Educational theme | Diagnostic approaches and risk stratification in T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive subtype of ALL which can be clinically presented with leukemic and/or lymphomatous manifestations.¹ According to the World Health Organization (WHO) classification, the current diagnostic approach for ALL relies on a combination of morphological, immunological, and genetic/cytogenetic-based assessments.²

Immunophenotypic-based diagnostic algorithms of ALL identify B- and T-cell lineage subtypes.^2,3 High-resolution genomic-based techniques have further characterized distinct T-ALL genetic subtypes and their association with the stages of thymocyte development; these represent key diagnostic biomarkers in T-ALL but often at a later stage of the process^2,3,4Accurate diagnosis of ALL subtypes can inform personalized therapy options and is important for optimal risk stratification.²

Our previous educational theme article focused on the biological heterogeneity, incidence, and prognostic relevance of T-ALL. In this education theme, we explore the diagnostic approaches and risk stratification of T-ALL.

Immunophenotyping diagnostics

Multi-channel flow cytometry (MFC) is the standard procedure used for the diagnosis and subclassification of the immunophenotypic features of ALL. New MFC strategies have been developed by the Euro Flow Consortium to achieve higher accuracy and reliability of diagnostic tests.²

Through immunophenotyping, CD1a, CD2, cytoplasmic and membrane/surface CD3, CD4, CD5, CD7, and CD8 have been identified as T-cell specific markers.^1,2 Positive expression of cytoplasmic CD3 and CD7 is commonly seen, with variable expression of the others.¹ CD10 antigens are observed in up to 25% of T-ALL cases in a non-specific manner and expression of CD34, alongside myeloid markers CD33 and CD13, has also been observed in T-ALL.²

T-ALL subtypes associated with thymocyte differentiation stages include pro-T-ALL, pre-T-ALL, cortical T-ALL, mature T-ALL, and the more recent early T-precursor (ETP-ALL).^1,2 Each of these subtypes can be identified based on unique immunological features.²  

The immunological profile of immature T-ALLs (ETP-ALL, near- ETP, and pro-T) show marginal differences in their antigen expression; however these subsets are not well identified due to the presence of overlapping phenotypic features with other ALL subtypes.^3,4

ETP-ALL has signature characteristics with a lack of CD1a and CD8 expression, weak CD5 expression, and at least one myeloid and/or stem cell marker.³ Table 1 details the immunological diagnosis of each T-ALL subtype in relation to the stage of differentiation.

Table 1. Immunophenotypic classification of T-ALL subtypes based on differentiation stages*

Genetics/cytogenetics-based diagnostics

Cytogenetics and fluorescence in situ hybridization enabled the identification of prevalent chromosomal translocations in the initial stages of T-ALL genomic characterization.⁴  however, determining the prognostic value of these aberrations was challenging due to their low incidences in the specific T-ALL cohorts analyzed.⁴ Common T-ALL aberrations detected through cytogenetics usually involve 14q11 breakpoints, such as t(10;14) (q24;q11) and t(11;14)(p13;q11).²

The application of novel genomic techniques such as gene expression profiling, comparative genomic hybridization arrays, and next-generation sequencing has led to a more comprehensive overview of the genetic subtypes of T-ALL at diagnosis and elucidated associations between specific genetic alterations and stages of thymocyte development.⁴

Initial studies, involving the use of karyotyping alongside gene expression analyses, identified a link between chromosomal rearrangements and overexpression of the transcription factor genes TAL1, TAL2, LYL1, LMO1, LMO2, TLX1, and TLX3.⁴

