Diagnostics

Acute lymphoblastic
leukemia (ALL)

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

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, so that you can inform yourself in more detail.

ALL: Classification

According to the WHO classification, ALL, together with lymphoblastic lymphoma, is assigned to the lymphoid precursor neoplasms of the B- or T-cell type, with the detection of more than 25% blasts in the bone marrow differentiating ALL from lymphoblastic lymphoma (Swerdlow et al. 2017). Classification into subgroups (Fig. 1) is primarily based on cyto- and molecular genetic criteria (Alaggio et al. 2022). Clinical relevant is furthermore the immunophenotypic classification according to EGIL, which is the basis for the classification of immunological subtypes by the GMALL study group, which are associated with specific clinical and cytogenetic aberrations.

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 bone marrow, together with cytochemistry, is part of the standard diagnostics of B-ALL. It is used to confirm the diagnosis by assessing the degree of infiltration of blasts in the bone marrow and to monitor the hematological response to therapy.

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

Immunophenotyping allows the categorization of ALL into the B- or T-cell lineage and by degree of differentiation, 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 1: 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 aberration with t(9;22) or t(4;11), which are defined as unfavorable prognostic factors in the GMALL study group, has therapeutic relevance in B-ALL. Patients with hypodiploid karyotype also have an unfavorable prognosis, whereas hyperdiploid ALL is associated with a good prognosis.

Table 2: 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::MLLT4	< 1%	unfavorable
t(8;14)(q11;q32)	Pre-B-ALL	IGH::CEBPD	< 1%	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%	unfavorable
t(14;19)(q32;q13)	Pre-B-ALL	IGH::CEBPA	< 1%	unfavorable
t(14;20)(q32;q13)	Pre-B-ALL	IGH::CEBPB	< 1%	unfavorable
t(17;19)(q22;p13)	Pre-B-ALL	TCF3::HLF	< 1%	unfavorable
9p	Pre-B-ALL	CDKN2A	15%	-

* improved through more intensive therapy

Table 3: Ploidy groups in ALL (according to Heim & Mitelman 2015)

Ploidy group	Chromosome number	Phenotype
near haploidy	25-29	c-, Pre-B-ALL
low hypodiploidy	30-39	c-, Pre-B-ALL
high hypodiploidy	42-45	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.

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 4: 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.

More than 60% 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). 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 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. 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 5: 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 6: 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 7: 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 8: 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).

ALL: Prognosis and Therapy

The GMALL study divides ALL into standard and high-risk groups based on the following factors: Leukocyte count, subtype, late CR, cyto/molecular genetic aberrations, and MRD. For an overview, see the Onkopedia guideline ALL.

MRD status during and after therapy influences event-free survival and overall survival (Brueggemann et al. 2006, Berry et al. 2017, O'Connor et al. 2017). MRD is a highly significant prognostic factor and allows early detection of relapse. Based on it, the rapid adaptation of the therapeutic strategy takes place. Quantification of MRD can be performed by molecular genetics or immunophenotyping.

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

Alaggio R et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia 2022;36(7):1720-1748.

Bene MC et al. Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia 1995;9(10):1783-1786.

Berry DA et al. Association of Minimal Residual Disease With Clinical Outcome in Pediatric and Adult Acute Lymphoblastic Leukemia: A Meta-analysis. JAMA Oncol 2017;3(7):e170580.

Brüggemann M et al. Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia. Blood 2006;107(3):1116-1123.

Flex E et al. Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia. J. Exp. Med 2008;205(4):751-758.

Grossmann V et al. The molecular profile of adult T-cell acute lymphoblastic leukemia: mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL. Genes Chromosomes Cancer 2013;52(4):410-422.

Heim S, Mitelman F. Cancer Cytogenetics: Chromosomal and Molecular Genetic Aberrations of Tumor Cells. Wiley Blackwell 2015; 4th.

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.

Moorman AV et al. Karyotype is an independent prognostic factor in adult acute lymphoblastic leukemia (ALL): analysis of cytogenetic data from patients treated on the Medical Research Council (MRC) UKALLXII/Eastern Cooperative Oncology Group (ECOG) 2993 trial. Blood 2007;109(8):3189-3197.

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

O'Connor D et al. Use of Minimal Residual Disease Assessment to Redefine Induction Failure in Pediatric Acute Lymphoblastic Leukemia. J Clin Oncol 2017;35(6):660-667.

Onkopedia Leitlinie „Akute Lymphatische Leukämie“ 2022; DGHO 2022.

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.

Swerdlow SH et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer (IARC) 2017; 4th.

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.

Van Vlierberghe P et al. PHF6 mutations in T-cell acute lymphoblastic leukemia. Nat Genet 2010;42(4):338-342.

Zenatti PP et al. Oncogenic IL7R gain-of-function mutations in childhood T-cell acute lymphoblastic leukemia. Nat Genet 2011;43(10):932-939.

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

Chromosome analysis in B-ALL

Table 2: Recurrent chromosomal aberrations in adult ALL

Table 3: Ploidy groups in ALL (according to Heim & Mitelman 2015)

Fluorescence in situ hybridization (FISH) in B-ALL

Molecular genetics in B-ALL

Table 4: Common recurrent fusion genes in B-precursor ALL

T-ALL: Diagnostic methods and their relevance

Cytomorphology in T-ALL

Immunophenotyping in T-ALL

Table 5: Classification of T-precursor ALL by immunophenotype according to EGIL

Chromosome analysis in T-ALL

Table 6: Subgroup-defining recurrent chromosomal aberrations in T-ALL

Table 7: Further recurrent balanced chromosomal aberrations in T-ALL

Fluorescence in situ hybridization (FISH) in T-ALL

Molecular genetics in T-ALL

Table 8: Frequent recurrent fusion genes in T-ALL

ALL: Prognosis and Therapy

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Acute lymphoblastic
leukemia (ALL)