Cyclin D2 expression in Diffuse Large Cell Lymphoma (DLBCL).

Background DLBCL is a common and aggressive type of lymphoma. Up to a third of patients fail to respond or relapse following standard therapy. For these patients outcomes remain poor despite novel treatments such as CAR-T cells and bispecific antibodies.

Better tools to select patients most in need of novel therapies at diagnosis, integrating molecular, cellular pathways and the microenvironment into existing scoring systems are required. The D type cyclins are proteins involved in cell cycle control. Cyclins D1 and D3 are potent oncogenes in B cell lymphomagenesis but the roles of Cyclin D2 are less clear.

In germinal centre (GCB) type DLBCL, Cyclin D3 alone is expressed; inactivation of cyclin D3 results in loss of proliferation.

However, we found unexpected patterns of cyclin D expression in activated B cell (ABC) DLBCL cell lines, with co-expression of cyclin D2 and cyclin D3 in half. In co-expressing cell lines, loss of cyclin D2 diminishes proliferation, whilst loss of cyclin D3 has no impact.

Moreover, publicly available RNASeq data suggest that over-expression of cyclin D2 may be linked to a markedly poor prognosis, with patients expressing CCND2 at a higher level than CCND3 having a median progression free survival (PFS) of 1.55-years compared to 8.39-years in all groups. Particularly compelling is the poor PFS in high CCND2 patients with otherwise good prognoses.

In this proposal I will validate these data using advanced multiplex immunohistochemistry.

Aim My aims are to determine whether there is co-expression of cyclin D2 and cyclin D3 proteins in ABC-DLBCL using archived patient samples; determine variability of expression and correlate expression with outcome.

Methods My initial step will be to optimise antibodies for use in a multiplex system using 2 panels I have specifically designed incorporating cyclin D2 and D3, markers of cell cycle phases, and B and T cell markers. I will undertake staining of 50 ABC and 50 GCB archival DLBCL tumour samples.

I will link protein expression from multiplex experiments to an anonymised dataset of patients’ disease characteristics and survival outcome.

Use of results These results will add to existing data we have generated from cell linesof cyclin D co-expression and single cell RNA sequencing datasets .

The information will allow us to generate preliminary data regarding the role of cyclin D2 and D3 as a prognostic biomarker in DLBCL.