Leica bond

Automated immunohistochemistry on all samples was performed at CMH. FFPE sections were stained with a monoclonal antibody to PTB, clone SH54 (ThermoFisher Scientific, Waltham, MA, USA), using a Leica Bond autostainer (Leica Biosystems, Buffalo Grove, Illinois) and a polymer refine detection system. After heat antigen retrieval, the antibody was applied at a 1:100 dilution in a low pH buffer. Deparaffinized slides were incubated with a primary antibody, secondary antibody, and a polymer conjugate. A single brown color staining was visualized by incubation with DAB chromogen. To visualize the characteristic granular nuclear staining, the hematoxylin counterstain step was omitted. Breast carcinoma tissue served as a positive control. Stained sections were analyzed and graded for the extent of staining, which was reported as a percentage of stained cells in viable areas (from 0 to 100%). PNC staining prevalence was determined by 3 independent reviewers. Immunohistochemistry was performed on 48 paraffin-embedded tissue blocks, including 39 from the primary untreated tumor, 5 after neo-adjunctive treatment, and 4 from metastatic sites.

Brain tumor metastases from primary lung (n = 46), breast (n = 27), melanoma (n = 46) and colorectum (n = 21) tumors were selected from those collected at Dunedin and Hamilton hospitals. Following surgery, the tumors were either fixed in 10% neutral buffered formalin or snap-frozen in liquid nitrogen and stored at −80 °C. Fixed tissue was processed into paraffin wax and embedded. Formalin-fixed paraffin-embedded tumors were cut on a microtome into either 4 µm (immunohistochemistry) or 5 µm (RNAscope) tissue sections that were placed onto coated slides (Leica Bond, Leica Biosystems, Wetzlar, Germany). Paraffin-embedded formalin-fixed tissues were available for all tumors and additional frozen tissue was also available for 51 tumors. Matching primary tumors were available for 24 cases (7 breast, 6 colorectal, 7 lung and 3 melanoma). The clinicopathological data associated with all samples are outlined in Table 1. The inclusion criteria were brain tumor metastases with tumor tissue removed between 2010 and 2016 diagnosed as breast, lung, colorectal or lung metastases with only tissue from the first brain tumor metastasis analyzed in cases with recurrent brain tumors excised. Ethical approval (reference LRS/10/09/037 and MEC/08/02/061) was obtained in New Zealand and all procedures followed institutional guidelines. All individuals provided written informed consent.

Specimens from resected (intracranial) brain metastasis tissue (n = 64) and available matched extracranial tumor tissue (n = 44, comprising 9 resected primary lung tumors, 9 lung biopsies and 26 lymph node biopsies) were considered for analysis. Each case was reviewed by a pathologist or neuropathologist. FFPE tissue sections of 2–3 µm thickness were used for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). IHC staining for PD-L1 was performed using a Leica Bond immunostainer (Leica Biosystems) according to manufacturer’s protocols. Briefly, tissue sections were deparaffinized, rehydrated, and heat-induced antigen retrieval was performed by incubation in CC1 mild buffer (Ventana Medical Systems) for 30 min at 100 °C. Subsequently, tissue sections were incubated with the primary monoclonal antibody rabbit anti-PD-L1 (Clone E1L3N, Cell Signaling, #13684) at 1:200 for 60 min followed by incubation with HRP-conjugated secondary antibody (Leica Biosystems) for 32 min, DAB incubation and counterstaining of nuclei with hematoxylin. Tonsil served as positive control. PD-L1 expression was quantified by assessing the tumor proportion score (TPS), i.e. assessing the percentage of tumor cells with positive membranous staining relative to all vital tumor cells. Only cases with at least 100 evaluable tumor cells were included [25 (link)].

Multiplexed immunofluorescence iterative staining of the CRC TMAs was performed as previously described [19 (link)] using the Cell DIVE™ technology (Cytiva, Issaquah, WA; formerly GE Healthcare). This involves iterative staining and imaging of the same tissue section with 60+ antibodies and is achieved by mild dye oxidation between successive staining and imaging rounds. In total, there were 13 staining rounds using the antibodies described above and DAPI was imaged in each round. The Leica Bond (Leica Biosystems) was used for antibody staining and the IN Cell 2200 was used for imaging. Staining and image recording was repeated twice for S6 due to staining failure. Exposure times were set to fixed values for all images of a given marker.

Immunohistochemistry (IHC) was performed on automated Leica Bond immunostainers (Leica Biosystems, Buffalo Gove, IL,) as described previously [6 (link)]. Briefly, formalin-fixed paraffin-embedded tissue sections cut at 4-5 μm thickness were de-paraffinized and subjected to antigen retrieval prior to incubation with an anti-FOXP3 monoclonal antibody (BioLegend, San Diego, CA. USA). Stains were visualized with 3,3′-diaminobenzidine and hematoxylin counterstain and interpreted by a board-certified pathologist [24 (link)]. The intensity of tissue infiltration by Tregs (FOXP3⁺) was expressed as number of positive cells per microscopic 40x high-power field evaluated on an Olympus BX41 light microscope (Leica Biosystems, Wetzlar, Germany).