Formaldehyde-fixed, paraffin-embedded tissue sections of 3µm thickness from tumor resection or biopsy were used for immunohistochemistry. Monoclonal mouse anti-human primary antibody for p16INK4A (1:50; clone: G175-405; BD Biosciences; Cat. No. 550834), HLA-ABC (1:100; clone: EMR8-5; Abcam; Cat. No. AB70328), CD20 (1:250; clone L26; Dako; Cat. No. M0755) and MUM1 (1:1000; clone: MUM1p; Dako; Cat. No. M725929-2) were applied. Detection of primary antibodies was achieved by secondary goat anti-mouse antibody conjugated with a HRP-labelled polymer (Dako EnVision+) and peroxidase activity was visualized by diaminobenzidine tetrahydrochloride. Hematoxylin was used as nuclear counterstaining. Detection of HLA-ABC expression in ≥ 70% of tumor cells counted as high MHC-I expression, strong cytoplasmic and nuclear staining was regarded positive for p16INK4A expression.
Mum1p
Manufactured by Agilent Technologies
Sourced in Denmark
The MUM1p is a lab equipment product manufactured by Agilent Technologies. It is a specialized instrument designed for specific laboratory applications. Due to the technical nature of this product, a detailed and unbiased description cannot be provided without the risk of making interpretations or extrapolations. Therefore, a comprehensive description for this product is not available at this time.
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Lab products found in correlation
Pg b6p, by Agilent Technologies (4 mentions)
Benchmark xt, by Roche (3 mentions)
Mib 1, by Agilent Technologies (2 mentions)
Ber h2, by Agilent Technologies (2 mentions)
G175 405, by BD (1 mentions)
Clone l26, by Agilent Technologies (1 mentions)
Emr8 5, by Abcam (1 mentions)
Goat anti mouse igg hrp conjugate, by Proteintech (1 mentions)
Cd3 polyclonal, by Agilent Technologies (1 mentions)
Bcl2 124, by Agilent Technologies (1 mentions)
Cd20 l26, by Leica (1 mentions)
Cd10 56c6, by Leica (1 mentions)
Cd79a jcb117, by Agilent Technologies (1 mentions)
Sp4 r, by Roche (1 mentions)
Bcl 2 oncoprotein, by Agilent Technologies (1 mentions)
C myc, by Abcam (1 mentions)
Ab20034, by Abcam (1 mentions)
A0398, by Agilent Technologies (1 mentions)
M0814, by Agilent Technologies (1 mentions)
M7259, by Agilent Technologies (1 mentions)
14 protocols using mum1p
1
Immunohistochemical Analysis of Tumor Markers
2
Immunohistochemical Profiling of Lymphoma Cells
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Cultured lymphoma cell lines were fixed overnight at room temperature in 10% neutral buffered formalin and then embedded into paraffin blocks. Sections of 5 µM thickness were cut. Sections were deparaffinized and blocked with hydrogen peroxide, followed by antigen retrieval through steam treatment in Tris-EDTA buffer (pH 9) for 20 minutes. Slides were washed with PBST and then blocked with UV light for 5 minutes. Antibody dilutions (in Tris-BSA buffer) for immunohistochemistry were as follows: anti-BCL6 (Dako, clone PG-B6p, #M7211) 1∶400; anti-CD10 (Leica, clone 56C6, #CD10-270-CE-S) 1∶50; anti-FOXP1 (Cell Signaling # 2005S) 1∶200; anti-GCET1 (Abcam, clone RAM341, #ab6889) 1∶100; anti-MUM1 (Dako, clone MUM1p, #M7259), 1∶1,500. Staining with the primary antibodies was for 30 minutes, followed UV antibody enhancer for 10 minutes and UV polymer treatment for 15 minutes. Slides were incubated with DAB substrate solution for 10 minutes and counterstained with Hematoxylin for 5 minutes. The slides were coversliped and scored by a hematopathologist (results in S1 File table D).
3
Immunohistochemical Staining of Lymphoma Markers
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Staining with anti‐CD20, CD10, BCL‐6, and MUM‐1 antibodies was performed as described previously.36 Briefly, mouse anti‐CD20 (L26; dilution 1:200; Invitrogen), mouse anti‐CD10 (56C6; dilution 1:1; Invitrogen), mouse anti‐BCL‐6 (D‐8; dilution 1:75; Santa Cruz), and mouse anti‐MUM‐1 (MUM1p; dilution 1:10; Dako) antibodies were used as the primary antibody in a 1‐hour reaction at room temperature and a goat anti‐mouse IgG HRP conjugate (dilution 1:500; ProteinTech) was then used as the secondary antibody.
