title: Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP.
abstract: Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma.
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK rittakoutinho@gmail.com.
authors.forename: Rita
authors.initials: R
authors.lastname: Coutinho
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Clear
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Mazzola
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK Department of Histopathology, Barts Health NHS Trust, Royal London Hospital, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Owen
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Greaves
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Wilson
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK.
authors.forename: Janet
authors.initials: J
authors.lastname: Matthews
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK Department of Histopathology, Barts Health NHS Trust, Royal London Hospital, UK.
authors.forename: Abigail
authors.initials: A
authors.lastname: Lee
authors.affiliation: Department of Hematology, Portuguese Institute of Oncology, Lisbon, Portugal.
authors.forename: Rute
authors.initials: R
authors.lastname: Alvarez
authors.affiliation: Department of Hematology, Portuguese Institute of Oncology, Lisbon, Portugal.
authors.forename: Maria Gomes
authors.initials: MG
authors.lastname: da Silva
authors.affiliation: Department of Pathology, Portuguese Institute of Oncology, Lisbon, Portugal.
authors.forename: José
authors.initials: J
authors.lastname: Cabeçadas
authors.affiliation: Department of Histopathology, Barts Health NHS Trust, Royal London Hospital, UK.
authors.forename: Donna
authors.initials: D
authors.lastname: Neuberg
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK Department of Histopathology, Barts Health NHS Trust, Royal London Hospital, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Calaminici
authors.affiliation: Department of Hemato-Oncology, Barts Cancer Institute, Queen Mary University, London, UK.
authors.forename: John G
authors.initials: JG
authors.lastname: Gribben
mesh_terms: D000465:Algorithms; D058846:Antibodies, Monoclonal, Murine-Derived / Q000008:administration & dosage; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage*; D057205:Automation, Laboratory; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology*; D003520:Cyclophosphamide / Q000008:administration & dosage; D004317:Doxorubicin / Q000008:administration & dosage; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D016403:Lymphoma, Large B-Cell, Diffuse / Q000188:drug therapy / Q000235:genetics / Q000276:immunology* / Q000401:mortality; D015999:Multivariate Analysis; D011241:Prednisone / Q000008:administration & dosage; D011379:Prognosis; D000069283:Rituximab; D016019:Survival Analysis; D046888:Tissue Array Analysis; D059016:Tumor Microenvironment / Q000235:genetics / Q000276:immunology*; D014750:Vincristine / Q000008:administration & dosage
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D017252:CD3 Complex; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C571759:R-CHOP protocol; D000069283:Rituximab; D014750:Vincristine; D004317:Doxorubicin; D003520:Cyclophosphamide; D011241:Prednisone