Patient tumor tissues
Whole section cuts of formalin-fixed, paraffin-embedded (FFPE) tumor tissue of 14 surgically resected lung adenocarcinoma patients of the University Hospital Zurich were used. One patient (Case N°11) was represented by two samples. In total, 15 surgical specimens were analyzed. Three specimens (Case N°12, 13 and 14) were used for protocol evaluation. The elaborated protocol was applied to the series of 12 other samples. As control for ALK-negative samples, we included a tissue microarray (TMA) of 196 surgically resected non-small cell lung carcinoma (NSCLC) patients. The Ethical Commission of the Canton of Zurich approved the study under reference number KEK ZH-Nr. 29–2009/14.
Blocking solutions, primary antibodies and detection systems used for the MTP-IF
To determine the optimal blocking solution the following reagent were tested: bovine serum albumin (BSA) 1% (Life Technologies), horse serum 2.5% (Vector Laboratories), Top Block (Lubioscience) and Sudan Black B (Sigma-Aldrich).
Mouse anti-human ALK antibody clone 5A4 (Novocastra) was used with a dilution of 1/10 in PBS-Tween (PBST) 0.05% (Fischer Scientific). Mouse anti-human pan-cytokeratin (pan-CK) antibody cocktail AE1/AE3 (Dako) was diluted 1/100.
Reagents used for the detection step included the goat anti-mouse IgG (H + L) highly cross-adsorbed Alexa Fluor 647 (Life Technologies, 1/40), ImmPRESS system with horseradish peroxidase (HRP)-coupled secondary antibody (Vector Laboratories, ready-to-use) and tyramide signal amplification (TSA) kit with Alexa Fluor 647 labelled tyramide diluted 1/100 in amplification buffer according to manufacturer recommendations (Life Technologies).
For nuclear counterstaining, DAPI was included in the SlowFade Gold Antifade mounting solution (Life Technologies, RTU).
Tissue slide pre-processing for MTP-IF
Two μm thick FFPE tumor whole sections were mounted on glass slides and manually (OFF-chip) de-paraffinized by heating at 65 °C for 10 min followed by 10 min incubation with dewaxing solution (Histoclear, National Diagnostic); after being rehydrated with decreasing concentrations of ethanol (100, 95, 70 and 40% v/v, Fischer Chemical) down to tap water, slides underwent heat-induced antigen retrieval step in TRIS/EDTA solution pH 9 (Dako) at 95 °C for 30 min. Slides were cooled-down for 20 min in the antigen retrieval solution, washed and kept in PBS until the staining process with the microfluidic device started.
Microfluidic setup
Glass slides were inserted into the MTP device, which contains a clamping system that interfaces the microfluidic chip with the tissue sample via an elastomeric gasket, forming a chamber of reaction of 100 μm height. Reagents were sequentially delivered into the chamber, incubated and washed over the surface of the tissue cuts on a one-second time resolution, as previously described [7] and detailed in Fig. 1b. The reagent delivery system (RDS) consisted of a set of valves used to deliver reagents from pressurized reservoirs of 50 mL and 1.5 mL to the reaction chamber. The microfluidic design allows for rapid paratope-epitope interactions, thus short incubation times are possible for immuno-stainings. A graphical user interface installed in a computer was used to control all protocol steps.
Immunofluorescence staining on the MTP device
For direct pan-CK IF, both primary and secondary antibodies (IgGAF647) were incubated for 4 min each; other protocol steps were as in Fig. 1c. Total duration of the staining was 10 min.
Detailed protocol and total staining time for direct ALK IF using the fluorophore-conjugated secondary antibody (IgGAF647) are shown in Fig. 1c.
For TSA mediated ALK indirect IF, the HRP-conjugated secondary antibody was incubated for 8 min, followed by fluorescently labelled tyramide incubation for 2 min. Other protocol steps were as detailed in Fig. 1c.
Negative controls were done by replacing the primary antibody with the antibody diluent PBST 0.05%; all other steps of the negative controls correspond to the same staining conditions of the experiment.
ALK chromogenic IHC staining and scoring
Two μm thick sections of representative tumor blocks were used. The Refine 30/30 HRP protocol was performed on a Leica Bond-Max automated IHC platform as following: mounted tissue cuts were dewaxed in a 60 °C oven, rehydrated and boiled for 30 min for heat induced epitope retrieval (HIER) at pH 8 Tris-buffered EDTA. Primary antibody 5A4 was diluted 1/10 and incubated for 30 min at room temperature (RT). Thereafter, the Refine post-primary antibody linker was applied for 15 min at RT, followed by the Refine polymer coupled to HRP for another 15 min at RT. Diaminobenzidine (DAB) was incubated for 4 min, followed by hematoxylin for 15 min. Chromogenic immunoreactivity was H-scored, whereby whole section tumor areas with semi-quantitative intensities 0, 1, 2 or 3 were multiplied by individual frequencies of stained cells and then summed up (range 0 to 300). As previously described in the European Thoracic Oncology Platform (ETOP) ALK 001 study, a threshold H-score value higher than 120 was thereafter used to define ALK positivity [11, 12].
ALK fluorescent in-situ hybridization
Four μm thick sections were incubated with a dual color break apart FISH probe for chromosome 2p23 (LSI 3’-ALK Spectrum-Orange and 5’-ALK Spectrum-Green, Vysis/Abbott Molecular) according to the manufacturer’s protocol. For each case, 100 non-overlapping nuclei were evaluated using a fluorescence microscope (Zeiss Axioskop) with a 100-fold magnification oil objective. Z-stacks of 20 images with 0.5 μm step distance were merged. Positive ALK FISH was defined as > 15% cells with either break-apart or isolated 3′ signals. Isolated 5′ signals were considered negative.
Image acquisition
The brightfield images of the chromogenic ALK IHC stainings were digitalized on a NanoZoomer Digital Pathology scanner (Hamamatsu, Japan). For IF we used a Zeiss AxioImager M2 m microscope and an Olympus slide scanner VS120-L100. Each image was taken in both DAPI (Alexa350) and Cy5 (Alexa647) channels. For all images within a figure, the same parameters of acquisition, such as filter set, exposure time, and filter intensities were used.
Image analysis
Image quantification was done using custom written ImageJ macros. For quantification of the IF signal, 25 representative regions of interest (ROI) were selected both on tumor epithelia and on stroma areas. For the tumor epithelia ROIs, which contain both ALK positive and negative areas, the pixels containing positive signals were selected in ImageJ by the Huang automatic thresholding before recording the mean fluorescence intensity. For the stroma areas, where the un-specific signal is reported to appear is various structures, the mean fluorescence intensity of whole ROI was recorded, independently of the expression level and cell type to include fluorescent signal of any origin. The average signal and corresponding standard deviation for tumor epithelia and stroma areas were calculated by averaging the mean intensities from all 25 ROIs.
Statistical analysis
MTP-derived IF values were correlated with chromogenic IHC and break-apart FISH using the Kendall’s tau-b test. A p-value of ≤0.05 was considered significant.