Nsolver version 4

The raw expression data from the Digital Analyzer were normalized using nSolver version 4.0 (NanoString Technologies, Seattle USA). Briefly, the expression data were background corrected by using the geometric mean of the negative controls. The data then were normalised with the geometric mean of the five housekeeping genes. Median of expression values of examined genes was set as threshold. Expression values below median were defined as “low expression” and values above median as “high expression”.
Categorical data were compared using Chi-square or Fisher’s exact tests. Asymmetrical numeric data (IMPC vs. IBC-NST) were analyzed by Mann–Whitney test. Kaplan–Meier analysis was performed using DMFS as the endpoint. DMFS intervals were determined as the time period from initial diagnosis to the time of diagnosing distant organ metastasis. The comparison of survival functions for different strata was assessed with the log-rank statistics. Multivariate analysis of prognostic factors was performed using Cox's regression model.
Statistical significance was confirmed when p-values were < 0.05. Statistical analysis was performed using Statistica 13.5 software (TIBCO Software Inc, Palo Alto, CA).
To compare our results of the prognostic impact of selected genes (based on DMFS) with a large database the KM Plotter Online Tool, a publicly available database was selected^{40 (link),41} .

The gene expression analysis was performed at PhileKorea Technology (Daejeon, Korea) on a NanoString nCounter gene expression platform by Auto Immune profiling panel and Host Response advanced panel (NanoString Technologies, WA, USA). A custom code set consisting of a 773 gene panel related to infectious diseases, and 56 pathway in Host response panel, and a 750 gene panel related to autoimmune and autoinflammatory diseases in Auto Immune profiling panel was used in this study. nSolver (version 4.0, NanoString Technologies) was used for differential gene expression analysis of nCounter analysis data. All reporter code count files passed the default QC settings, and by default, all raw gene expression data was normalized in the Positive Control and Housekeeping normalization steps. Fold changes were calculated by averaging normalized lanes from controls.

Human PBMCs were isolated from heparinized venous blood using Lymphoprep (1114545; Axis-shield) in accordance with the manufacturer’s instructions. Total RNA from PBMCs was extracted using TRIzol reagent (15596026; Invitrogen) in accordance with the manufacturer’s instructions, followed by RNA quantification and assessment using QIAxpert (Qiagen). Nanostring nCounter Human Immunology gene expression assays for PBMCs were performed at PhileKorea Technology (Daejeon, South Korea), and the data were analyzed by nSolver version 4.0 (NanoString Technologies) as described previously.^{32 (link)}

Nanostring nCounter Human Immunology gene expression assays and Human miRNA expression assays were performed at PhileKorea Technology (Daejeon, South Korea) using the NanoString nCounter GX Human Immunology Kit V2 (NanoString Technologies, Inc., Seattle, WA, USA) and nCounter Human miRNA Assay Kit V3 (NanoString Technologies), respectively. nSolver, version 4.0 (NanoString Technologies), was utilized for differential gene expression analyses from nCounter assay data. Based on the nSolver 4.0 Analysis Software User Manual, all reporter code count files from nCounter instruments passed the default QC settings. All raw gene expression data were normalized in positive control normalization and housekeeping normalization steps by default. Fold changes were computed by taking the mean of the normalized lanes from healthy controls, and false discovery rate (FDR)-adjusted p-values were calculated using the Benjamini-Yekutieli procedure.

LDH serum level and NLR were assessed at baseline; NLR was recorded after 3 months of treatment with anti-PD-1 ICI. Blood samples from enrolled patients were collected at baseline to conduct a gene profile analysis. RNA from PBMCs was extracted using RNA blood mini-Kit (Qiagen). Purified RNA was used for hybridization and underwent to gene profiling analysis on NanoString nCounter through PanCancer IO 360™ panel, characterized by human genes associated with immune activation, inflammation and control of the cell cycle. Gene data were normalized using nSolver Version 4.0 Software; NanoString. Counts were normalized to External RNA Controls Consortium (ERCC) technical controls and 30 housekeeping genes.

RNA from FFPE tumor tissues was extracted using RNeasy FFPE Kit (Qiagen). Purified RNA (100 ng) was used for hybridization and subjected to gene-profiling analysis on NanoString nCounter Sprint Profiler™ through PanCancer IO 360 panel (Nanostring Technologies), characterized by 20 internal references and 770 human genes involved in the interplay between tumor microenvironment and immune response.
Gene data were normalized and analyzed using nSolver Version 4.0 Software; volcano plots were visualized using nSolver Advanced Analysis Version 2.0.115 and R Version 3.3.2 Software (Nanostring Technologies). Due to limited sample sizes, statistical correction for multiple comparisons resulted in a null result for all comparisons. Reported p-values are uncorrected values.