Formalin-fixed paraffin-embedded tissue was collected from the archives of the pathology division of our hospital. Hematoxylin–eosin archival slides were revised by an expert pathologist and the diagnosis was confirmed in all cases. The most representative slide of each sample was selected for IHC in cases with available material. Specifically, three-micron-thick serial paraffin sections of each case were processed by IHC using an automated immunostainer (Ventana BenchMark Ultra AutoStainer, Ventana Medical Systems, Tucson, AZ, USA) with antibodies against p53 (clone DO-7, catalogue number 7800-2912,Ventana Medical Systems, Tucson, AZ, USA), ER (clone SP1, catalogue number 790-4324, Ventana Medical Systems, Tucson, AZ, USA), PgR (clone 1E2, catalogue number 790-2223, Ventana Medical Systems, Tucson, AZ, USA), and the MMR status, evaluating the protein expression of MLH1 (clone M1, catalogue number 760-5091, Ventana Medical Systems, Tucson, AZ, USA), PMS2 (clone EPR3947, catalogue number 760-5094, Ventana Medical Systems, Tucson, AZ, USA), MSH2 (clone G219-1129, catalogue number 760-5093, Ventana Medical Systems, Tucson, AZ, USA), MSH6 (clone 44, catalogue number 760-5092, Ventana Medical Systems, Tucson, AZ, USA). Appropriate positive controls were included for each IHC run.
Clone m1
Manufactured by Roche
Sourced in Switzerland
The Clone M1 is a compact and versatile laboratory equipment designed for various experimental applications. It functions as a thermal cycler, capable of accurately controlling and cycling temperatures to facilitate nucleic acid amplification processes such as PCR (Polymerase Chain Reaction). The equipment offers precise temperature regulation and programmable cycling parameters to support a wide range of molecular biology protocols.
Automatically generated - may contain errors
Lab products found in correlation
Clone g219 1129, by Roche (7 mentions)
Clone epr3947, by Roche (4 mentions)
Clone 44, by Cell Marque (4 mentions)
Clone 4b5, by Roche (3 mentions)
Clone 138g6, by Cell Signaling Technology (3 mentions)
Clone mrq28, by Cell Marque (3 mentions)
Clone 44, by Roche (2 mentions)
Clone sp93, by Roche (2 mentions)
Clone a16 4, by Roche (2 mentions)
Ep38y, by Abcam (2 mentions)
Clone sp44, by Roche (2 mentions)
Clone do 7, by Roche (1 mentions)
Clone 1e2, by Roche (1 mentions)
Benchmark ultra autostainer, by Roche (1 mentions)
Clone sp1, by Roche (1 mentions)
Benchmark ultra, by Roche (1 mentions)
Clone 44, by BD (1 mentions)
Optiview dab detection kit, by Roche (1 mentions)
G219 1129, by Cell Marque (1 mentions)
Miseq dx platform, by Illumina (1 mentions)
11 protocols using clone m1
1
Immunohistochemical Analysis of Formalin-Fixed Paraffin-Embedded Tissue
2
Tumor Imaging and MMR Status Assessment
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Tumor imaging was conducted at baseline, every 8 weeks until the primary analysis for the study, and then every 12 weeks thereafter. Response was assessed by blinded independent central review (BICR) per RECIST version 1.1. Adverse events (AEs) were graded per the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0, and were monitored throughout the study and for 30 days (120 days for serious AEs) after treatment discontinuation.
Tumor tissue was collected from all enrolled patients for determination of MMR status by central assessment by pathologist evaluation before randomization. Mismatch repair was assessed in archived tumor tissue from the most recent surgery/biopsy or from a fresh biopsy if no archival tumor tissue was available. Automated immunohistochemistry staining and chromogenic labeling of the MMR proteins MLH1, MSH2, MSH6, and PMS2 on the Ventana Benchmark Ultra was performed using mouse and rabbit antibodies (Roche Diagnostics). Specifically, the MLH1 (clone M1, mouse monoclonal, Ventana, Cat# 790–5091), PMS2 (clone A16‐4, mouse monoclonal, Ventana, Cat# 790–5094), MSH2 (clone G219‐1129, mouse monoclonal, Ventana, Cat# 790–5093), and MSH6 (clone SP93, rabbit monoclonal, Ventana, Cat# 790–5092) antibodies were used to perform immunohistochemistry staining.
