The Clinical Impact of the Cell-of-Origin Classification and the MYC+/BCL2+ Double Expresser Status in DLBCL Treated within Prospective Clinical Trials of the Dshnhl
Authors: Staiger Annette M. , Ziepert Marita , Horn Heike , Scott David W. , Barth Thomas F. E. , Bernd Heinz-Wolfram , Feller Alfred C. , Klapper Wolfram , Hummel Michael , Stein Harald , Lenze Dido , Hansmann Martin-Leo , Hartmann Sylvia , Moeller Peter , Cogliatti Sergio , Lenz Georg , Truemper Lorenz H. , Loeffler Markus , Schmitz Norbert , Pfreundschuh Michael , Rosenwald Andreas , Ott German 
Published: October 2019

Abstract
The Lymph2Cx Cell-of-origin (COO) assay enables the identification of molecular GCB and ABC subtypes of diffuse large B-cell lymphomas (DLBCL) using RNA from formalin-fixed paraffin-embedded (FFPE) samples. In order to explore the prognostic power of this COO assay within the frame of prospectively randomized clinical trials for the first time, we retrospectively performed COO analyses in patient samples from two clinical trials (RICOVER-60: n=360 and R-MegaCHOEP: n=92) of the German High Grade Lymphoma Study Group (DSHNHL) using the Nanostring nCounter®-platform. Immunohistochemistry was done for MYC, FOXP1, LMO2, GCET1, CD5, CD10, BCL2, BCL6, IRF4/MUM1 protein expression, and fluorescence in-situ hybridization was performed for MYC, BCL2 and BCL6 genes with break-apart probes. COO classification was successfully obtained from 414 samples (326/360 (91%) and 88/92 (96%) FFPE samples from the RICOVER60 and R-MegaCHOEP trials, respectively). In the RICOVER-60 study (patients aged > 60 and <80 years), 42% of tumors were classified as ABC-DLBCL, 44% as GCB-DLBCL, and 14% as unclassified. In the R-MegaCHOEP-trial (patient age 18 to 60 years) 27% were of ABC-type, 60% of GCB-type, and 13% were unclassified. Molecular classification provided by the Lymph2Cx correlated well with IPI factors and stringently with the presence or absence of genetic alterations. Upon FISH analysis, 41/44 (93%) BCL2 translocations and all double or triple hit constellations involving MYC/BCL2 or MYC/BCL2/BCL6 were encountered in the GCB group. A higher proportion of MYC breaks also clustered in GCB DLBCL (14% in GCB vs. 3% in ABC) in RICOVER-60 (p=0.005). In contrast, BCL6 translocations were equally distributed in the RICOVER-60 trial while a higher proportion of BCL6 translocations was observed in the ABC-subtype in the R-MegaCHOEP trial.
In RICOVER-60, the ABC-DLBCL group was associated with inferior clinical outcome compared with the GCB subtype (p=0.041, p=0.022 and p=0.034 for EFS, PFS and OS, respectively). A significant survival difference was also seen in CHOP-treated patients. This effect, however, was completely abrogated in R-CHOP treated patients with no significant difference observed in EFS, PFS and OS. This result was confirmed in multivariable analyses adjusted for IPI factors. We found the same results for all RICOVER-60 patients after adjustment for IPI factors (EFS: HR=1.1, PFS: HR=1.2, OS: HR=1.2 for ABC vs. GCB) and after adjustment for IPI factors, bulky disease, gender and age > 70 years. In keeping with this result, no survival differences were observed between ABC-DLBCL and GCB-DLBCL patients in the R-MegaCHOEP trial for EFS, PFS and OS, and this was also confirmed in multivariable analysis (data not shown).
In the RICOVER-60 trial, patients with concomitant over-expression of both BCL2 (50-100%) and MYC (40-100%) (MYC+/BCL2+ DE DLBCL) showed significantly inferior survival in all R-CHOP treated patients and especially in the GCB subgroup compared with non-expressers [MYC-/BCL2- DLBCL; MYC-/BCL2+] (p<0.001;<0.001 for EFS, p<0.001;0.003 for PFS and p<0.001;0.004 for OS, respectively), but not in the ABC group. When nonMYC+/BCL2+ DE DLBCL were separately analyzed, interestingly, the outcome of ABC DLBCL patients was inferior to that of GCB DLBCL patients and MYC+/BCL2+ DLBCL had a significantly inferior outcome in comparison with ABC non-MYC+/BCL2+ DLBCL (Figure 1: p=0.015, p=0.029 and p=0.008 for EFS, PFS and OS, respectively) and GCB. These results could be confirmed in a multivariable model adjusted for IPI factors for the comparison of dual MYC+/BCL2+ expressers with ABC(non-MYC+/BCL2+) patients, but was not significant for the ABC versus GCB group within the non-MYC+/BCL2+ for EFS (HR=1.6, p=0.301), PFS (HR=1.7, p=0.272)and OS (HR=1.3, p=0.604).
In summary, COO profiling alone failed to identify prognostic subgroups, whereas MYC/BCL2 double expression was highly predictive of poor survival. These data from prospective randomized trials demonstrate that older patients and young high-risk patients show significant differences in molecular profiles and, furthermore, indicate that complete subtyping using different molecular methods will be necessary to predict survival under current standard therapy. Only comprehensive molecular analysis of individual patient samples will allow identification of optimal therapy including new molecules.

Disclosures
Scott: NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies. Klapper:Roche, Novartis, Amgen, Takeda: Research Funding.