title: BCL2 antibodies targeted at different epitopes detect varying levels of protein expression and correlate with frequent gene amplification in diffuse large B-cell lymphoma.
abstract: Patients with aggressive, BCL2 protein-positive (+) diffuse large B-cell lymphoma (DLBCL) often experience rapid disease progression that is refractory to standard therapy. However, there is potential for false-negative staining of BCL2 using the standard monoclonal mouse 124 antibody that hinders the identification of these high-risk DLBCL patients. Herein, we compare 2 alternative rabbit monoclonal antibodies (E17 and SP66) to the 124 clone in staining for BCL2 in formalin-fixed, paraffin-embedded DLBCL tissues. Overall, in 2 independent DLBCL cohorts, E17 and SP66 detected BCL2 expression more frequently than 124. In the context of MYC expression, cases identified as BCL2 (+) with SP66 demonstrated the strongest correlation with worse overall survival. The 124 clone failed to detect BCL2 expression in the majority of translocation (+), amplification (+), and activated B-cell DLBCL cases in which high levels of BCL2 protein are expected. Using dual in situ hybridization as a new tool to detect BCL2 translocation and amplification, we observed similar results as previously reported for fluorescence in situ hybridization for translocation but a higher amplification frequency, indicating that BCL2 amplification may be underreported in DLBCL. Among the discrepant cases, phosphorylation of BCL2 at T69 and/or S70 was more common than in the concordant cases and may contribute to the 124 false negatives, in addition to previously associated mutations within the epitope region. The accurate detection of BCL2 expression is important in the prognosis and treatment of DLBCL particularly with new anti-BCL2 therapies.
authors.affiliation: Department of Pathology, University of Arizona, Tucson, AZ 85724.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Kendrick
authors.affiliation: Department of Pathology, University of Arizona, Tucson, AZ 85724.
authors.forename: Lucas
authors.initials: L
authors.lastname: Redd
authors.affiliation: Ventana Medical Systems, Inc., Tucson, AZ 85755.
authors.forename: Andrea
authors.initials: A
authors.lastname: Muranyi
authors.affiliation: Ventana Medical Systems, Inc., Tucson, AZ 85755.
authors.forename: Leigh A
authors.initials: LA
authors.lastname: Henricksen
authors.affiliation: Ventana Medical Systems, Inc., Tucson, AZ 85755.
authors.forename: Stacey
authors.initials: S
authors.lastname: Stanislaw
authors.affiliation: Department of Biostatistics, University of Nebraska Medical Center, Omaha, NE 68198.
authors.forename: Lynette M
authors.initials: LM
authors.lastname: Smith
authors.affiliation: Department of Pathology, University of Manitoba, Winnipeg, MB, Canada R3A 1R9.
authors.forename: Anamarija M
authors.initials: AM
authors.lastname: Perry
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198.
authors.forename: Kai
authors.initials: K
authors.lastname: Fu
authors.affiliation: Department of Pathology, City of Hope Medical Center, Duarte, CA 91010.
authors.forename: Dennis D
authors.initials: DD
authors.lastname: Weisenburger
authors.affiliation: Department of Pathology, University of Wuerzburg, Wuerzburg, Germany 97070.
authors.forename: Andreas
authors.initials: A
authors.lastname: Rosenwald
authors.affiliation: Department of Clinical Pathology, Robert-Bosch-Krankenhaus, and Dr. Margarete Fischer-Bosch-Institu fur Klinische Pharmakologie (IKP), 70376 Stuttgart, Germany.
authors.forename: German
authors.initials: G
authors.lastname: Ott
authors.affiliation: Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4E6.
authors.forename: Randy D
authors.initials: RD
authors.lastname: Gascoyne
authors.affiliation: Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892.
authors.forename: Elaine S
authors.initials: ES
authors.lastname: Jaffe
authors.affiliation: Department of Pathology, Hospital Clínic, Barcelona, Spain 08028.
authors.forename: Elías
authors.initials: E
authors.lastname: Campo
authors.affiliation: Department of Pathology, The Norwegian Radium Hospital, University of Oslo, Oslo, Norway 0310.
authors.forename: Jan
authors.initials: J
authors.lastname: Delabie
authors.affiliation: Department of Clinical Pathology, Oregon Health & Science University, Portland, OR 97239.
authors.forename: Rita M
authors.initials: RM
authors.lastname: Braziel
authors.affiliation: Department of Molecular Pathology and Laboratory Medicine, Cleveland Clinic, Cleveland, OH 44195.
authors.forename: James R
authors.initials: JR
authors.lastname: Cook
authors.affiliation: Department of Molecular Pathology and Laboratory Medicine, Cleveland Clinic, Cleveland, OH 44195.
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Tubbs
authors.affiliation: Metabolism Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892.
authors.forename: Louis M
authors.initials: LM
authors.lastname: Staudt
authors.affiliation: Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4E6.
authors.forename: Wing Chung
authors.initials: WC
authors.lastname: Chan
authors.affiliation: Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4E6.
authors.forename: Christian
authors.initials: C
authors.lastname: Steidl
authors.affiliation: Department of Pathology, University of Arizona, Tucson, AZ 85724; Ventana Medical Systems, Inc., Tucson, AZ 85755.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Grogan
authors.affiliation: Department of Pathology, University of Arizona, Tucson, AZ 85724. Electronic address: lrimsza@pathology.arizona.edu.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Rimsza
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor / Q000032:analysis*; D018985:Epitopes, B-Lymphocyte / Q000032:analysis; D005784:Gene Amplification; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics* / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis*; D011817:Rabbits; D046888:Tissue Array Analysis
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D018985:Epitopes, B-Lymphocyte; D019253:Proto-Oncogene Proteins c-bcl-2
keywords: BCL2 amplification and translocation; BCL2 phosphorylation; Diffuse large B-cell lymphoma; Dual in situ hybridization (dual ISH); E17 and SP66 BCL2 antibodies
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