The study consisted of formalin-fixed paraffin-embedded (FFPE) metastatic melanoma samples from 17 patients. All of the patients were diagnosed with stage IV metastatic melanoma and went on to receive first line immunotherapy with anti-CTLA-4 monotherapy. Patient information is summarized in Table 1. Experimental methods used in this study were approved and performed in accordance with the University of Arkansas for Medical Sciences (UAMS) Institution Review Board policies and procedures (study #204543). All samples used in the study were from deidentified archived human tissue biopsies with a waiver for informed consent.
FFPE Tissue Processing.
For each tumor, we used one 10 µm FFPE slide for proteomic studies. The tumor region on each slide was demarcated by a dermatopathologist. The tissue was deparaffinized by heating the slides to 65°C for 4.5 minutes, then placed in xylene for 1 minute, 100% ethanol for 30 seconds, 95% ethanol for 15 seconds, and rinsed in deionized water 3 times. The tissue was collected from the demarcated region and placed in lysis buffer (100 mM Tris, 2% SDS, pH 7.6). Tumor area was measured prior to deparaffinization using ImageJ and used to normalize volume of lysis buffer. The samples were heated for 30 minutes at 95°C followed by water bath sonication for 5 minutes on high with a Diagenode Bioruptor. After sonication sample were incubated overnight at 65°C.
Filter Aided Sample Preparation (FASP) and Tandem Mass Tag (TMT)-labeling.
Each sample was prepared for digestion using 100 µL of the patient FFPE tissue solution. Protein was reduced using TCEP (tris(2-carboxyethyl)phosphine hydrochloride) at a final concentration of 10 mM for 30 minutes at 37°C. Samples were diluted in 11 times the volume of 8 M urea in 100 mM Tris, pH 8.5 (UA buffer) and protein collected by passage through a FASP filter (30,000 MWCO, Sartorius Vivacon 500 DNA Concentrator VN01H22). Protein immobilized on the filters was treated with 100 µL of 50 mM iodoacetamide and incubated in the dark at room temperature for 20 minutes. Following the incubation, the filters were washed with UA buffer and 50 mM TEAB (tetraethylammonium bicarbonate). Trypsin (1 µg) was added to each filter and incubated at 37°C overnight. The following day, peptides were collected by centrifugation, and formic acid was added to a final concentration of 0.1%. The samples were desalted using a Sep-Pak (Waters) according to the manufacturer’s directions and then lyophilized. Lyophilized peptide samples were re-suspended in 28 µL of 100 mM TEAB. A pooled reference sample was created by mixing 3 µL of each sample. The TMT labels (TMT 10plex Isobaric Label Reagent Set, 0.8 mg, Thermo Scientific) were prepared, and subsequent steps were completed according the manufacturer’s directions. Labeled samples and the pooled reference were combined into 3 respective batches (Table 1).
Off-Line Fractionation and In-Line Liquid Chromatography Mass Spectrometry (LC-MS).
Each of the TMT-labeled batches were fractionated off-line with basic pH reverse-phase chromatography into 36 fractions using a 100 x 1.0 C18 column (Acquity BEH, Waters) and UHPLC system (UltiMate 3000, Thermo) with a 40-minute gradient from 99:1 to 60:40 buffer A:B ratio (buffer A: 0.1% formic acid, 0.5% acetonitrile; buffer B: 0.1% formic acid, 99.9% acetonitrile). The fractions of each batch were then combined into 12 super fractions using a concatenation scheme (1 + 13 + 25, 2 + 14 + 26, etc.) (18). The 12 super fractions of each batch were then resolved in-line using reverse phase resin (Jupiter Proteo resin, Phenomenex) in a 200 x 0.075 mm column using a UPLC system (nanoAcquity, Waters) coupled to a Thermo Orbitrap Fusion Tribrid mass spectrometer. Peptides were eluted with a 60-minute gradient from 97:3 to 67:33 buffer A:B ratio and ionized by electrospray (2.15kV) followed by mass spectrometric analysis using multi-notch MS3 parameters(19). MS data were acquired using the Fourier Transformation Mass Spectrometry (FTMS) analysis in top-speed profile mode with a resolution of 240,000 and a range of 375 to 1500 m/z. Collision-induced dissociation (CID) activation was performed with a normalized collision energy of 35.0. MS/MS data were then acquired using the ion trap analyzer in centroid mode with a range of 400-2000 m/z. Up to 10 MS/MS precursors were selected for HCD activation with a normalized collision energy of 65.0 using synchronous precursor selection. MS3 reporter ion data was acquired using the FTMS analysis in profile mode at a resolution of 60,000 and a range of 100-500 m/z.
