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T-lineage acute lymphoblastic leukemia (T-ALL) was first described over 4 decades ago. It was initially identified by the capacity of T-ALL cells to form rosettes when incubated with sheep erythrocytes. Rosetting with sheep erythrocytes was used at that time to distinguish T lymphocytes from other cells. Rosette formation is thought to be due to binding of CD2 on the surface of T cells to a CD58 homologue on the surface of the sheep red blood cells.

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The clinical features of childhood T-ALL are well established. T-ALL also occurs in adult patients where represents approximately 25% of ALL

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T-ALL cells appear to be generally more resistant to chemotherapy than B-lineage ALL in vitro and in vivo

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Modern treatment of leukemia is based on the implementation of risk-adapted therapy. It is important to determine relative risk of relapse to avoid over- or under-treatment.

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This study from the Pediatric Oncology Group illustrates well the lack of strong correlations between presenting clinical features and response to therapy in childhood T-ALL as compared to B-lineage ALL. The top panels show event-free survival curves in ALL patients divided according to lineage (B vs T) and risk (based on age plus white blood cell count) (top left); lineage and age (top right). While in B-lineage ALL these factors identify groups of patients with a distinct prognosis, in T-ALL they did not. The bottom panels show event-free survival curves for patients with T-ALL according to white blood cell count (WBC; bottom left) or immunophenotypic subclassification (bottom right). This was based on the main stages of thymic differentiation (i.e., early stage, CD1 negative, CD3 negative; intermediate stage, CD1 positive, CD3 negative or positive; and mature stage, CD1 negative, CD3 positive). Please note that there is no linear correlation between prognosis and WBC or successive stages of differentiation.

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Some immunophenotypic features of T-ALL had been correlated with prognosis in previous studies but none is sufficiently predictive of outcome to trigger changes in treatment intensity

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Genetic features of T-ALL that may have a prognostic impact. However, the correlations reported appeared to be insufficiently strong to trigger changes in treatment intensity.

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In conclusion, none of known prognostic features for B-lineage ALL are clinically useful for T-ALL. Likewise, immunophenotypic and genetic parameters do not allow reliable prediction of outcome.

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Early treatment response as measured by MRD has prognostic significance in childhood T-ALL. Curves show event-free survival (left) and cumulative incidence of relapse (right) for patients with T-ALL classified as standard-risk (SR), intermediate risk (IR) and high risk (HR) according to the MRD-based classification developed by the AIEOP-BFM group (please see publication for details).

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Early T-cell precursors (ETPs) are recent immigrant from the bone marrow into the thymus. They retain multi-lineage differentiation potential, and have a distinct gene expression profile when compared with more differentiated T cell precursors.

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This commentary suggested a revised outline of thymic T-cell differentiation in which multipotent progentiors that have recently entered the thymus (i.e., ETP) can differentiate to T cells but retain the capacity to differentiate into other lineages

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To determine whether there was a subset of T-ALL with ETP features we performed this study.

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Gene expression and immunophenotypic features of ETP-ALL. Top left: Unsupervised clustering analysis of diagnostic bone marrow samples from 55 pediatric patients with T-ALL, using a set of genes differentially expressed in ETP. One cluster had a gene expression profile resembling that of ETP cells. Top right: Flow cytometric contour dot plots illustrating the immunophenotypes of normal thymocytes, and representative cases of ETP and typical T-ALL. Bottom left: The heat map shows percentages of positive leukemic cells for each of the listed markers among the 139 St Jude T-ALL cases studied at diagnosis; gray indicates missing data. Each row represents one case and each column the percentage of positive leukemic cells with the indicated marker. Bottom right: Heat map of the top 150 differentially expressed genes, ranked by P value.

