A total of 48 patients with recurrent or advanced NSCLC who initiated treatment with nivolumab at Gunma University Hospital (Maebashi, Japan), Hidaka Hospital (Takasaki, Japan), and National Hospital Organization Shibukawa Medical Center (Shibukawa, Japan) between February 2016 and November 2017 were included in this study. The inclusion criteria were i) pathologically confirmed NSCLC, ii) recurrent or advanced stage, iii) eligibility for nivolumab treatment following initial chemotherapy, and iv) Eastern Cooperative Oncology Group performance status of 0–2. Plasma samples and clinical data from patients included in a previous study were used (18). Plasma samples from 11 healthy volunteers were used as the controls.

The follow-up period for censored cases ranged from 1.3 to 36.5 months (median: 12.4 months). miRNA microarray analysis was performed using total RNA extracted from OAA1-enriched plasma samples collected before and 1 month post-treatment from two individuals who developed PD despite receiving nivolumab. Patient consent was obtained using the opt-out method. Healthy volunteers were hospital staff aged ≥20 years who provided written informed consent after receiving an explanation of the study. Individuals with a history of malignant disease, acute infection, or regular use of immunosuppressive medications were excluded. Additional exclusion criteria included a bleeding tendency, severe anemia, coagulation disorders, current use of anticoagulant or antiplatelet drugs, pregnancy or breastfeeding, poor physical condition on the day of blood sampling (e.g., fever or presyncope), or any other condition judged inappropriate by the investigators.

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board for Clinical Research at Gunma University Hospital (Maebashi, Japan; approval no. HS2023-094).

OAA1, which contained two amino acid substitutions of OAA and an additional linker sequence at the C-terminal region for covalent immobilization, and OAA1-immobilized columns on monolithic silica were prepared according to our previous report (19).

Histochemical analysis of lectin was performed on formalin-fixed paraffin-embedded sections of lung cancer tissue to detect tumor-specific high-mannose glycans using OAA1. OAA1 was labeled with biotin using the biotin-labeling kit-NH₂ (final concentration: 1.3 mg/ml) [Dojindo Molecular Technologies, Kumamoto, Japan]. Non-specific binding sites were blocked by incubating the sections with 1% bovine serum albumin (Sigma-Aldrich)/phosphate-buffered saline (PBS) for 1 h at room temperature. The sections were then incubated with biotinylated OAA1 diluted in PBS (×800) for 1 h at room temperature. After washing, the slides were treated with the VECTASTAIN Elite ABC Kit (Vector Laboratories, Inc.) for 30 min. The color was developed using a diaminobenzidine (DAB) substrate solution, and the sections were lightly counterstained with hematoxylin and mounted. Negative controls were prepared by omitting the lectin incubation step.

We obtained 28 sections consisting of resected specimens (n=20) and needle biopsies (n=8) from patients for whom clinical samples were obtained.

For immunohistochemistry, 4-µm sections were cut from the paraffin blocks of each sample. Each section was mounted on a silane-coated glass slide, deparaffinized in xylene, rehydrated through graded ethanol to water, and incubated with 0.3% hydrogen peroxide for 30 min at room temperature to block endogenous peroxidase activity. After rehydration through a graded series of the ethanol treatments (90% for 1 min, 80% for 1 min, and 60% for 1 min), the sections for CD8 staining were heated in boiling water using Immunosaver (Nisshin EM Co., Ltd.) for 45 min at 98–100°C for antigen retrieval. For PD-L1 staining, Universal HIER antigen retrieval reagent (cat. no. ab208572; Abcam) at 120°C for 20 min in an autoclave. Non-specific binding sites were blocked by incubating the sections with Protein Block Serum-Free (Agilent Technologies) for 30 min at room temperature. Samples were incubated overnight at 4°C with the following primary antibodies: PD-L1 (E1L3N Rabbit mAb 1:200; cat. no. 13684; Cell Signaling Technology, Inc., Danvers, MA) and CD8 (cat. no. ab4055; 1:1,000; Abcam). The primary antibody was visualized using the Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei Biosciences, Inc.). Chromogen 3,3-diaminobenzidine tetrahydrochloride was used as a 0.02% solution in 50 mM citric acid-ammonium acetate buffer (pH 6) containing 0.005% hydrogen peroxide. The sections were lightly counterstained with hematoxylin and mounted.