Tumor tissue was collected from all enrolled patients for determination of MMR status by central assessment by pathologist evaluation before randomization. Mismatch repair was assessed in archived tumor tissue from the most recent surgery/biopsy or from a fresh biopsy if no archival tumor tissue was available. Automated immunohistochemistry staining and chromogenic labeling of the MMR proteins MLH1, MSH2, MSH6, and PMS2 on the Ventana Benchmark Ultra was performed using mouse and rabbit antibodies (Roche Diagnostics). Specifically, the MLH1 (clone M1, mouse monoclonal, Ventana, Cat# 790–5091), PMS2 (clone A16‐4, mouse monoclonal, Ventana, Cat# 790–5094), MSH2 (clone G219‐1129, mouse monoclonal, Ventana, Cat# 790–5093), and MSH6 (clone SP93, rabbit monoclonal, Ventana, Cat# 790–5092) antibodies were used to perform immunohistochemistry staining.
3
Immunohistochemical Evaluation of MMR Status
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MMR status was evaluated independently, on full stained slides using Ready-to-Use monoclonal antibodies against MLH1 (clone M1, Ventana/Roche, Basal, Schweiz), MSH6 (clone SP93, Ventana/Roche, Basal, Schweiz), MSH2 (clone G219-1129, Ventana/Roche, Basal, Schweiz) and PMS2 (clone A16-4, Ventana/Roche, Basal, Schweiz).
4
Mismatch Repair Protein Immunohistochemistry
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Formalin-fixed, paraffin-embedded tumors were stained for MLH1, MSH2, MSH6, and PMS2 proteins. Mismatch repair protein loss is defined as the absence of nuclear staining in neoplastic cells but positive nuclear staining in lymphocytes and normal adjacent colonic epithelium (16 (link)). Primary monoclonal antibodies against MLH1 (clone M1, Ventana, prediluted), MSH2 (clone G219-1129, Ventana, prediluted), MSH6 (clone 44, Ventana, prediluted), and PMS2 (clone EPR3947, Ventana, prediluted) were applied.
5
Immunohistochemical Detection of DNA Mismatch Repair Proteins
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Immunohistochemistry for MSH2 (clone 25D12, Novocastra (Biosystems Switzerland, Muttenz, Switzerland), dilution 1 : 100; or Clone G219-1129, Roche Ventana Medical Systems, ready-to-use), MSH-6 (clone 44, Becton Dickinson, Allschwil, Switzerland, dilution 1 : 500; or Clone EP49, Epitomics (LabForce, Nunningen, Switzerland), dilution 1 : 50), MLH1 (clone G168-15, Becton Dickinson, dilution 1 : 100; or clone M1, Roche Ventana Medical Systems, ready-to-use) and PMS2 (clone A16-4, Becton Dickinson, dilution 1 : 300; or Clone EPR3947, Roche Ventana Medical Systems, ready-to-use) was performed using the Ventana Benchmark system or using the OptiView DAB Detection Kit (Ventana Medical Systems) followed by counterstaining with haematoxylin.
Any nuclear staining in the tumour cells was considered as ‘positive'. Complete absence of immunoreactivity in the presence of a positive internal control (lymphocytes) was scored as ‘negative'.
Any nuclear staining in the tumour cells was considered as ‘positive'. Complete absence of immunoreactivity in the presence of a positive internal control (lymphocytes) was scored as ‘negative'.
6
Immunohistochemistry and In Situ Hybridization for Mismatch Repair and EBV in Cancers
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One representative FFPE block of the cancer region in each case was chosen for IHC and ISH analysis. Unstained 4-um thick tissue sections were tested by IHC antibodies to MLH1 (Clone M1, ready-to-use; Roche), PMS2 (Clone EPR3947, ready-to-use; Roche), MSH2 (Clone 219-1129, ready-to-use; Roche) and MSH6 (Clone 44, ready-to-use; Roche) for detection of MMR status, as well as chromogenic ISH with EBV-encoded RNA (EBER, Ventana) probe to prove EBV infection, using Benchmark automated staining device (Ventana Medical Systems, Roche, Switzerland) according to the manufacturer' instructions. All IHC and ISH stained sections were reviewed and scored independently by two professional digestive pathologists (WLF and WRF) without knowledge of previous clinical or pathological parameters.
The slides were evaluated as follows: at least one of the MMR proteins (MLH1, MSH2, MSH6, and PMS2) with complete loss of nuclear reactivity in tumor cells but consistently preserved nuclear staining in background non-tumor cells was taken as d-MMR(aberrant expression). When the tumor cells demonstrated intact nuclear immunostaining of all four MMR proteins, the tumor was judged as p-MMR (normal expression). For EBER, tumors with strong blue-black nuclear staining were considered positive.
