The methods for extracting tumor DNA and testing for MSI have been described elsewhere [13 (link),14 (link)]. In short, MSI testing was performed using the Bethesda panel. In this test of five microsatellite markers (BAT-25, BAT-26, D2S123, D5S346, and D17D250), instability of two markers indicated high MSI (MSI-H), instability of one marker indicated low MSI (MSI-L), and no signs of instability indicated microsatellite stable (MSS). Tumors showing MSI-H were subjected to IHC testing for four biomarkers (MLH1, MSH2, MSH6, and PMS2) in the pathological sample using antibody cross-linkage (MLH1 [mouse monoclonal primary antibody, prediluted, clone M1, Roche, Basel, Switzerland], MSH2 [mouse monoclonal primary antibody, 1:200, clone G219-1129, Cell Marque, Darmstadt, Germany], MSH6 [mouse monoclonal primary antibody, 1:100, clone 44, Cell Marque], and PMS2 [mouse monoclonal primary antibody, prediluted, clone ERP3947, Roche]). Germline genetic testing was done by direct sequencing (Illumina MiseqDX platform, Illumina, San Diego, CA) with optional addition of multiplex ligation-dependent probe amplification of genomic DNA of the target gene isolated from peripheral blood leukocytes. Variants were then clinically curated by a clinician.
G219 1129
Manufactured by Cell Marque
Sourced in United States, United Kingdom
G219-1129 is a lab equipment product. It is a device used for performing laboratory tasks and analysis. The core function of this product is to assist in the execution of various experiments and tests within a laboratory setting. No further details are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Clone 44, by Cell Marque (2 mentions)
Mrq 28, by Cell Marque (2 mentions)
Miseq dx platform, by Illumina (1 mentions)
Clone m1, by Roche (1 mentions)
Bond max autoimmunostainer, by Leica (1 mentions)
Clone es05, by Leica (1 mentions)
Bond 3, by Leica (1 mentions)
Bond refine polymer, by Leica (1 mentions)
Ultrasystem, by Roche (1 mentions)
Pd l1 ihc 22c3 pharmdx assay, by Agilent Technologies (1 mentions)
Bond max system, by Leica (1 mentions)
Antibody es05 clone, by Leica (1 mentions)
7 protocols using g219 1129
1
Tumor DNA Extraction and MSI Testing
2
Immunohistochemical Evaluation of MMR Proteins
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IHC was performed on tissue microarray tumor sections using antibodies against MLH1 (clone ES05; 1: 200 dilution; Leica Biosystems, Melbourne, Australia) and MSH2 (clone G219-1129; 1: 500 dilution; CELL Marque; Rocklin, CA, USA). IHC staining procedures were conducted using a Bond-max autoimmunostainer (Leica Biosystems, Melbourne, Australia) using Bond™ Polymer refine detection, DS9800 (Vision Biosystems, Melbourne, Australia). MLH1 and MSH2 proteins were determined to be preserved in a case when nuclear staining in the tumor cells of the case was observed. Loss of expression of MMR proteins (MLH1- or MSH2-) was determined when nuclear staining in the tumor cells was not observed. In the present study, MMR-deficient or MSI tumors was defined as the loss of MLH1 and/or MSH2 expression.
3
Immunohistochemical Mismatch Repair Protein Analysis
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Immunohistochemical staining of MLH1 (G168-728, Cell Marque, CA, USA), PMS2 (MRQ-28, Cell Marque, CA, USA), MSH2 (G219-1129, Cell Marque, CA, USA), and MSH6 (PU29, Leica Biosystems, Nussloch, Germany) was performed to confirm the expression of mismatch repair proteins. Staining was performed in representative blocks (whole sections) of primary tumors. The cases showing loss of expression in all tumor cells were defined as negative. The nuclei of normal epithelium and inflammatory cells were regarded as internal controls. The tumors positive for all four immunohistochemical stains were classified as microsatellite stable (MSS). Those that gave negative results for one or more of the four immunohistochemical stains were classified as MSI.
4
Immunohistochemical Evaluation of MMR and MSI
5
Immunohistochemical Staining for MMR Proteins
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Immunohistochemical staining for MLH1, MSH2, MSH6, and PMS2 was performed according to the manufacturer’s instructions, with primary antibodies against MLH1 (mouse monoclonal primary antibody, prediluted, clone M1; Ventana, Tucson, AZ, USA), MSH2 (mouse monoclonal primary antibody, 1:200, clone G219-1129; Cell Marque, Rocklin, CA, USA), MSH6 (mouse monoclonal primary antibody, 1:100, clone 44; Cell Marque), and PMS2 (mouse monoclonal primary antibody, prediluted, clone ERP3947; Ventana) used for staining using a Benchmark XT staining and ULTRA system (Ventana).
6
Comprehensive Tumor Biomarker Profiling
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MSI status of tumor tissue was determined by immunohistochemistry (IHC) for MLH1 (antibody: ES05 clone; 1:100 dilution; Novocastra, UK) and MSH2 (clone G219-1129; 1:500 dilution; CELL Marque; Rocklin, CA, USA) in formalin-fixed paraffin-embedded tissue sections. Preservation of MLH1 and MSH2 protein expression was determined by the presence of nuclear staining in the tumor cells; loss of MMR protein expression (i.e., MLH1-or MSH2-) was determined by absence of nuclear staining in the tumor cells. In the present study, MSI-H tumors were indicative of the loss of MLH1 and/or MSH2 expression.
PD-L1 expression was assessed by using PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies; Santa Clara, CA, USA) [3] . Combined positive score (i.e., the number of PD-L1 staining cells divided by the total number of viable tumor cells) ≥ 1% indicated PD-L1 positivity.
For determination of EBV status, in situ hybridization for EBV-encoded small RNA was performed by using Leica BOND-MAX system (Leica Biosystems; Melbourne, Australia) [23] . Strong signals within most tumor cells were considered EBV positive.
PD-L1 expression was assessed by using PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies; Santa Clara, CA, USA) [3] . Combined positive score (i.e., the number of PD-L1 staining cells divided by the total number of viable tumor cells) ≥ 1% indicated PD-L1 positivity.
For determination of EBV status, in situ hybridization for EBV-encoded small RNA was performed by using Leica BOND-MAX system (Leica Biosystems; Melbourne, Australia) [23] . Strong signals within most tumor cells were considered EBV positive.
7
Immunohistochemistry for MMR Protein Analysis
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To characterize the MMR system, IHC was performed using 4-μm-thick paraffin tissue sections. The manufacturers and incubation conditions for primary antibodies are summarized in Table 1 . Sections were incubated for 15 minutes with antibodies against MLH1 protein (1:200, ES05, Leica, Newcastle upon Tyne, UK), MSH2 protein (1:100, G219-1129, Cell Marque, Rocklin, CA, USA), MSH6 protein (1:50, 44, Cell Marque), and PMS2 (1:50, MRQ-28, Cell Marque).
Tumors were considered deficient in MLH1, MSH2, MSH6, and PMS2 expression when there was a complete absence of detectable nuclear staining in neoplastic cells. Intact nuclear staining of the adjacent non-neoplastic epithelium, stromal cells, or lymphocytes served as an internal positive control (Appendix 1 ).
Whole sections were stained and reviewed for cases that showed a loss of expression on a microarray.
Tumors were considered deficient in MLH1, MSH2, MSH6, and PMS2 expression when there was a complete absence of detectable nuclear staining in neoplastic cells. Intact nuclear staining of the adjacent non-neoplastic epithelium, stromal cells, or lymphocytes served as an internal positive control (
Whole sections were stained and reviewed for cases that showed a loss of expression on a microarray.
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