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This slides summarizes the immunophenotypic criteria that define ETP-ALL. Leukemic blasts must have unequivocal T cell markers: they must express both CD7 and either cytoplasmic or surface CD3. Myeloperoxidase must be absent. CD1a and CD8 must also be absent on leukemic cells (<10% positive blasts). CD5 expression must be dim when compared to that of normal T lymphocytes (residual normal T lymphocytes commonly present in the diagnostic samples can be used as a reference). Thus, CD5 mean fluorescence intensity (MFI) on leukemic cell should be at least 1 log lower than that of normal T lymphocytes and/or CD5 should be expressed in <75% of leukemic blast cells. Finally, leukemic cells must express stem cell- or myeloid-associated markers (>10%) Important: Early-stage T-ALL according to historic classifications (see slide 6) is not ETP-ALL.

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Gene Set Enrichment Analysis (GSEA) determines whether the expression of a previously defined set of genes is statistically associated with a biological state. GSEA was applied to gene expression data of 89 T-ALL cases (55 from St Jude, 34 from AIEOP) , 12 of which had the ETP-ALL immunophenotype. There was a highly significant enrichment of the molecular signature of murine ETP cells in the ETP-ALL cases.

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Patients with ETP-ALL in this study tended to be older and had a lower presenting leukocyte count than non-ETP T-ALL patients

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Prevalence of MRD during the early phases of therapy is significantly higher in patients with ETP than in those with typical T-ALL. MRD levels were measured by flow cytometry (St Jude) or by polymerase chain reaction amplification of antigen-receptor genes (AIEOP). Horizontal bars indicate median values, if above 0.01%.

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Prednisone response in T-ALL patients enrolled in the AIEOP ALL-2000 study (data available for 94 patients). Leukemic lymphoblasts were counted in peripheral blood on day 8 of treatment with prednisone alone. Patients with ETP-ALL had a significantly poorer response (P = 0.0039). Bar indicates median value (if above 1 x 103 blasts per mm3).

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Patients with ETP-ALL had a dismal outcome in two independent cohorts, St Jude and AIEOP. Kaplan-Meier plots of (a, d) overall survival, (b, e) event-free survival, and (c, f) the cumulative incidence of remission failure or hematologic relapse in patients with typical T-ALL (gray line) versus ETP-ALL (black line) treated on either St. Jude (a-c) or AIEOP protocols (d-f). The event-free survival curves start at the end of remission induction (day 43 for St. Jude and day 33 for AIEOP patients). Outcome estimates at 10 and 2 years of follow-up are shown; P values are from the log-rank test.

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The prognostic significance of ETP-ALL was examined in relation to that of other known prognostic factors in T-ALL, including age, sex, race, central nervous system involvement, presence of a mediastinal mass, use of different treatment protocol, as well as minimal residual disease (MRD) at the end of remission induction therapy (day 43). In a univariate analysis, only ETP status and MRD were significant predictors of outcome. In a multivariate analysis, only ETP status remain significant. These results show that ETP status is a strong and independent predictor of outcome in pediatric T-ALL.

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Myeloid antigen expression in the absence of ETP phenotype had no prognostic significance in this study. Table shows event-free survival (EFS) rates (± standard error, SE) for ETP-ALL cases compared to those non-ETP T-ALL subdivided according to expression of myeloid-antigen expression.

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ETP-ALL has a higher expression of some transcription factors such as LMO1, LYL1 and ERG but there is considerable overlap with non-ETP T-ALL. Thus, the level of expression of these factors is not sufficient to identify ETP-ALL.

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Among T-ALL patients studied at St Jude with data available on transcription factor expression (measured by Affymetrix U133 genechip; n = 55), the prognostic significance of their expression was compared ETP status as defined by flow cytometry. Kaplan-Meier curves of 5-year overall survival (OS) are shown. (a) OS of patients with ETP-ALL (black line) compared to that of those with typical T-ALL (grey line) among the 55 patients studied with genechips. OS according to levels of LMO1 (b, e), ERG (c, f) and LYL1 (d, g) expression. In the analyses shown in b, c, and d we used the median level of expression to subdivide the patient population into two groups. In the analyses shown in e, f, and g, cases with the highest levels of expression of each transcription factor (black lines) were compared to the remaining cases (grey lines). The top quartile cut-off was used for LMO1 (e) and ERG (f) following the approach used by Baldus et al. (J Clin Oncol 24: 4714-4720, 2006) for ERG. For LYL1 (g), we used a more stringent cut-off level, as only the highest levels of LYL1 expression identify a distinct subset of T-ALL according to Ferrando et al. (Cancer Cell 1; 75-87, 2002); a signal of 2000 by genechip was selected as a cut-off level based on the data distribution. All 4 patients who failed therapy in the LYL1 >2000

