A Molecular Classifier Increased the Accuracy of Lymphoma Diagnosis By Expert Pathologists in the Diffuse Large B-Cell Lymphoma Gained Trial from the Lysa
Authors: Moslemi Amine , Bobée Victor , Blanc-Durand Paul , Drieux Fanny , Desmots-Loyer Fabienne , Briere Josette , Casasnovas Olivier , Canioni Danielle , Ghesquieres Herve , Gaulard Philippe , Oberic Lucie , Damotte Diane , Morschhauser Franck , Martin Antoine , Tilly Hervé , Thieblemont Catherine , Ribrag Vincent , Hermine Olivier , Haioun Corinne , Lamy Thierry , Fest Thierry , Jardin Fabrice , Le Gouill Steven , Itti Emmanuel , Ruminy Philippe , Molina Thierry Jo 
Published: November 2021

Abstract
Introduction
We (Bobée et al, 2020) have recently described a new molecular classifier combining gene expression profiling of 137 genes and machine learning able to diagnose B-cell lymphomas. As this tool can be easily performed from formalin fixed paraffin embedded tissue, we proposed to evaluate how it may be useful after the process of central review by expert pathologists in a LYSA diffuse large B-cell lymphoma (DLBCL) clinical trial, the GAINED trial (Le Gouill et al, 2021).
Methods
The GAINED trial was a multicenter randomized phase 3 trial comparing obinutuzumab to rituximab followed by a PET-driven consolidation in patients with DLBCL. Histological inclusion criteria were : DLBCL, high grade B-cell lymphoma double/triple hit (DH/TH) or high grade NOS. 670 patients were included and 646 underwent a central review of the biopsy by at least two LYSA pathologists. The central review performed immunostaining of full slides by anti-CD20, CD5, CD10, BCL6, MUM1, MYC, BCL2 if sufficient material was available as well as RNA extraction for the molecular classifier, the nanostring Lymph2CX and DNA extraction for targeted NGS. The molecular classifier was able to deliver 8 different signatures: DLBCL GCB, DLBCL ABC, primary mediastinal large B-cell lymphoma (PMBL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma/lymphoplasmacytic lymphoma, CLL/lymphocytic Lymphoma, T-cell rich signature. We compared classification by the histopathology review and by the molecular classifier. Discordant cases were reviewed both by two histopathologists (JB, TJM) and the molecular biologist (PR) and a reconciliation diagnosis was proposed
Results
The central histopathological review alone identified 580 patients with DLBCL or high grade B-cell lymphoma and 40 patients with a lymphoma subtype not eligible for the trial. Among the 470 patients evaluated by the molecular classifier, the reconciliation diagnosis identified 292 DLBCL NOS with 40 associated with a small B-cell component, 13 High grade DH/TH, 9 high grade NOS, 109 PMBL, 16 T-cell rich large B-cell Lymphoma (TCRBCL), 5 DLBCL EBV+ NOS. 29 cases did not fit with inclusion criteria and were mainly FL (3A or 3B), MCL, Nodular lymphocyte Predominant Hodgkin Lymphoma (NLPHL). Among them, the classifier helped to identify one case of CD5- MCL initially diagnosed as DLBCL with CD5- marginal zone lymphoma as well as one case of CD10+ follicular T-cell lymphoma (T-cell rich signature with Rho A mutation) initally diagnosed as FL 3B. Among 235 patients with GCB/ ABC subtyping, the correlation with Nanostring was 97.4% for the molecular classifier and 86,4% for the Hans algorithm. Although the main aim of the pathology review was not dedicated to differentiate DLBCL NOS from PMBL but to validate histological inclusion criteria, after reconciliation 22 DLBCL NOS were reclassified as PMBL. Overall, among the 109 PMBL , PET-CT at baseline in 108 cases evaluable found in 93 cases (86%) a mediastinal mass unique (37) or with satellite lymph nodes(56), in 12 cases (11%) a mediastinal mass with extramediastinal localization and in only 3 cases (3%) the absence of mediastinal mass. This observation fits well with the known anatomical distribution of PMBL and with the rare non mediastinal lymphomas with a PMBL signature. However, the molecular classifier was able to identify 8 signatures and could not recognize other lymphoma subtypes such as High grade B-cell Lymphomas NOS or DH/TH. In addition, 14 follicular lymphomas in our series comprising 5 grade 3A and 6 grade 3B were diagnosed molecularly mainly as DLBCL GC or rarely ABC . Although the TCRBCL were all included in the T-cell signature lymphoma, this signature included high grade B-cell lymphoma, DLBCL NOS, PMBL, DLBCL EBV+, NLPHL.
Conclusion
Overall, the molecular classifier in addition to the expert pathologic review increased accuracy of diagnostic by the pathologists particularly for difficult or rare cases, as well as it strongly helped to identify PMBL and GC/ABC DLCBL. However, the pathologist should be aware of the limit of the tool when dealing with DLBCL differential diagnosis, particularly for the identification of FL3A, FL3B or for cases with a T-cell rich microenvironment such as TCRBCL.

Disclosures
Bobée: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. . Drieux: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties.. Casasnovas: ROCHE: Consultancy, Research Funding; TAKEDA: Consultancy, Research Funding; Gilead/Kite: Consultancy, Research Funding; BMS: Consultancy; Janssen: Consultancy; Amgen: Consultancy. Ghesquieres: Mundipharma Research Limited: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Roche: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Takeda: Other: Travel, accommodation, expenses. Morschhauser: abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; epizyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; jannssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation. Thieblemont: Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Hospira: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: travel compensation, Research Funding; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation. Ribrag: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Epizyme: Honoraria, Research Funding; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; PharmaMar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Argen-X: Research Funding; Astex Pharmaceuticals: Research Funding; Infinity Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MSD Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Haioun: Takeda: Honoraria, Research Funding; Servier: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Miltenyi: Honoraria, Research Funding. Jardin: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. . Ruminy: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. .