CD44 Expression Profile Varies According to Maturational Subtypes and Molecular Profiles of Pediatric T-Cell Lymphoblastic Leukemia

Abstract

CD44 is a glycoprotein expressed in leucocytes and a marker of leukemia-initiating cells, being shown to be important in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). In this study, we have (i) identified the aberrant antigenic pattern of CD44 and its isoform CD44v6 in T-ALL; (ii) tested the association with different T-cell subtypes and genomic alterations; (iii) identified the impact of CD44 status in T-ALL outcome. Samples from 184 patients (123 T-ALL and 61 AML; <19 years) were analyzed throughout multiparametric flow cytometry. Mutations in N/KRAS, NOTCH1, FBXW7 as well as STIL-TAL1 and TLX3 rearrangements were detected using standard molecular techniques. CD44 expression was characterized in all T-ALL and AML cases. Compared with AML samples in which the median fluorescence intensity (MFI) was 79.1 (1-1272), T-ALL was relatively low, with MFI 43.2 (1.9-1239); CD44v6 expression was rarely found, MFI 1 (0.3-3.7). T-ALL immature subtypes (mCD3/CD1a^neg) had a lower CD44 expression, MFI 57.5 (2.7-866.3), whereas mCD3/TCRγδ^pos cases had higher expressions, MFI 99.9 (16.4-866.3). NOTCH1 ^mut and STIL-TAL1 were associated with low CD44 expression, whereas N/KRAS ^mut and FBXW7 ^mut cases had intermediate expression. In relation to clinical features, CD44 expression was associated with tumor infiltrations (p = 0.065). However, no association was found with initial treatment responses and overall survival prediction. Our results indicate that CD44 is aberrantly expressed in T-ALL being influenced by different genomic alterations. Unraveling this intricate mechanism is required to place CD44 as a therapeutic target in T-ALL.

Keywords: CD44; CD44/CD44v6; NOTCH1; T-cell leukemia; acute myeloid leukemia.

Figure 1

Cellular expression of CD44 in T-ALL, AML, and UCB analyzed by M-FCM. SSC × CD45 (above) and SSC x CD44 (middle) dot plots with leukemic blasts and UCB precursor cells in black and lymphocytes in dark gray. CD44 fluorescence intensity in histograms below, with isotype controls in gray lines, blasts and UCB precursor cells in black and lymphocytes in dark gray. T-ALL, T-cell acute lymphoblastic leukemia; AML, Acute myeloid leukemia; UCB, Umbilical Blood Cord; M-FCM, multiparametric flow cytometry; SSC, side scatter.

Figure 2

CD44 expression according to T-ALL and AML subtypes and immunophenotypic features. (A) Comparison of CD44 expression among T-ALL, AML, and UCB. Scatter dot plot with median and interquartile range of CD44 MFI. (B) Comparison of CD44 expression among AML subtypes. Minimally differentiated AML (M0-M1, n = 12), AML with granulocytic differentiation (M2-M3, n = 17), myelomonocytic (M4-M5, n = 27) and megakaryoblastic (M7, n = 6). (C) Comparison of CD44 expression in minimally differentiated CD7 positive and CD7 negative AML. (D) Comparison of CD44 expression between immature T-ALL (ETP and pre-T-ALL, n = 42) and mature T-ALL (cortical and mature T-ALL, n = 79). (E) Comparison of CD44 expression in TCRαβ positive (n = 18), TCRδγ positive (n = 11) and TCR negative mature (T-III+T-IV, n = 43) and immature (ETP+T-II, n = 41) cases. T-ALL, T-cell acute lymphoblastic leukemia; AML, Acute myeloid leukemia; UCB, Umbilical cord blood; MFI, Median Fluorescence Intensity; ^*p < 0.05, ^**p < 0.01.

Figure 3

CD44v6 expression profile in T-ALL. (A) Scatter dot plots with median and interquartile ranges of CD44v6 percentage of leukemic blasts in T-ALL (on left) and CD44v6 MFI in T-ALL (on right). (B) Dot plot CD44v6 × CD7 demonstrating cases of T-ALL with leukemic blasts negative for CD44v6 (on left) and positive for CD44v6 (on right). MFI, median fluorescence intensity; T-ALL, T-cell acute lymphoblastic leukemia.

Figure 4

CD44 expression according to patients' clinical characteristics. (A) Scatter dot plots with median and interquartile ranges of CD44 MFI in T-ALL and AML according to WBC. Twenty-six T-ALL cases had low WBC (<50 × 10⁹/L) and 97 had high WBC (>50 × 10⁹/L), p = 0.877; 30 AML cases had low WBC and 31 high WBC, p = 0.702. (B) CD44 MFI according to the presence of organ and/or CNS infiltration. In T-ALL, 12 cases did not have the presence of organ infiltration, while 110 had, p = 0.0646; in AML, 23 cases did not present organ infiltration, while in 38 it was present, p = 0.214. (C) CD44 MFI according to prednisone response on D8 in T-ALL. Seventeen cases had good prednisone response and 18 cases poor response, p = 0.228. (D) CD44 MFI according to occurrance of relapse in T-ALL patients. Sixteen cases presented relapsed T-ALL and 46 no relapse, p = 0.278. (E) Survival analysis of CD44 expression in T-ALL. Kaplan-Meier estimates for the probability of overall survival for different levels of CD44 expression, p = 0.436. All p-values calculated by the Mann-Whitney or Log Rank test. T-ALL, T-cell acute lymphoblastic leukemia; AML, Acute myeloid leukemia; MFI, Median Fluorescence Intensity; CNS, Central nervous system; WBC, White blood cell count; pOS, probability of overall survival.

Figure 5

CD44 expression according to status of genomic alterations in T-ALL. Scatter dot plots with median and interquartile ranges of CD44 MFI in T-ALL. Cases with NOTCH1 mutations and STIL-TAL1 (−), n = 36; NOTCH1 WT, STIL-TAL1(+) cases, n = 12; STIL-TAL1(+) and NOTCH1 mutated cases, n = 5; NOTCH1 WT and STIL-TAL1(−) cases, n = 34; FBXW7 mutated, n = 19; FBXW7 WT, n = 84; TLX3 positive, n = 6; TLX3 negative, n = 108; N/KRAS mutated, n = 13; WT, n = 108. MFI, median fluorescence intensity; mut, mutation; WT, wild type; (+), positive cases; (−), negative cases; ^*p < 0.05.