The slides were evaluated as follows: at least one of the MMR proteins (MLH1, MSH2, MSH6, and PMS2) with complete loss of nuclear reactivity in tumor cells but consistently preserved nuclear staining in background non-tumor cells was taken as d-MMR(aberrant expression). When the tumor cells demonstrated intact nuclear immunostaining of all four MMR proteins, the tumor was judged as p-MMR (normal expression). For EBER, tumors with strong blue-black nuclear staining were considered positive.
7
Tumor DNA Extraction and MSI Testing
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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.
8
Immunohistochemical Profiling of Cancer Biomarkers
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Immunohistochemistry (IHC) was performed using a Ventana XT automated stainer (Ventana) with antibodies for MutL homolog 1 (MLH1, ready to use, clone M1, Roche, Basel, Switzerland), MutS protein homolog 2 (MSH2, ready to use, clone G219-1129; Roche), MutS homolog 6 (MSH6, 1:100, clone 44; Cell Marque, Rocklin, CA, USA), postmeiotic segregation increased 2 (PMS2, 1:40, clone MRQ28; Cell Marque), epidermal growth factor receptor 2 (HER2, ready to use, clone 4B5; Roche), MET (ready to use, clone SP44; Roche), epidermal growth factor receptor (EGFR, 1:100, clone EP38Y; Abcam, Cambridge, UK), phosphatase and tensin homolog (PTEN, 1:100, clone 138G6; Cell Signaling, Danvers, MA, USA), and p53 (1:300, clone DO7; Novocastra, Newcastle, UK). IHC was performed in all cases as previously described [14] .
9
Immunohistochemical Staining Protocol
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IHC was performed with a Ventana XT automated staining instrument. Antibodies recognizing the following targets were used: MutL homolog 1 (MLH1, ready to use, clone M1, Roche, Basel, Schweiz), MutS protein homolog 2 (MSH2, ready to use, clone G219-1129, Roche), MutS homolog 6 (MSH6, 1:100, clone 44, Cell Marque, Rocklin, CA), postmeiotic segregation increased 2 (PMS2, 1:40, clone MRQ28, Cell Marque), HER2 (ready to use, clone 4B5, Roche), EGFR (1:100, EP38Y, Abcam, Cambridge, UK), c-MET (ready to use, clone SP44, Roche), PTEN (1:100, clone 138G6, Cell signaling, Danvers, MA), and p53 (1:300, DO7, Novocastra, Newcastle, UK). Sections were deparaffinized using EZ Prep solution (Ventana Corporation, Tucson, AZ). CC1 standard (pH 8.4 buffer containing Tris/borate/EDTA) was used for antigen retrieval and blocked with inhibitor D (3% H2O2) for 4 min at 37°C. Slides were incubated with primary antibody for 40 min at 37°C, followed by a universal secondary antibody for 20 min at 37°C. Slides were incubated in streptavidin-horseradish peroxidase (SA-HRP) D for 16 min at 37°C, after which the substrate, 3,3′-diaminobenzidine tetrahydrochloride (DAB) H2O2, was added for 8 min, followed by hematoxylin and bluing reagent counterstaining at 37°C.
10
Immunohistochemistry Profiling of Biomarkers
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IHC was performed on a Ventana XT automated staining instrument (Ventana Medical Systems, Tucson, AZ, USA). The following target-specific antibodies were used according to the manufacturer's instructions and a previous study [20 (link)]: MutL homolog 1 (MLH1, ready to use, clone M1, Roche, Basel, Switzerland), MutS protein homolog 2 (MSH2, ready to use, clone G219-1129, Roche), MutS homolog 6 (MSH6, 1:100, clone 44, Cell Marque, Rocklin, CA, USA), postmeiotic segregation increased 2 (PMS2, 1:40, clone MRQ28, Cell Marque), ERBB2 (ready to use, clone 4B5, Roche), EGFR (1:100, EP38Y, Abcam, Cambridge, UK), c-MET (ready to use, clone SP44, Roche), PTEN (1:100, clone 138G6, Cell Signaling, Danvers, MA, USA), and p53 (1:300, DO7, Novocastra, Newcastle, UK). Epstein-Barr virus-encoded small RNAs (EBER) in situ hybridization (ISH) was performed using a Ventana Benchmark ISH system and ISH iView kit (Ventana Medical Systems, Tucson, AZ, USA).
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