Anti pd l1 antibodies clone 22c3

TMB was calculated by counting somatic mutations, including coding base substitutions and indel mutations per megabase (muts/Mb) of genome examined, and by excluding known hotspot mutations in oncogenic drivers and known germline alterations within the single nucleotide polymorphism database (dbSNP) (11 (link)). Using previous studies as a guide, the threshold for high TMB was set as ten.
Immunohistochemical staining for FFPE tissue sections was performed using anti-PD-L1 antibodies (clone 22C3, Cat # M3653, DAKO, Agilent, CA, USA). Dilutions (28-8 1:300; 22C3 1:50) of primary antibodies were used for antigen detection. All slides were counterstained with hematoxylin. The PD-L1 tumor proportion score (TPS), which is the percentage of tumor cells showing partial or complete membrane staining, was determined; and samples were classified as negative, low-positive, or high-positive (TPS of <1%, 1%–49%, and ≥50%, respectively) (12 (link)).

All resected specimens for IHC were formalin‐fixed, paraffin‐embedded (FFPE). Tumor subtypes are determined through obtaining ER, PR, HER2, and ki67 IHC staining. Staining for all markers was carried out according to the standard laboratory techniques. FFPE tissue sections for ER, PR, HER2, and Ki67 were stained, respectively. Ki67 ≥ 20% is defined as high (Ki67‐H) and Ki67 < 20% is defined as low (Ki67‐L). ER or PR expression were identified positive if >1% of tumor nuclei were strongly stained in accordance with the 2010 ASCO/CAP guidelines.¹⁶ Additionally, HER2 status was detected by fluorescence in situ hybridization.¹⁷Immunohistochemical staining of FFPE tissues were performed by anti‐PD‐L1 antibodies (clone 22C3, Cat#M3653, DAKO). Dilutions (22C3 1:50) of the primary antibodies were used for antigen detection. All slides were counterstained with hematoxylin. PD‐L1 level was reported as CPS which was defined as the number of PD‐L1‐positive cells divided by the total tumor cells, multiplied by 100. CPS <1 is defined as PD‐L1‐negative; 1 ≤ CPS <10 is defined as PD‐L1‐low (PD‐L1‐L); CPS ≥10 is defined as PD‐L1‐high (PD‐L1‐H).