Page 204 - 2022_01-Haematologica-web

T. Braun et al.
healthy donor T cells (Online Supplementary Figure S10A). GSEA of associated mRNA showed an enrichment of genes of the HALLMARK E2F TARGET (NES=-3.52, q<0.0001) and of the TNFA SIGNALING VIA NFKB gene sets (NES=- 2.08, q=0.01, both miR-29a-3p-correlated mRNA) in low miR-29 expressing T-PLL (Online Supplementary Figure S10B). Furthermore, we identified 79 putative-target mRNA of the miR-29a-3p / miR-29b-1-5p / miR-29c-3p cluster in T-PLL by the above described strategy (Online Supplementary Figure S10C). Indicating an association of reduced miR-29 species with aberrant survival signaling, surface TCR activation markers were elevated in T-PLL with low miR-29a-3p (Online Supplementary Figure S10D), namely CD38 (mean expression 43.1% vs. 12.9%, P=0.02, MWW) and CD69 (mean 27.9% vs. 0.89%, P=0.02, MWW). A more active disease state as indicated by lower platelet counts (mean 110 G/L vs. 186 G/L, P=0.15, MWW) and higher LDH serum levels (mean 1,850 U/L vs. 708 U/l, P=0.06, MWW) at sampling was linked to low miR-29b-1- 5p expression (Online Supplementary Figure S10E and F). Lower miR-29c expression tended to be associated with a higher incidence of effusions (six of 12 over one of ten with high miR-29c-3p-expression; P=0.07, Fisher’s exact test, Online Supplementary Figure S11A). In addition, genomic ATM deletions were more frequent in cases with low miR- 29b-1-5p expression (nine of 11 vs. one of 11 with high miR-29b-1-5p; P=0.002, Fisher’s exact test, Online Supplementary Figure S11B, Online Supplementary Table S11 with summary of clinical data).
A combinatorial miR-based overall survival score for T-cell prolymphocytic leukemia
Based on the observed correlation of miR expression with clinical parameters, we aimed to establish a prognostic score that stratifies T-PLL patients according to miR expression levels. In order to identify best candidates in an unbiased fashion, we first associated miR expression levels with OS for all miR detected in at least 80% of T-PLL samples (n>36 cases) and compared T-PLL patients with highest expression levels (upper tertile) to those with lowest expression of the respective miR (lower tertile; Online Supplementary Table S12 with summary of clinical data). In this analysis, miR-98-3p (median OS in high vs. low expression: 16.3 months vs. 29.4 months, P=0.0008, log-rank, Figure 5A), miR-200a-3p (52.7 months vs. 19.1 months, P=0.001, log-rank, Figure 5B), miR- 223-3p (14.9 months vs. 26.0 months, P=0.001, log-rank, Figure 5C), and miR-424-5p (14.4 months vs. 26.0 months, P=0.0007, log-rank, Figure 5D) were most significantly cor- related with OS. We subjected a training model composed of these four miR (miR-98-3p, miR-200a-3p, miR-223-3p, miR-424-5p) and of selected factors that had shown to be of prognostic relevance in T-PLL (e.g., WBC counts, TCL1A mRNA level)1,11,30 to parameter shaving by recursive parti- tioning. This algorithm identified optimum individual thresholds stratifying OS in the randomly created 22-case training set. Using these cutoffs, three miR (200a-3p, miR- 223-3p, miR-424-5p) and TCL1A expression remained as most significant discriminators for OS. Multiple combinato- rial scores of these four parameters were built and for these scores optimum thresholds for discrimination of OS were calculated (recursive partitioning, see Methods). Best sepa- ration was obtained using a miR-exclusive 3-tier score: miR- 200a-3p fc<2.21, miR-223-3p fc≥9.8, miR-424-5p fc≥0.91; relative to healthy donor T cells) with a cutoff of ≥2 sum points (Table 2). Finally, we verified the miROS-T-PLL score
in the 22-case validation set (P=0.0004, log-rank) and in the total cohort of 44 T-PLL (median OS high vs. low miROS-T- PLL: 14.4 months vs. 29.4 months, P<0.0001, log-rank, Figure 5E).
In order to identify variables underlying (as potential con- founders) the miR-based prognostic separation, we associ- ated the expression of miR, which we used for the score, as well as the miROS-T-PLL score itself, with genomic, mRNA expression, immunophenotypic, and clinical data (Online Supplementary Tables S13 and S14). We did not detect signif- icant differences in the distribution of these parameters between the groups determined by expression of the three miR or by the miROS-T-PLL score, further validating the newly established score.
Discussion
Here, we report a pilot analysis of cellular miR expression in a cohort of 46 T-PLL patients. We identified 34 miR to be significantly deregulated in comparison to PB-derived T cells from age-matched healthy donors. These miR includ- ed those which had already been reported as altered in T- cell acute lymphoblastic leukemia (T-ALL, e.g., miR-223- 3p)35 and in CTCL (e.g., miR-29 and miR-200).17,23 They also contained miR that had not been described in the neoplastic context (e.g., miR-10395-5p). The global profiles of deregu- lated miR in T-PLL showed a rather uniform pattern across the analyzed cases. Together with the integrated informa- tion from transcriptome sequencing, this set of data allows for the first time insights into miR-based regulatory net- works in T-PLL.
It is important to mention, that four of the 34 differential- ly expressed miR presented with low CPM values (<1), either being unspecific background in the sequencing tech- nology or representing biological relevant miR expressed at low levels. In addition, for two of the small-RNA identified to be differentially expressed in T-PLL (miR-6724-5p, miR- 5699-3p), miR-base36 assigned questionable confidence in their annotation, although their expression was previously reported in other entities (e.g., bladder cancer).37
In line with T-PLL’s phenotype of augmented TCR activa- tion,1,5,11 our comparative profiling revealed a resemblance of T-PLL’s miR-ome to the one of TCR-activated healthy donor-derived T cells. A specific remodeling of the miR repertoire upon TCR activation had been shown,38 howev- er, analysis of full spectrum miR expression by small RNA sequencing upon TCR activation in healthy donor-derived pan-T cells had not been reported before. We identified here previously unknown miR (e.g., upregulation of miR- 18a-5p) to be altered upon TCR activation in addition to those that had been described (miR-17-5p or miR-150- 5p).31,39 We conclude that constitutive TCR activation shapes the characteristic miR-ome of T-PLL cells.
As hallmarks of T-PLL’s miR-ome, we identified miR-223- 3p, miR-21, the miR-29 family, and the miR-200c/-141 clus- ter as significantly deregulated. These miR have previously emerged as either onco miR or tumor-suppressive miR in other T- or B-cell malignancies.23,40–43 We further identified putative target signatures, potentially mediating the postu- lated effects of prosurvival signaling and aberrant DNA damage responses (e.g., downregulation of FOXO1 upon miR-223 upregulation). Limiting, the postulated target genes are, although predicted through multiple robust algo- rithms, based on associations without proven biological
196
haematologica | 2022; 107(1)