4
Immunohistochemical Evaluation of B-Cell Lymphoma
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The B‐cell lymphoma diagnosis was made according to the 2017 WHO classification. Two hematopathologists reviewed the BM slides for the histopathological detection of BMI. Immunohistochemical staining was performed using monoclonal antibodies against the following antigens: CD3 (polyclonal, 1:200; Dako), CD5 (SP19, RTU; Roche), CD10 (56C6, 1:200; Novocastra), CD20 (L26, 1:4; Novocastra), CD79a (JCB117, 1:200; Dako), BCL2 (124, 1:200; Dako), BCL6 (GI191E/A8, RTU; Roche), cyclin D1 (SP4‐R, RTU; Roche), FMC7 (4C7, 1:100; Novocastra), Ki‐67 (MIB‐1, 1:100; Dako), and MUM‐1 (MUM1p, 1:400; Dako).
5
Immunohistochemical and FISH Analyses of DLBCL
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Immunohistochemical and FISH analyses were performed by using standard protocols. FFPE sections were stained with primary antibodies against CD5 (sp19, Roche), CD10 (56C6; DAKO), BCL6 (PG‐B6P; DAKO), BCL2 (124; DAKO), MYC (Y69, Epitomics), and IRF4/MUM1 (MUM1P; DAKO) on a BenchMark XT automated immunostainer (Ventana Medical Systems). A positive result was concluded if ≥30% of tumor cells were stained with antibodies against CD10, BCL6, and MUM1. GCB and non‐GCB subtypes of DLBCL were determined by the Hans algorithm.7 High‐level expression of BCL2 or MYC was defined, respectively, in that >50% and >40% of tumor cells were positive. Detection of breakpoints affecting the BCL2, MYC (LSI dual‐color break‐apart probes; Vysis‐Abbott), and IRF4 (LSI dual‐color break‐apart probes; Zytovision) was performed by using FISH. A positive result was concluded when ≥15% of tumor cells demonstrating split signals. Epstein–Barr virus (EBV)‐encoded small RNA (EBER) was detected by in situ hybridization.
6
Immunohistochemical Analysis of Lymphoid Neoplasms
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Excised tissue specimens were fixed in 10% formalin and embedded in paraffin wax. Serial sections from the paraffin blocks were stained with hematoxylin and eosin. Immunohistochemical analysis was performed according to standard procedures. The antibodies used are as follows: CD3 (1:50, PS1; Leica Biosystems, Newcastle upon Tyne, UK), CD5 (1:50, 4C7; Leica Biosystems), CD10 (1:50, 56C6; Leica Biosystems), CD20 (1:100, L26; Leica Biosystems), CD30 (1:30, Ber-H2; Dako, Glostrup, Denmark), BCL2 oncoprotein (1:50, 124; Dako), BCL6 oncoprotein (1:100, LN22, Leica Biosystems), multiple myeloma oncogene 1 (MUM1; 1:50, MUM1p; Dako), cyclin D1 (1:100, DCS-6; Nichirei Biosciences Inc., Tokyo, Japan), Ki-67 (1:100, MIB-1; Dako), and c-MYC (1:200, Y69; Abcam plc., Cambridge, UK). Positivity was evaluated using cut-off values of 40% for MYC and 20% for others.
7
Immunohistochemistry Assessment of Cell of Origin
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Cell of origin was assessed by immunohistochemistry according to the Hans algorithm.[11 (link)] Based on the 3 markers, CD10 (56C6, Novacastra/Leica microsystems, Wetzler, Germany), BCL6 (PG-B6p, DAKO, Glostrup, Denmark) and MUM1(MUM1p, DAKO, Glostrup, Denmark), according to the Hans classifier, the subgroups are as follows: GCB type (CD10+; CD10− BCL6+ MUM1−), nonGCB type (CD10− BCL6+ MUM1+; CD10− BCL6−). All biopsies were reviewed by 3 pathologists. The conditions for all the antibodies and their evaluation followed the guidelines recommended by the Lunenburg Lymphoma Biomarker Consortium.[12 (link)]
8
Quantitative Immune Cell Profiling
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Quantification of immune cells (T cells, cytotoxic T cells, macrophages, plasma cells, and neutrophils) was assessed by IHC staining on tissue microarrays and automatic quantification by QuPath63 (link). CD45Ro (Ventana; 790-2930; Mouse monoclonal (UCHL-1)) for total immune cells, CD3 (Ventana; 790-4341; Rabbit monoclonal (2GV6)) for total T cells, CD8 (NOVO; PA0183; Mouse monoclonal (4B11)) for cytotoxic T cells, Foxp3 (Abcam; ab20034; Mouse monoclonal (236A/E7)) for Tregs, CD68 (DAKO; M0814; Mouse monoclonal (KP1)) for macrophages, CD163 (NOVO; NCL-CD163; Mouse monoclonal (10D6)) for M2 macrophages, MUM1 (DAKO; M7259; Mouse monoclonal (MUM1P)) for plasma cells, and MPO (DAKO; A0398; Rabbit polyclonal) for neutrophils were used as cell type markers. Four-μm-thick glass slides were stained using Ventana BenchMark XT and OptiView universal DAB staining kit (Ventana #760-700). Staining procedure of automatic stainer is antigen retrieval (100 °C for 24 min in citrate buffer), peroxidase inhibition (37 °C for 4 min in 3% H2O2), primary antibody (37 °C for 16 min), Linker (HQ linker; 37 °C for 8 min), polymer amplification (HRP multimer; 37 °C for 8 min), chromogen by DAB (37 °C for 8 min), counterstaining by hematoxylin (37 °C for 8 min), and post counterstain (37 °C for 4 min).
