Small Nucleolar RNA Expression Profiling Identifies Potential Prognostic Markers in Peripheral T-Cell Lymphoma.
Authors: Ysebaert Loic , Valleron Wilfried , Berquet Laure , Fataccioli Virginie , Quelen Cathy , Martin Antoine , Parrens Marie , Lamant Laurence , de Leval Laurence , Gisselbrecht Christian , Gaulard Philippe , Brousset Pierre 
Published: October 2019

Abstract
Abstract 2678

Introduction:
Peripheral T-cell Lymphomas (PTCL) are types of rare and heterogeneous Non-Hodgkin's Lymphoma (NHL) that, in general, are associated with a poor clinical outcome. Discovery of new prognostic tools is thus a current and major challenge. In a cohort of 122 cases of PTCL collected from a multicentric T-cell lymphoma consortium (TENOMIC), we analyzed the expression of 80 non-coding small nucleolar RNAs (snoRNA) using high-throughput quantitative PCR (Fluidigm). SnoRNAs belong to the non-coding RNA family, and participate to diverse biological processes, most importantly ribosomal RNA maturation.

Patients and methods:
PTCL samples (n=122, 32 ALCL (22 ALK+, 10 ALK-) and 90 non-ALCL cases) were collected from TENOMIC. For each case, a consensus diagnosis was made by a panel of expert hemopathologists (some of the patients participated in a GELA-sponsored clinical trial, the others benefited from a national review protocol for T-cell lymphomas). Total RNA from sorted healthy donor-derived T-lymphocytes (n=35) and tumor samples were extracted (Trizol), integrity was verified using Agilent NanoChip, and reverse transcribed and assessed for snoRNA expression levels using The Fluidigm high-throughput quantitative PCR method. Medical cases were used to calculate progression-free and overall survival (PFS and OS) in 78 non-ALCL patients (26 PTCL-NOS, 46 AITL, 6 other rare entities) who received CHOP or CHOP-like regimen first-line.

Results:
First, we used unsupervised hierarchical clustering to show that, irrespectively from cell of the origin of the T cells, PTCL cells had a significant down-regulation of snoRNA expression in 63% of the snoRNA tested. In particular, there was a clear delineation between AITL, a tumor of T-follicular Helper (TFH) origin, and normal TFH cells from healthy donors. Among PTCL entities, ALCL had a specific snoRNA profile, but unsupervised clustering was not able to distinguish ALK- from ALK+ patients. However, a supervised comparison identified snoRNA U3 as a discriminant marker that sorted ALK+ from ALK- ALCL samples. Unsupervised or supervised snoRNA clustering failed to distinguish AITL from the other subgroups of PTCL.
Second, we assessed prognostic impact of snoRNA expression in 78 non-ALCL patients. Characteristics of the cohorts were as follows: median age 65y/74y, Ann-Arbor stage III-IV in 94%/88%, elevated LDH in 76.7%/73.1%, IPI score 3–5 in 70%/61%, respectively. Although the snoRNA expression profiles of AITL and other PTCL subtypes appeared very similar, unsupervised clustering revealed the over-expression of 8 snoRNA in a subgroup of 31 patients with 45% OS at 5y (vs 47 patients with 18% OS at 5y). In AITL and PTCL-NOS cases, median OS was 26mo and 13mo, respectively. These thresholds were used in a supervised analysis to try to identify a specific prognostic snoRNA signature. A three snoRNA set was found significantly over-expressed in patients with the best prognosis, both in AITL patients (HBII-239, U59B, U90 impacted both on OS and PFS), and in PTCL-NOS patients (HBII-239, HBII-438A, U80 impacted on OS). Very interestingly, these signatures were not correlated neither with IPI or IPI-T scores, nor with overall response rates after CHOP. Lastly, we investigated the prognostic impact of HBII-239 snoRNA as a single marker. HBII-239 over-expression was statistically associated with prolonged PFS and OS in the entire cohort of 78 patients.
Third, we investigated the impact of HBII-239 over-expression on cellular pathways in the FEPD cell line. This snoRNA is a precursor for microRNA-768, which is processed from the 3p strand of HBII-239. The FEPD cell line had a weaker expression of miR-768-3p as compared to patients' samples. Transfection of a miR-768-3p microRNAmimic induced a significant reduction of proliferation rate (as assessed by an MTS assay), due to a block at the G1/S checkpoint. In patients with low HBII-239 level, it is therefore expected that proliferation rate of lymphomatous cells would be increased, translating into decreased OS.

Conclusions:
Our study showed that snoRNAs are differentially regulated in normal compared to malignant T-cell populations. Besides a global down-regulation of these molecules, specific signatures may have a prognostic significance in PTCL. The different snoRNAs that can be regarded as potential biomarkers in these tumors may play a direct role in different cellular pathways.

Disclosures:
No relevant conflicts of interest to declare.