All tumors were evaluated for mismatch repair (MMR) deficiency and microsatellite instability (MSI) via immunohistochemical approach in a Clinical Laboratory Improvement Amendments-approved laboratory for clinical care. Immunohistochemical staining was performed using an automated immunostainer (Leica Bond-III; Leica Biosystems, Buffalo Grove, IL) and Bond Refine PolymerTM biotin-free 3,3’-diaminobenzidine (DAB) detection kit. Antibodies included MLH1 (mouse anti-human antibody clone ES05, Leica, Buffalo Grove, IL), MSH2 (mouse anti-human antibody clone G219–1129, Cell-Marque, Rocklin, CA), MSH6 (mouse anti-human clone 44, Cell-Marque, Rocklin, CA) and PMS2 (mouse anti-human antibody clone MRQ-28, Cell-Marque, Rocklin, CA). Bright signal intensity in greater than 1% of tumor cells was considered positive for protein expression[12 (link), 13 (link)].
Mrq 28
Manufactured by Cell Marque
Sourced in United States
The MRQ-28 is a laboratory equipment product offered by Cell Marque. It is designed for performing various analytical tasks within a laboratory setting. The core function of the MRQ-28 is to facilitate precise and efficient data collection and analysis, but its specific intended use is not provided.
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5 protocols using mrq 28
1
Immunohistochemical Evaluation of MMR and MSI
2
Evaluating Microsatellite Instability Status
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The MSI status was determined using MSI analysis and/or immunohistochemical staining for MLH1 (1:100, clone ES05, Novocastra; Leica Biosystems, Seoul, Korea), MSH2 (1:400, clone G219-1129, Novocastra; Leica Biosystems), PMS2 (1:100, clone MRQ-28; Cell Marque, Rocklin, CA, USA), and MSH6 (1:200, clone 44; Cell Marque). MSI analysis was performed using the Bethesda panel or pentaplex panel. The Bethesda panel consisted of two mononucleotide repeat markers (BAT25 and BAT26) and three dinucleotide repeat markers (D2S123, D5S346, and D17S250), whereas the pentaplex panel contained five mononucleotide repeat markers (NR21, NR24, NR27, BAT25, and BAT26). Normal and tumor allele patterns were compared for each marker. Cases with ≥2, ≥1, and ≥0 positive markers were classified as MSI-H, MSI-low (MSI-L), and MSS, respectively. MMR immunostaining results were recorded as retained or loss of nuclear expression. Retained nuclear expression was considered normal/MMR-proficient, and loss of nuclear expression was abnormal/MMR deficiency.
3
Mismatch Repair Protein Immunohistochemistry
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Mismatch repair protein immunohistochemistry was performed on the primary diffuse gastric tumor using the standard streptavidin-biotin-peroxidase procedure. Primary monoclonal antibodies against MLH1 (clone G168-728, 1:200, BD PharMingen, San Diego, CA, USA 1:200), MSH2 (clone FE11, 1:100, Oncogene Research Products, Cambridge, MA, USA), MSH6 (clone 44, 1:200, BD Transduction, San Jose, CA, USA) and PMS2 (clone MRQ-28, 1:10, Cell Marque, Rocklin, CA, USA) were applied to formalin-fixed, paraffin embedded sections four microns thick. The sections were deparaffinized in xylene, and rehydrated through graded alcohols to distilled water before undergoing antigen retrieval by heat treatment in either citrate solution pH 6.0 (MLH1, PMS2, and MSH2) or EDTA solution pH 9.0 (MSH6). An automated detection using a Leica Bond Autostainer (Leica, Buffalo Groove, IL, USA) was employed. Normal expression was defined as nuclear staining within tumor cells, using infiltrating lymphocytes as positive internal control. Negative protein expression was defined as complete absence of nuclear staining within tumor cells in the face of concurrent positive labeling in internal non-neoplastic tissues.
4
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.
5
Immunohistochemical Evaluation of MMR Proteins
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