Mass Spectrometry. Proteins were identified and MS3 reporter ions were quantified using MaxQuant (Version 188.8.131.52, Max Planck Institute) with a parent ion tolerance of 3 ppm, a fragment ion tolerance of 0.5 Da, and a reporter ion tolerance of 0.001 Da, fixed modifications of carbamidomethyl on cysteine, variable modifications of oxidation on methionine and N-terminal acetylation, and 3 missed cleavages possible with trypsin. The MS3 reporter ion intensities were normalized using the Cyclic Loess normalization function from the limma Bioconductor package (20).
Random Forest. A random forest was generated using the 4,776 proteins identified across all samples (21). The model was built using Salford Random Forest Predictive Modeler. The parameters for the random forest model were tuned to optimize model fit and specified as Target Type: Classification/Logistic Binary, Number of trees to build: 100000, N predictors: Exactly 500, Seed: 17395, Testing: Out of bag data used for testing (22). The top 483 (Score ≥ 0.04) most important features were used for further analysis.
Reactome Pathway Analysis. Reactome pathway analysis was used to identify pathways associated with the top 483 most important proteins from the random forest (23). The 281 proteins elevated in samples from complete responders were analyzed separately from the 202 proteins decreased. ReacFoams were generated using Reactome pathway analysis and are based on Voronoi tessellation.
Differential Protein Abundance. Significance was calculated on the top 483 most important protein features identified from the random forest model using the moderated t-test. Proteins were considered differentially abundant with a q-value < 0.05 and a fold change ≥ 2. Cluster heatmaps were constructed using hierarchical clustering with a variance minimization (Ward) algorithm to calculate the distances between points. Classification of patients into high, moderate, and low groups was based on innate immune protein abundance. For each innate immune protein, the median abundance was calculated on the normalized intensity. High abundance proteins were proteins with a normalized intensity above the median value. The number of high abundance proteins were summed for each patient and the patients were classified based on the percentage of high abundance proteins (PHAP) into the high (PHAP > 66.7%), moderate (PHAP = 33.3% - 66.7%), or low (PHAP < 33.3%) categories.
Slides were cut from FFPE patient tissue blocks to a thickness of 4 µm and processed for IHC. Antigen retrieval was perform using citrate buffer, pH 6.0 (Dako Target Retrieval) with incubation in a decloaking chamber (Biocare) for 20 minutes followed by a 30-minute cool down and rinse in deionized water. Peroxidase block (Dako Peroxidase Block) was applied for 10 minutes and rinsed with TBST (tris-buffered saline with tween 20, Dako). Non-specific binding was blocked by applying protein block (Dako Protein Block) for 10 minutes and rinsed with TBST. Primary antibody against AZU1 (Prestige Antibodies: HPA055851) was applied at a 1:100 dilution (diluted in Dako Antibody Diluent with background reducing components) and incubated at room temperature for 1 hour and rinsed with TBST. Secondary antibody, biotinylated goat anti-rabbit (Vector Laboratories: BA-1000) was applied at a 1:400 dilution (diluted in TBST) and incubated at room temperature for 30 minutes and rinsed with TBST. Immunodetection reagent (Vector Elite ABC Kit, Vector Laboratories) was applied and incubated at room temperature for 30 minutes and rinsed with TBST. DAB chromogen (Dako DAB+) was applied and incubated for 3 minutes and rinsed in running tap water. Slides were counterstained with hematoxylin 2 (Richard-Allan Scientific) for 1 minute and rinsed in running tap water. Slides were mounted with permanent mounting media. IHC results were analyzed by a dermatopathologist, blinded to response status, based on the presence or absence of staining in four regions: within tumor, at the tumor-stroma interface, within areas of tumor necrosis, and at the tumor-necrosis interface. A Fisher’s exact test was used to test for significance between patients with complete response and those with disease progression for each region.