Combined use of genomic techniques has revealed distinct mutation patterns of T-ALL differentiation stages.⁴ For example, molecular-cytogenetic and sequencing studies revealed a higher occurrence of CDKN2AB deletions (such as NOTCH1, FBXW7, PTEN, and RPL10) and translocations in T-related transcription factors (such as TAL1, LMO1, LMO2, MYB, NKX2.1, and TLX1) in typical T-ALL compared with rarer occurrence in immature T-ALL.³ Moreover, the age distribution (pediatrics vs adulthood) of genetic aberrations within the rarer ETP-ALL subtype has been characterized using whole genome sequencing.^3,4 One analysis found that mutations involved in the RAS signaling pathway, hematopoietic development, and histone modifier genes were typical of childhood ETP-ALL; conversely, DNMT3A alterations were more frequent in adult patients and those aged >60 years.^3,4

Whilst the immunological characteristics of immature T-ALL have been identified, the genetic basis and clinical relevance of pro-T and near-ETP types remain unclear.⁴

Risk stratification of T-ALL

In adult patients with T-ALL, measurable residual disease (MRD)-based pediatric protocols have been a useful tool for precise risk stratification.³ A study by the German ALL Group (GMALL) found that adult patients with standard-risk ALL (33% of whom had T-ALL) and a rapid decline in their MRD status within the first month of therapy, experienced no relapses at the 3-year mark.¹ A further 2009 GMALL study, which included 744 patients aged 15–55 years with T-ALL, indicated early T-ALL and mature T-ALL as high-risk subgroups.¹

In a Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL) study, it was found that patients with T-ALL who had higher MRD levels (≥10⁻⁴), harbored N/K-RAS mutations and/or PTEN gene alterations, and lacked the NOTCH1/FBXW7 mutation were at a higher risk of relapse; 59% of the patients with T-ALL were represented in this high-risk subgroup.¹

Given the uncertainty of the distinct clinical and prognostic relevance of immature T-ALL subsets and the lack of information surrounding their classification, they have been deemed as high-risk subgroups in both adults and children.³ The ETP-ALL subgroup was once defined as a high-risk subgroup with poor prognostic outcomes; however, recent studies have shown that this is not always the case.¹

Patients with the T-ALL related translocation t(8;14) with breakpoints at q24;q11, often present with aggressive and lymphomatous manifestations.²

Relapse-specific mutations in T-ALL

Although intensive chemotherapy treatments have been essential for increasing survival rates in children with T-ALL (up to 93%), adult survival rates are still very low (<50%). Across all ages there remains a poor prognosis for T-ALL patients with primary-resistant or relapsed leukemia, primarily because of the disease's progression and the dearth of effective treatments. Ongoing research to better understand relapse mechanisms and find potential new treatment targets using multi-omics techniques has helped to identify private and shared variants in matched-diagnosis, germline, or remission and relapse DNA samples.⁴

In a large cohort (N = 175) of patients with both B-cell precursor (BCP)-ALL (n = 129) and T-ALL (n = 46), the most prominent relapse-specific genetic lesions linked to chemotherapy resistance in ALL were mutations in NT5C2 (12%). Additionally, SETD2 (2.3%), NR3C1 (1%), WHSC129 (4.6%), WT1 (6.2%), and CREBBP9 (9.7%) gene mutations were more common in relapsed samples. TP53 was found to be diagnosed more frequently in adults than in pediatric patients. In a similar study examining 103 pediatric ALL triplets (87 with BCP and 16 with T-cell ALL), relapse-specific somatic alterations were found to be enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) that were all involved in the drug response.⁴

Conclusion

There is currently no consensus approach for the diagnosis and management of T-ALL. Although immunophenotyping is presently part of the immediate diagnostic approach for T-ALL, genetic-based assessments at a later stage can facilitate a better diagnosis of the T-ALL molecular landscape and aid in risk stratification. For example, implementation of routine screening to identify high-risk patients with a T-ALL-specific gene expression profile could have a clinical benefit. Additionally, relapse in T-ALL should be managed by appropriate genomic testing to increase survival.

There are challenges in the characterization of rarer subtypes of T-ALL such as immature T-ALL that still need to be elucidated. Further research into the genomic characterization of T-ALL will improve diagnostic approaches, aid in relapse management, and optimize therapies for patients in the clinical setting.