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S.J. Erkeland et al.
some 14q32 in close proximity to the T-cell receptor-a/d (TRA/TRD) regulatory region on chromosome 14q11, or more rarely to T-cell receptor-b (TRB) regulatory elements located on 7q35, resulting in overexpression of TCL1.6 TCL1 is an adapter protein of 14 kDa that functions in kinase complexes and enhances T-cell receptor-mediated pro-survival signaling.7 The gene encoding mature T-cell proliferation-1 (MTCP1), is highly homologous to TCL1 and is involved in the less frequent translocations t(X;14)(q28;q11) and t(X;7)(q28;q35).5 The total incidence of translocations involving TCL1 and MTCP1 is around 90%.5 The high incidence of TCL1 and MTCP1 transloca- tions strongly suggest that this family of proteins plays a key role in the pathogenesis of T-PLL. In full agreement, different TCL1 and MTCP1 transgenic mouse models demonstrate a role for TCL1 and MTCP1 in the initiation of malignant transformation to overt leukemia similar to T-PLL,8-10 albeit with a long latency of 12-20 months. These data indicate that secondary oncogenic events are required for full oncogenic transformation of the mature T cells. Identification of co-operating leukemia genes in the Em-TCL1 transgenic mouse with a Sleeping Beauty trans- poson-mediated mutagenesis screen revealed increased nuclear factor (NF)-kB signaling as a collaborating event in TCL1-mediated leukemogenesis.11 The role for small non- coding RNA in the pathogenesis of T-PLL has not been investigated yet.
MicroRNA (miRNA) are an abundant class of small non- coding RNA of 19-24 nucleotides, which guide the RNA- induced silencing complex (RISC) to reverse and partially complementary sequences in the 3’-untranslated regions (3’-UTR) of mRNA and control gene expression through translational inhibition and mRNA destabilization.12 Aberrant expression, biogenesis and activities of miRNA are hallmarks of human cancer, including malignant hema- tological disorders.13-17 Numerous miRNA have been iden- tified as diagnostic biomarkers for human leukemia.16 Some miRNA are potent oncogenic drivers of leukemoge- nesis, while others have critical tumor-suppressing activi- ties.18-22
Here, we characterized a cohort of 31 T-PLL cases by cytomorphology, immuno-phenotyping and immuno- genetic analysis, as well as mRNA and miRNA profiling to identify novel mechanisms that contribute to leukemic transformation. We revealed that the expression of miRNA is aberrant, though often heterogeneous, in T-PLL. Additionally, we present the first evidence that aberrant expression of miR-200c/141 affects TGFb-controlled mechanisms in T-PLL that may contribute to its pathogen- esis.
Methods
Patient cohort
The patient cohort consisted of 31 well-defined T-PLL cases diagnosed between 1985 and 2011. Sampling was done at the moment of diagnosis. Diagnosis of T-PLL is based on a combina- tion of clinical features, morphology, cytogenetics and immuno- phenotype. The cohort consisted of males (62%) and females (38%) with a median age at diagnosis of 66 years (range, 41-89 years) (Table 1). In our cohort, 52% of the T-PLL patients were CD4+ (n=14), 33% were CD4+ CD8+ (n=9), 11% were CD8+ (n=3), 4% were CD4–CD8– (n=1) and one was not determined. All patients were characterized for known aberrations in genes such
as ATM, TP53, TCL1 and IGH. Use of T-PLL patient samples was approved by the Erasmus MC Medical Ethics Committee (MEC- 2015-617). A cohort of 31 T-PLL cases was characterized morpho- logically, cytogenetically and immunophenotypically. We could isolate high quality RNA from 23 T-PLL patients for gene expres- sion profiling. The average RIN value of the T-PLL samples was 8.7 with a standard deviation of 0.9. Of these, the RNA of 21 T-PLL cases was sufficient for miRNA expression profiling. All RNA samples resulted in high-quality cDNA libraries. Four addi- tional T-PLL samples (T-PLL44, 46, 48 and 49) were used for vali- dation experiments and Western Blotting.
Normal T-cell subsets
Peripheral blood from healthy donors were obtained from buffy coats (Sanquin) with approval by the Erasmus MC Medical Ethics Committee (MEC-2016-202). For gene expression profiling, peripheral blood mononuclear cells (PBMC) were first isolated by Ficoll-Paque (density 1.077 g/mL, Pharmacia). CD4+ T cells were labeled with anti-CD3-PerCP-Cy5.5 (cat.#340948) and anti-CD4- PE-Cy7 (cat.#557852) and CD8+ T cells were labeled with anti- CD3-PerCP-Cy5.5 (cat.#340948) and anti-CD8-APC-H7 (cat.#560273)(BD Biosciences). Naïve T cells (CD27+ and CD45RA+), memory (CD27+ and CD45RO+ and effector (CD27– and CD45RA+) T-cell subsets were labeled with, anti-CD45RA-PE (cat.# 555489), anti-CD27-APC (cat.#558664), anti-CD45RO-FITC (cat.#555492) and anti-CD45RA-PE cat.#555489) antibodies (BD Biosciences) (Online Supplementary Table S1). In order to prevent antibody-mediated activation of T cells, all cell populations were kept on ice during antibody staining. Cells were sorted at 4°C and directly in TRIzol (Thermofisher) to prevent cell activation, cell death and/or RNA degradation. Cells fractions were sorted on a FACS Aria II cell sorter (BD Biosciences).
Data availability: i) GSE147930: microarray gene expression data; ii) GSE147931: mRNA sequencing data; iii) GSE147932: small RNA sequencing data.
For a detailed description of the immunophenotyping and cyt- morphological analysis, fluorescence in situ hybridization analysis and cytogenetics, T-cell receptor gene rearrangement analysis, Lentiviral miRNA expression vectors and transduction of Jurkat and HeLa cells, gene expression profiling as well as RNA sequenc- ing and data analysis see the Online Supplementary Appendix.
Results
Cytomorphologic, immunophenotypic and immunogenetic analysis of T-cell prolymphocytic leukemia
As T-PLL is a heterogeneous disease,23 we first charac- terized our T-PLL cohort with respect to clinical, morpho- logical, phenotypical and molecular features (Table 1). Cytomorphologic analysis resulted in three different T- PLL subgroups: i) cells with small lymphocytic cell mor- phology (64%); ii) cells with large blast-like morphology (10%); iii) cells with equivocal morphology (26%) (Online Supplementary Figure S1A). We determined deletions in TP53 (24% of cases tested) and ATM (64% of cases test- ed) as well as translocations involving TCL1 located on 14q32 (67% of cases tested). However, we did not observe any correlation between cell morphology and different parameters including known aberrations in genes, e.g., ATM, TP53, and TCL1. In order to determine the cell of origin of T-PLL, we performed flowcytometric immunophenotyping of T-PLL samples using the EuroFlow T-CLPD panel24 (www.euroflow.org) taking flow
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haematologica | 2022; 107(1)