ROQUIN/RC3H1 alterations are not found in angioimmunoblastic T-cell lymphoma

Abstract

Angioimmunoblastic T-cell Lymphoma (AITL) is one of the most frequent T-cell lymphoma entities. Follicular helper T lymphocytes (TFH) are recognized as the normal cellular counterpart of the neoplastic component. Despite a clonal T-cell feature and few described recurrent cytogenetic abnormalities, a driving oncogenic event has not been identified so far. It has been recently reported that in mice, heterozygous inactivation of Roquin/Rc3h1, a RING type E3 ubiquitine ligase, recapitulates many of the clinical, histological, and cellular features associated with human AITL. In this study we explored whether ROQUIN alterations could be an initial event in the human AITL oncogenic process. Using microarray and RT-PCR analyses, we investigated the levels of ROQUIN transcripts in TFH tumor cells purified from AITL (n = 8) and reactive tonsils (n = 12) and found similar levels of ROQUIN expression in both. Moreover, we also demonstrated that ROQUIN protein was expressed by AITL TFH (PD1+) cells. We then analysed ROQUIN coding sequence in 12 tumor cell-rich AITL samples and found no mutation in any of the samples. Finally, we analysed the expression of MiR101, a putative partner of ROQUIN involved in the modulation of ICOS expression and found similar levels of expression in tumor and reactive TFH. Altogether, this study shows that neither alteration of ROQUIN gene nor deregulation of miR101 expression is likely to be a frequent recurrent event in AITL.

Figure 1. Illustrative case of AITL rich in tumor cells.

Diffuse proliferation of large neoplastic cells surrounded by inflammatory cells (plasma cells, eosinophils) and vascular hyperplasia (hematoxylin-eosin, original magnification ×20) (A). Numerous neoplastic cells highlighted by positivity for T_FH markers CXCL13 (B), ICOS (C) and PD1(D) (immunoperoxidase, original magnification ×20).

Figure 2. ROQUIN expression in human reactive and tumoral lymphoid samples.

Levels of ROQUIN transcripts determined by gene expression profiling of 17 AITL tumor tissue samples and 2 AITL cell suspensions enriched in tumor cells (≥50%) samples as previously reported . (probe-set 228996_at): ROQUIN transcripts level is slightly higher in enriched tumor cell sample (P = 0,0067 unpaired t-test) (A). ROQUIN mRNA levels determined by quantitative RT-PCR in reactive tonsils; total extract (n = 1), CD4⁺ (n = 2), CD8⁺ (n = 2), or CD19⁺ (n = 2) lymphocytes. Results were normalized by HPRT and compared to reactive purified TFH cells as calibrator: reactive CD4- CD8- and CD19-positive subsets display heterogeneous levels of ROQUIN mRNA (B). ROQUIN mRNA levels [(228996_at) probeset] in purified reactive (n = 12) and neoplastic T_FH cells (n = 8). T_FH cells were purified from 12 reactive tonsils and 8 AITL lymph nodes, RNA was extracted and whole genome expression was analysed on HG-U133 plus 2.0 Affymetrix GeneChip arrays. Similar levels of ROQUIN transcript are observed (C).

Figure 3. Immunohistochemical detection of ROQUIN in AITL.

Among the many cells showing a cytoplasmic granular staining for ROQUIN (brown), a few are PAX5-positive large cells (B-immunoblasts) (pink arrow) whereas most of them are small to medium-sized PAX5-negative lymphoid cells forming small aggregates, corresponding to neoplastic cells of AITL (black arrows) (A). In addition, these aggregates of medium-sized ROQUIN- positive cells (brown, granular staining) co expressed the T_FH-associated marker PD1 (red, membrane staining) (B). Double immunohistochemistry, original magnification ×250.

Figure 4. Expression of ICOS and miR101 expression in human reactive and neoplastic T_FH cells.

Analyses of ICOS expression (210439_at). The level of ICOS mRNA expression is maintained even in the presence of ROQUIN transcripts both in human reactive and tumor T_FH cells (A) Level of miR101 (has-miR-101) is low and similar in both tumor and reactive T_FH cells (p = 0,8 unpaired t-test, NS) (B).