RQ-PCR Provides a Superior Alternative to Immunohistochemistry In Defining Prognostic Groups In DLBCL, and Predicts Treatment Failure with CHOP-R
Authors: Worrillow Lisa , Barrans Sharon , Crouch Simon , Care Matthew , Smith Alex , Patmore Russell , Tooze Reuben , Roman Eve , Jack Andrew 
Published: October 2019

Abstract
Abstract 2484
Diffuse large B cell lymphoma (DLBCL) is a heterogenous disease, which has been subclassified into germinal centre (GCB) and activated B-cell (ABC) type using gene expression profiling. This has been shown to separate DLBCL into distinct prognostic sub-groups in patients treated with either CHOP or CHOP-R therapy. A number of published immunohistochemistry algorithms have attempted to replicate this subclassification using a limited number of markers, however, despite being widely adopted, the reproducibility of the algorithms has proven difficult, possibly due to the subjective nature of interpreting immunohistochemistry results.
The aim of this study was therefore to evaluate the most widely accepted immunohistochemistry algorithms, and validate the results using RQ-PCR on RNA extracted from paraffin sections in a large series of well characterised formalin fixed paraffin embedded (FFPE) biopsies of R-CHOP treated DLBCLs.
RNA was extracted using the Ambion Recoverall extraction kit. Applied Biosytems Taqman probes were used to evaluate gene expression of CD10, BCL6, GCET1, FOXP1 and IRF4. RQ-PCR was run on an Applied Biosystems 7500Fast cycler, and results were calculated using the deltadeltaCt method, using PGK1 as the reference housekeeper gene and either RAJI cell line or commercial RNA as the standard.
Using the Hans criteria, 130/277 (47%) presentation DLBCL biopsies were classified as GCB and 147/277 (53%) were ABC. Further classification of a subset of cases using the Choi algorithm showed concordant results in 48/61 (78.7%) cases, with 1 (1.6%) case classified as GCB using Hans and ABC using Choi, and 12 (19.7%) cases classified as ABC using Hans and GCB using Choi.
RQ-PCR data showed excellent correlation with immunohistochemistry for all genes incorporated into the algorithms (CD10, p<0.0001; BCL6, p=0.0003; GCET1, p=0.009; FOXP1, p<0.0001; and IRF4, p=0.008). RQ-PCR defined positivity was characterized by inspecting the distribution histograms of the range of values for each gene, and applying density smooths. Mixture distributions were identified and the minimum values of these plots were used to define the cut-points. Patients were then re-classified using RQ-PCR data to define GCB/ABC status. For the Hans classification, 50/79 (63%) patients were GCB and 29/79 (37%) were ABC. Of these 51/77 (66%) cases resulted in the same sub-classification using RQ-PCR compared to immunohistochemistry, with 26/77 (34%) cases classified differently. For the Choi algorithm, 49/79 (62%) patients were GCB and 30/79 (38%) were ABC, with 23/37 (62%) cases were classified the same using RQ-PCR and immunohistochemistry, and 14/37 (38%) classified differently.
In univariate Kaplan-Meier survival analysis, there was no difference in outcome when comparing either Hans (n=170, p=0.7) or Choi (n=60, p=0.3) algorithms using immunohistochemistry, however using RQ-PCR data to define GCB and ABC subgroups, both algorithms showed a poorer survival in the ABC subgroup. For the Hans algorithm, 6 month overall survival (OS) was 76% in the GCB group compared to 55% in patients classified as ABC (p=0.06), and similarly for the Choi algorithm, 6 month OS was 77% in the GCB group and 53% for patients classified as ABC (p=0.03).
This data supports the use of gene expression algorithms to classify DLBCL patients into clinically relevant prognostic groups, with patients classified as ABC exhibiting an inferior outcome compared to the GCB group.
RQ-PCR provides a quantitative method to determine expression and eliminates the subjective element associated with interpreting immunohistochemistry. Using RQ-PCR to define prognostic subgroups in DLBCL provides a realistic alternative to gene expression profiling, which is currently not applicable to the majority of diagnostic laboratories. Patients classified as ABC type using this approach show early treatment failure with CHOP-R, and alternative therapies should be considered in this group.

Disclosures:
No relevant conflicts of interest to declare.