Clone ep49

PD-L1 status was estimated by IHC staining of FFPE tissue sections using anti-PD-L1 antibodies clone 22C3 (DAKO, cat#m3666) or 28–8 (Abcam, Cat#ab205921) [29 (link), 44 (link)]. The specimen was considered PD-L1 positive expression when the combined positive score (CPS) was either ≥ 1 [45 (link)–47 (link)]. IHC analysis was conducted to detect MMR-related proteins, including MLH1 (clone ES05, cat#IR079, DAKO), PMS2 (clone EP51, cat#IR087, DAKO), MSH2 (clone FE11, cat#IR08561-2, DAKO), and MSH6 (clone EP49, cat#IR086, DAKO). Tumors were classified as dMMR when the expression of at least one MMR protein was lost and as MMR-proficient when all four MMR proteins had positive nuclear staining in tumor cells [48 (link)].

All parental tumor tissues and PDX tissues fixed in 10% neutral buffered formalin were processed routinely and embedded in paraffin wax. The sections (3 μm thick) were stained with hematoxylin and eosin (HE) and histopathologically evaluated. Paraffin-embedded sections were also used for immunohistochemistry analysis, and primary antibodies against human pancytokeratin (prediluted; clone AE1/AE3, Dako, Glostrup, Denmark), PAX8 (1:50; clone PAX8R1, Abcam, Cambridge, MA, USA), estrogen receptor (ER) (prediluted; clone SP1, Ventana Medical Systems, Tucson, AZ, USA), TP53 (prediluted; clone DO7, Dako), ARID1A (1:2000; rabbit polyclonal, Sigma, St. Louis, MO, USA), PAX2 (1:200 dilution; clone EPR8586, Abcam), PTEN (1:200; clone D4.3, Cell Signaling Technology, Beverly, MA, USA), PMS2 (prediluted; clone EP51, Dako), and MSH6 (prediluted; clone EP49, Dako) were used for all cases. Primary antibodies against human CD45 (prediluted; clone M0701, Dako) were optionally used for several cases.

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).

Archival formalin-fixed paraffin-embedded (FFPE) tumor specimens with the pathological report, which included HER2 status, were retrospectively collected for IHC analysis, if residual samples were available. IHC analysis, such as for PD-L1, MMR, and chromogenic in situ hybridization for EBV-encoded RNA (EBER-ISH) using fluorescein-labeled peptide nucleic acid probes (EBV PNA Probe/Fluorescein, Agilent), were performed on FFPE tumor samples and assessed by an established pathologist.
For PD-L1 evaluation, IHC staining was performed using PD-L1 IHC 28-8 pharmDx (Dako). The level of PD-L1 protein expression was determined using the combined positive score (CPS), which was calculated as the number of PD-L1-stained cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells and multiplied by 100. Tumor PD-L1 positivity was defined as CPS ≥ 1%.
MMR status was determined by IHC for the following proteins; anti-mutL homolog 1 (MLH1; Clone ES05, Agilent Technologies), anti-mutS homolog 2 (MSH2; Clone FE11, Agilent Technologies), anti-postmeiotic segregation increased 2 (PMS2; Clone EP51, Agilent Technologies), and anti-mutS homolog 6 (MSH6; Clone EP49, Agilent Technologies), in FFPE samples. MMR-deficient (dMMR) was defined as a tumor that lacked staining for at least one of the MMR proteins.