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Poor early treatment response for ETP-ALL was also noted by COG investigators. Patients with ETP-ALL had a significantly higher prevalence of >5% blasts by morphology in bone marrow on days 15 and 28 of treatment, as well as a higher prevalence of MRD on day 29.

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Poor treatment response for ETP-ALL also noted by TCCSG investigators. A higher bone marrow blast count on day 14 (top left) and a higher peripheral blood blast count on day 15 (bottom left) was noted among ETP-ALL patients, who also had a worse event-free survival (top right)

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TCRg gene rearrangement and deletion occurs during normal T cell development in the thymus. The most immature thymocytes lack such rearrangements/deletions. This paper shows that T-ALL lacking biallelic TCRg deletions (presumably derived from more immature thymocytes) have a worse prognosis when compare the T-ALL cases with TCRg biallelic deletions

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Biallelic deletion of TCRG is much less frequent in ETP-ALL. Thus, the results reported by Gutierrez et al. could be due to the inclusion of ETP-ALL in the group lacking biallelic deletion of TCRG.

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SNP array analysis to identify genetic lesions in 11 ETP- and 43 typical T-ALL cases. (a) Number of genomic gains and losses. ETP-ALL cases had a higher number of genomic losses (P = 0.0134) and gains (P = 0.0005). (b) Size of genomic lesions. The sizes of both losses (P = 0.0068) and gains (P = 0.0027) were greater in ETP-ALL cases. These initial observations about the genetic complexity of ETP-ALL were expanded in a subsequent study based on whole genome sequencing (see following slides).

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ETP-ALL was the first leukemia subtype to be included in the Pediatric Cancer Genome Project (for more details on this project, please see Downing et al. Nat Genet. 44:619-22, 2012).

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Circos plots (a display used to visualize of variations in genome structure) show genetic alterations in four representative ETP-ALL cases. The plots depict structural genetic variants, including DNA copy number alterations, intra- and inter-chromosomal translocations, and sequence alterations. Loss-of-heterozygosity, orange; amplification, red; deletion, blue. Sequence mutations in RefSeq genes: silent single-nucleotide variants (SNVs), green; non-silent SNVs, brown; indels, red. Genes at structural variant breakpoints: genes involved in in-frame fusions, pink; others, blue.

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This study revealed that the genetic alterations in ETP-ALL are heterogeneous. However, 58% of cases displayed inactivating lesions in genes involved in hematopoietic development, which could explain the early block in differentiation characteristic of ETP-ALL. In addition, the overall mutational spectrum appeared to be more similar to that of AML than that of ALL.

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When compared to other biologic features of T-ALL leukemic cells, ETP status was the most important predictor of relapse in this study.

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ETP-ALL has also been identified in adult ALL. Whether ETP-ALL in adults predicts a worse outcome as compared to other T-ALL subtypes is not yet clear, as the overall prognosis for adult patients with T-ALL is poor.

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Unfortunately, investigators often label some leukemias as “ETP-ALL” when the criteria outlined in slide 15 are not met. For example, cases with myeloperoxidase expression should not be classified as ETP-ALL; these cases are likely to be AML or mixed-phenotype acute leukemia (MPAL). Other cases are labeled as “ETP-ALL” because of the lack of CD2 expression, or the lack of both CD4 and CD8 expression: these features per se do not warrant a diagnosis of ETP-ALL. Some key markers for the identification of ETP-ALL, such as CD1a or CD8, are often not included in diagnostic panels. It is important to use them is ETP-ALL is suspected.

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