Protocol detail

Determination of Microsatellite Instability in Tumors

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Mismatch repair/microsatellite status of tumors was determined by immunohistochemistry (IHC) and/or polymerase chain reaction (PCR) as described previously (Hirsch et al. 2018 (link)). Briefly, IHC was performed using the following primary antibodies: MLH1 (1:25; clone ES05, cat # M3640, Dako, Agilent Pathology Solutions, Agilent, Santa Clara, CA, USA), MSH2 (ready-to-use; clone FE11, cat # IR085, Dako), MSH6 (ready-to-use; clone EP49, cat # IR086, Dako), and PMS2 (1:50; clone EP51, cat # M3647, Dako). Detection was done using the EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse (cat # K5007, Dako). IHC stainings were validated by internal and/or external positive controls as well as negative control specimens. IHC stainings were evaluated by two pathologists (DH, TG). Microsatellite PCR of tumor and corresponding normal DNA was done using a panel of five mononucleotide markers (BAT25, BAT26, NR-21, NR-24, and MONO-27; cf. MSI Analysis System, Promega), and a panel of two mononucleotide (BAT25 and BAT26) and three dinucleotide markers [D5S346, D2S123, and D17S250; so-called Bethesda panel; (Boland et al. 1998 (link))]. Tumors were classified as MSI-H when two or more markers of either the Bethesda panel or the Promega panel showed an allelic size variation (i.e., a band shift compared with corresponding normal DNA).

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Hirsch D., Gaiser T., Merx K., Weingaertner S., Forster M., Hendricks A., Woenckhaus M., Schubert T., Hofheinz R.D, & Gencer D. (2020). Clinical responses to PD-1 inhibition and their molecular characterization in six patients with mismatch repair-deficient metastatic cancer of the digestive system. Journal of Cancer Research and Clinical Oncology, 147(1), 263-273.

Publication 2020

clone ES05 clone FE11 clone EP49 clone EP51 MSI Analysis System

Allelic Antibodies Clone Dinucleotide Immunohistochemistry stainings Microsatellite Mismatch repair Mlh1 Mouse Msh6 Pathologists Peroxidase Pms2 Polymerase chain reaction Promega Rabbit Tumors

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Variable analysis

independent variables

Mismatch repair/microsatellite status of tumors

dependent variables

Mismatch repair/microsatellite status of tumors as determined by immunohistochemistry (IHC) and/or polymerase chain reaction (PCR)

control variables

Internal and/or external positive controls for IHC stainings
Negative control specimens for IHC stainings

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