Gene Expression Analysis of Follicular Lymphoma Provides a Potential Rationale for Histological Grading Revision.
Authors: Piccaluga Pier Paolo , Califano Andrea , Klein Ulf , Agostinelli Claudio , Bellosillo Beartiz , Gimeno Eva , Serrano Sergi , Solè Francisco , Zang Yonghui , Falini Brunangelo , Zinzani Pier Luigi , Pileri Stefano A. 
Published: April 2020

Abstract
Background. Follicular lymphoma (FL) is usually classified as grade (G) I, II or III. Grade III is further subdivided into GIIIa and GIIIb, the latter almost exclusively consisting of CBs. However, this distinction is questioned. Recently, gene expression studies showed that clinical aggressiveness can be associated with specific molecular signatures partially independent from histological grade and that immune reactive cells can play a major role in the outcome determinism. However, to date, gene expression studies did not provide molecular rationale for histological grading and it is still debated if including FL GIIIb within FL or DLBCL. We studied the gene expression profile (GEP) of 43 FLs, 50 B-cell non-Hodgkin lymphomas (B-NHLs) of different histotypes, and 20 samples of normal B-lymphocytes in order to assess:
the relationship of FL with normal B-cells and other B-NHLs; whether FL is a unique disease; and whether FL GIIIb is closer to FL or diffuse large B-cell lymphoma of the germinal center B-cell type (GCB-DLBCL).
Methods. Forty-three FL cases were analyzed. Of these, 37 corresponded to cryopreserved tissue blocks and 6 to enriched neoplastic cells All the samples were obtained at the time of diagnosis, before treatment administration. In addition, samples of normal B-cell sub-populations including CB (N=5), centrocytes (CC, N=5), naïve, N (N=5), and memory cells, M (N=5) were studied. Finally, a panel of B-NHLs was analyzed including Burkitt’s lymphoma (BL, N=4), DLBCL, (N=16), mantle cell lymphoma (MCL, N=10), hairy cell leukemia (HCL, N=10), and B-cell chronic lymphocytic leukemia (B-CLL, N=10). Finally, in silico data concerning 37 cases of germinal center B-cell type (GCB) DLBCL were retrieved at http://www.ncbi.nlm.nih.gov/projects/geo/. For proper comparison with our samples, gene expression values were normalized “per gene” and “per chip”. For microarray analysis, fragmented cRNA was hybridized to HG-U133 2.0 plus microarray.
Results. First, we found that the molecular profile of FL is intimately related to that of normal germinal centre B-cells, irrespectively of the histological grade. However, interestingly, several cell programs are regulated as in memory cells. Secondly, we observed that FL has a relatively homogeneous GEP that is distinct from that of other B-NHLs and does not include discrete molecular subgroups. However, by further clustering samples according to signatures differentially expressed among FLs or in FL vs. DLBCL, we showed that GI-IIIa tumors tend to cluster together, while GIIIb FL constitutes a distinct subgroup. Finally, we found that the molecular signature of GIIIb FL is indeed closer to that of the other FLs than to the one of GCB-DLBCL. These data support the hypothesis that GIIIb FL belongs to FL rather than DLBCL, and sustain the possible revision of FL histological grading, with the simple distinction into FL (GI-IIIa) and FL/large cell (GIIIb).