A TaqMan Low-Density Array to Predict Outcome in Advanced Hodgkin's Lymphoma Using Paraffin-Embedded Samples

Abstract

Purpose: Despite major advances in the treatment of classic Hodgkin's lymphoma (cHL), f30% of patientsinadvancedstagesmayeventuallydie as resultof the disease,andcurrentmethods topredict prognosis are ratherunreliable.Thus, the applicationof robust techniques for theidentificationofbiomarkers associated with treatment response is essentialif new predictive tools are to be developed. Experimental Design: We used gene expression data from advanced cHL patients to identify transcriptional patterns from the tumoral cells and their nonneoplastic microenvironment, associated with lack of maintained treatment response. Gene-Set Enrichment Analysis was used to identify functionalpathways associated withunfavorable outcome that were significantly enrichedin either the Hodgkin's and Reed-Sternberg cells (regulation of the G 2 -M checkpoint, chaperones, histone modification, and signalingpathways) or the reactive cellmicroenvironment (mainly representedby specificT-cell populations and macrophage activation markers). Results:To explore the pathways identified previously, we used a series of 52 formalin-fixed paraffin-embedded advanced cHL samples and designed a real-time PCR-based low-density array that included the most relevant genes. A large majority of the samples (82.7%) and all selected genes were analyzed successfully with this approach. Conclusions:The results of this assay can be combined in a single risk score integrating these biologicalpathways associated with treatment response and eventually usedina larger series to develop a new molecular outcome predictor for advanced cHL.

A molecular risk score based on 4 functional pathways for advanced classical Hodgkin lymphoma

“…Thus, the selection of genes to be analyzed was determined by results previously obtained in 2 independent series of 29 and 52 advanced cHL patients. 10,11 The patients included in this study fulfilled stringent, previously described criteria (ie, age older than 16 years; advanced cHL; Ann Arbor stage IV, III, or IIB with bulky masses 1 ; proven HIV-negative status) who have been treated with a first-line standard chemotherapy regimen that included adriamycin-ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine) or ABVD variants-and for whom information was available about the achievement of complete remission (CR) and a follow-up of at least 12 months thereafter, which is a well-known and accepted surrogate indication of the course of the disease. With respect to the latter, patients were considered to have had a favorable course if they had achieved CR and maintained it for at least 12 months or with an unfavorable course if they had either not achieved CR or if they had once had it but had relapsed during the following 12 months.…”

“…The genes included in the assay initially were selected from 2 preliminary expression-profiling studies 10,11 that rendered a list of genes expressed by HRS and microenvironment cell subpopulations identified in unfavorable cHL patients. Selected genes were primarily chosen on the basis of their prognostic ability and capacity to represent biologic functions identified as relevant in cHL pathogenesis.…”

“…Selected genes were primarily chosen on the basis of their prognostic ability and capacity to represent biologic functions identified as relevant in cHL pathogenesis. 11 In addition, the strength and consistency of primer and probe performance also were taken into account. 11 The initial selection consisted of 30 genes, including genes expressed by the neoplastic cells involved in the cell cycle (G 2 /M), apoptosis, histones, chaperones, drug metabolism, and mitogen-activated protein kinase signatures, and from microenvironment genes expressed by different cellular or functional populations of T cells, monocytes, macrophages, and dendritic cells.…”

“…4 The clinical outcome of cHL has been found to be related to the expression of multiple biologic markers alone [5][6][7][8] or in combination, 9 expressed either by the tumor HRS cells, macrophages, regulatory T cells, or other nonneoplastic cell subpopulations. [10][11][12][13] Some of these previous analyses rely on array-based gene expression analyses, which use frozen tissue and in most cases can only provide retrospective information. Investigators of other studies have used immunohistochemical staining, with some inherent limitations to the reproducibility of the data thus generated.…”

Immunohistochemical markers for tumor associated macrophages and survival in advanced classical Hodgkin's lymphoma

“…Also, roles for Epstein-Barr virus (EBV) and age as confounding factors have been claimed, since both variables influence the host immune response and TAM composition. 12,16 We recently used GEP to identify specific genes associated with treatment failure in cHL patients 8,17 including gene signatures associated with reactive cells in the microenvironment. This gene signature was related to outcome in a selected series of advanced stage cHL patients using a different approach: RT-PCR analyses and integration of gene expression levels into a logistical regression model.…”