Accurate Diagnosis of Aggressive B Cell Non-Hodgkin Lymphomas Using Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissues
Authors: Scott David W. , Wright George W. , Williams Mickey , Lih Jason , Jaffe Elaine S. , Rosenwald Andreas , Campo Elias , Chan Wing (John) C , Connors Joseph M. , Smeland Erlend , Braziel Rita M. , Ott German , Delabie Jan , Weisenburger Dennis D. , Cook James R. , Greiner Timothy C. , Fu Kai , Walsh William , Gascoyne Randy D. , Staudt Louis M. , Rimsza Lisa M. 
Published: October 2019

Abstract

Background: Currently, diagnosis of aggressive B cell non-Hodgkin lymphomas (agg-B-NHL) uses a varying combination of morphology, immunophenotyping, cytogenetics, and/or other molecular techniques resulting in a fragmented, confusing diagnostic system. We sought to develop a multi-analyte gene expression signature assay that could consolidate the diagnostic process into a single platform to improve standardization and accuracy.
Methods: We used formalin-fixed, paraffin-embedded tissue biopsies, qualified by an expert Hematopathology review panel, tumor content of ≥60%, and confirmed B cell immunophenotype. Diagnostic categories included diffuse large B cell lymphoma (DLBCL) including the activated B cell-like (ABC), germinal center B cell-like (GCB) subtypes, unclassifiable (UNC) DLBCL, primary mediastinal B cell lymphoma (PMBCL), Burkitt lymphoma (BL), and mantle cell lymphoma (MCL). Using our previous GEP data, diagnostic signatures, nCounter system (Nanostring, Seattle, WA), and employing published procedures (Scott et al, Blood 2014); we designed probes to 800 genes with utility in distinguishing between these pathological entities. The training cohort comprised 107 unique cases, whose FFPET biopsies were independently assayed at the Molecular Characterization Laboratory, Frederick National Laboratory for Cancer Research (Frederick, MD) and the Centre for Lymphoid Cancer, BC Cancer Agency (Vancouver, BC). The resulting algorithm was locked down and applied to an independent cohort of 199 cases. The nucleic acids from FFPET biopsies from these cases were extracted and run across the two independent laboratories with 83 cases run at both laboratories to assess inter-laboratory performance. The gold standard by which the Nanostring classification was compared was based on Affymetrix gene expression profiling of matched frozen biopsies in the cases of ABC, GCB, and UNC DLBCL (Lenz et al. NEJM 2008) and on the pathological diagnosis by the Hematopathology review panel in the cases of BL, MCL, and PMBCL. The use of human tissues and clinical data for this study was approved by the University of Arizona Institutional Review Board in accordance with the Declaration of Helsinki.
Results: The final locked algorithm consisted of 297 gene probes including 47 housekeeping genes. Thirty-six cases from the training cohort were run again on the new lot of Nanostring code set to allow for cross code set calibration of the assay. The laboratory procedure and algorithm, together termed the "Lymph5Cx" test, consists of a hierarchical series of pair-wise comparisons. In the independent validation set, 257/282 assays (91.1%) yielded gene expression data of sufficient quality (total of 185 of the 199 cases). A classification summary is given in the Table below. In this cohort, 136 cases (82%) were correctly assigned while 11 cases (6.6%) were assigned incorrect diagnoses as follows: 6 BL assigned to GCB, 1 GCB labeled a PMBCL, and 4 PMBCL assigned to DLBCL subtypes. The Lymph5Cx test included categories of indeterminate results between two diagnostic entities and were declared borderline, as indicated in the Table. The agreement between the 2 laboratory sites was 71/72 (99%) of cases that yielded adequate gene expression data at both sites.
Conclusions: The Lymph5Cx test was robust and able to discriminate the often clinically difficult diagnostic categories of agg-B-NHL using a single methodology for cases with histologic and immunophenotypic features of an agg-B-NHL. Misclassification errors were low, suggesting that this test would be useful adjunct to current diagnostic methods. In addition, targetable pathways, as well as genes associated with known prognostic signatures in DLBCL (stromal) and MCL (proliferation) were quantified. Investigation into these latter aspects is on-going. Gene expression signature assays have become a useful clinical and research tool in the on-going area of precision therapeutics based on highly-defined molecular entities.
Table # cases % accurate % borderline % error ABC 26 76.9% 23.1% 0.0% GCB 27 88.9% 7.4% 3.7% BL 48 68.8% 19.8% 11.5% PMBL 30 80.0% 6.7% 13.3% MCL 34 100.0% 0.0% 0.0%

Disclosures
Scott: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Wright:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Williams:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Lih:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Jaffe:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Rosenwald:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Campo:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Chan:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Connors:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Smeland:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Braziel:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Ott:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Delabie:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Weisenburger:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Cook:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Greiner:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Fu:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Walsh:Nanostring: The author is a potential inventor on a patent application using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Gascoyne:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Staudt:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Rimsza:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties.