor ALL by immunophenotype according to EGIL

Chromosome analysis in B-ALL

Table 3: Recurrent chromosomal aberrations in adult ALL

Table 4: Ploidy groups in ALL (according to WHO 2022)

Fluorescence in situ hybridization (FISH) in B-ALL

Molecular genetics in B-ALL

Table 5: Common recurrent fusion genes in B-precursor ALL

T-ALL: Diagnostic methods and their relevance

Cytomorphology in T-ALL

Immunophenotyping in T-ALL

Table 6: Classification of T-precursor ALL by immunophenotype according to EGIL

Chromosome analysis in T-ALL

Table 7: Subgroup-defining recurrent chromosomal aberrations in T-ALL

Table 8: Further recurrent balanced chromosomal aberrations in T-ALL

Fluorescence in situ hybridization (FISH) in T-ALL

Molecular genetics in T-ALL

Table 9: Frequent recurrent fusion genes in T-ALL

ALL: Prognosis and Therapy

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Acute lymphoblastic
leukemia (ALL)