9
Immunohistochemical Profiling of PTLD Samples
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Primary diagnostic formalin fixed paraffin-embedded tissue samples containing representative PTLD were available from 52 patients; samples of these were included in a tissue microarray (TMA). Briefly, three 1-mm diameter tissue cores were identified in tumor areas, punched out and re-embedded in recipient blocks using a TMA master-01 (3DHISTECH Ltd., Budapest, Hungary).
Immunohistochemical stains were performed on 4 μm formalin fixed paraffin-embedded tissue sections according to standard antibody-specific protocols, optimized in house for use with the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson, AZ). For review of the classification of the tumors according to WHO 2008 criteria, a panel of markers was applied consisting of CD3 (polyclonal, Dako, Glostrup, Denmark), CD5 (SP19, Ventana Medical Systems), CD4 (SP35, Ventana), CD8 (SP57, Ventana), CD10 (SP67, Ventana), CD20 (L26, Ventana), CD79a (MRQ-48, Ventana), CD30 (Ber-H2, Dako) and MUM1 (MUM1P, Dako). The EBV antigens were visualized using antibodies to LMP (LMP clones CS.1-4, Dako) and EBNA2 (PE2; Abcam, Cambridge, UK). EBV-RNA (EBER) expression was identified by in-situ hybridization (ISH iView Blue Detection kit; Ventana). In each case, EBV status in the PTLD cell population was evaluated by the authors (S.H.D. and M.V.) and recorded as either positive or negative.
Immunohistochemical stains were performed on 4 μm formalin fixed paraffin-embedded tissue sections according to standard antibody-specific protocols, optimized in house for use with the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson, AZ). For review of the classification of the tumors according to WHO 2008 criteria, a panel of markers was applied consisting of CD3 (polyclonal, Dako, Glostrup, Denmark), CD5 (SP19, Ventana Medical Systems), CD4 (SP35, Ventana), CD8 (SP57, Ventana), CD10 (SP67, Ventana), CD20 (L26, Ventana), CD79a (MRQ-48, Ventana), CD30 (Ber-H2, Dako) and MUM1 (MUM1P, Dako). The EBV antigens were visualized using antibodies to LMP (LMP clones CS.1-4, Dako) and EBNA2 (PE2; Abcam, Cambridge, UK). EBV-RNA (EBER) expression was identified by in-situ hybridization (ISH iView Blue Detection kit; Ventana). In each case, EBV status in the PTLD cell population was evaluated by the authors (S.H.D. and M.V.) and recorded as either positive or negative.
10
Immunohistochemical Profiling of Lymphomas
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Using an automated immunohistochemical stainer (Bench-MarkXT, Ventana, Tucson, AZ, USA), sections of the TMA cut in 4-μm thickness were routinely processed for immunohistochemical stains for CD20 (1:500, H1, heat-induced antigen retrieval, BD Pharmingen, San Jose, CA, USA), BCL2 (1:100, 124, heat-induced antigen retrieval, Dako, Carpinteria, CA, USA), BCL6 (1:30, PG-B6p, heat-induced antigen retrieval, Dako), CD10 (pre-diluted, SP67, heat-induced antigen retrieval, Ventana), and MUM1 (1:100, MUM1p, heat-induced antigen retrieval, Dako). Cases were designated as positive when ≥30% of the tumor cells were immuoreactive for all antibodies except for CD20 [5 (link)]. Cases were classified into germinal center B-cell versus non-germinal center B-cell type according to Hans classification [6 (link)].
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