Table 1.
Relevant clinical and genetic data.
Figure 1.
Examples of aCGH and FISH analysis.
A) upper panel: genomic profile of chromosome 7 of four index cases with the indicated common deleted region (CDR) on 7p and common gained region (CGR) on 7q; lower left panel: gene content (Hg19) of the biallelically deleted 7p21 interval in case 4; lower right panel: zoomed CDR with the indicated selectively amplified region (SAR). B) Examples of interphase FISH validation of aCGH results performed in cases 1 (left panel) and 2 (right panel) with r(7). Applied probes: (a) RP11-99J06-SG/RP11-735O20-SO, (b) RP13-11C11-SG/RP11-807G04-SO, (c) RP11-513N08-SG/RP11-514N09-SO, (d) RP11-379L24-SO/RP11-16K22-SG,(e) RP11-269N18-SG/RP5-894A10-SO, (f) RP4-548K24-SG/RP11-135F23-SO.
Figure 2.
Mechanism underlying formation of r(7) in HSTL.
A) Partial karyotype showing r(7). B) Proposed model of the r(7) formation. Illegitimate somatic rearrangement of TCRB and TCRG in δγT-cells leads to the aberrant TCRB-TCRG lesion and consequently to the formation of r(7) and loss of the terminal 7p and 7q regions, respectively. This process is followed by a subsequent gain/amplification of 7q sequences (shown in red).
Figure 3.
Expression of CHN2/β2-chimerin.
A) Normalized values of all CHN2 probes in the used U133 array for the analyzed malignancies and normal controls. The observed expression differences were statistically significant, with FDR_BH <0.05 (Table S6). B) Expression values of CHN2 using the RNAseq data. FPKM (Fragments Per Kilobase of exon Model) is a measurement of transcript abundance in RNAseq experiments. C) Western blotting and densitometry of β2-chimerin. The bars represent the fold change of the normalized intensity of β2-chimerin (compared to β-actin) versus spleen. The number between parenthesis represent the number of samples.
Table 2.
Ingenuity Pathway Analysis: Most significant networks, functions and pathways associated to the top 401 genes differentially expressed in HSTL.
Figure 4.
Hierarchical clustering using the 24 gene signature for HSTL.
The MA data (A–E) and RNAseq data (F–H) show an accurate separation of the HSTL cluster from PTCL, AITCL, NK/TCL, nonmalignant spleen, normal T-cells, nonmalignant thymus and T-ALL.
Table 3.
List of genes comprising the HSTL signature.
Figure 5.
Morphology and ABCB1 expression in HSTL cases detected by IHC.
Immunohistochemical stainings (A–C, anti-CD3; D-F, anti-MDR1/ABCB1) of the typical intrasinusoidal spread (red arrows) by HSTL cells in the bone marrow (A/D, case 4) and spleen (B/E, case 6), respectively, compared to staining pattern in normal spleen (C/F). Pictures captured by Leica DFC290HD camera at 400X. Scale bar = 50 µm.
Figure 6.
Postulated model for the pathogenesis of HSTL.
(A) In resting T-cells, NFAT proteins are located in the cytoplasm and are associated with a large RNA-protein scaffold complex composed of the lincRNA NRON, a repressor of NFAT [30], and several additional proteins [31]. NFAT proteins are heavily phosphorylated through synergistic action of three different family of kinases, casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), and dual specificity tyrosine phosphorylation regulated kinase (DYRK) [29]. When T-cells are stimulated, TCR engagement triggers a rapid increase in intracellular calcium (Ca2+) and activation of RAC1, a GTPase which belongs to the RAS superfamily of small GTP-binding proteins [79]. The active, GTP-bound RAC1 binds to IQGAP (IQ-domain GTPase-activating protein) negatively regulating its binding affinity for other proteins and consequently, stimulating the disassembly of the NRON complex [31], [82], [83]. In parallel, the calcium increase leads to activation of calmodulin, a calcium-binding messenger protein, which activates of the phosphatase calcineurin. This enzyme dephosphorylates NFAT and promotes nuclear transport of activated NFAT by importins (KPNB1, CSE1L). In the nucleus, NFAT, in synergy with a numbers of other transcriptional regulators (e.g. FOS and JUN), participates in a transcriptional regulation of a wide range of genes involved in immune system responses and organs development [29], [66]. (B) We postulate that formation of i(7)(q10) or r(7) in γδT-cell triggers an aberrant expression of β2-chimerin which subsequently inactivates RAC1 by keeping it in a GDP-bound state. This prevents RAC1 binding with IQGAP resulting in a strengthening of the NRON complex and arrests the phosphorylated NFAT in the cytoplasm. Cytoplasmic retention of NFAT may be also attributed to the kinase LRRK2 (overexpressed in HSTL), which blocks the transport of NFAT to the nucleus [32], [33]. The significantly reduced nuclear level of NFAT leads to dysregulated transcription of responsive genes controlling cell-cycle, cell death and proliferation, and eventually, to malignant transformation and clonal proliferation of i(7)(q10)/r(7)-positive γδT-cells. The candidate causative genes include the MYC oncogene (↑ in HSTL), known to be repressed by NFAT [62] and the IKZF1 tumor suppressor gene (↓ in HSTL) which is activated by NFAT [66]. (C) Hierarchical clustering using NFAT-related genes, including components of the NRON complex. Note that all HSTL samples, except for DERL-2, form a distinct cluster apart from the activated γδT-cells.