Tissue sections were examined by two independent evaluators who were blinded to the patient data. The expression of PD-L1 was evaluated using a semiquantitative scoring method based on the percentage of stained cells: 1, ≤1; 2, 1–5; 3, 5–10; 4, 10–50; and 5, ≥50%. Tumors with a score >3 were graded as positive. CD8 expression was semi-quantitatively evaluated based on the extent of positive lymphocyte infiltration in the tumor specimens, and patients with >5% positive lymphocytes were defined as positive, based on previous studies (20–22).

To enrich for tumor-specific high-mannose glycan-containing molecules, plasma samples were subjected to lectin affinity capture using an OAA1 column. OAA1 was immobilized on a silica monolith matrix in the column. Briefly, each plasma sample was centrifuged at 10,000 × g for 10 min, and 50 µl of plasma was applied onto the pre-equilibrated OAA1 column. The column was centrifuged at 3,000 × g for 1 min to allow the sample to pass through. After binding, unbound plasma components were removed by washing the column with 400 µl of 10X D-PBS (−) (FUJIFILM Wako Pure Chemical Corporation) and D-PBS (−) (FUJIFILM Wako Pure Chemical Corporation). Glycan-containing molecules specifically bound to OAA1 were then eluted using 200 µl of QIAzol Lysis Reagent (QIAGEN), which was applied to a column and allowed to stand for 5 min at room temperature by centrifugation at 3,000 × g for 1 min.

Total RNA, including miRNA, was extracted from glycan-containing molecules in the plasma samples as follows: 40 µl of chloroform was added to each sample, and the tubes were shaken vigorously for 15 sec. The tubes were incubated at room temperature for 2 min, followed by centrifugation at 12,000 × g for 5 min. The upper aqueous phase was carefully transferred to a new tube, and an equal volume of isopropanol was added. After thorough vortex mixing, the miRNA was purified using the NucleoSpin miRNA Plasma kit (Macherey-Nagel, Germany) with DNA digestion treatment, according to the manufacturer's protocol. Without the OAA1 enrichment group, miRNA was directly extracted from plasma samples using the NucleoSpin miRNA Plasma kit with DNA digestion treatment, according to the manufacturer's protocol. The concentration and purity of the extracted RNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and miRNA samples were stored at −80°C until further analysis. The amount of RNA obtained from the plasma-derived, lectin-captured fraction was extremely low and consisted primarily of short RNAs such as miRNAs; therefore, reliable quantification using NanoDrop and quality assessment based on the RNA integrity number were difficult. In this study, RNA was extracted from 50 µl of plasma from each sample, and RNA quality was indirectly evaluated based on the reproducibility and stability of Ct values in the TaqMan miRNA assay.

For miRNA microarray analysis, to enrich tumor-specific high-mannose glycan-containing molecules, plasma samples were subjected to lectin affinity capture using OAA1 lectin columns, as previously described, with modifications to the sample volume and extraction protocol. Briefly, 1 ml of plasma was divided equally among five OAA1 columns (200 µl plasma per column), each containing OAA1 immobilized on a silica monolith matrix. After centrifugation of each plasma sample at 10,000 × g for 10 min, the plasma was applied to pre-equilibrated columns. The columns were centrifuged at 3,000 × g for 1 min to allow the samples to pass through. After washing, glycan-containing molecules bound to OAA1 were eluted by adding 200 µl of QIAzol Lysis Reagent to each column and incubating at room temperature for 5 min, followed by centrifugation at 3,000 × g for 1 min. The eluates from each column were combined, and total RNA, including miRNA, was extracted as follows: chloroform was added to the QIAzol eluate, and the mixture was vigorously vortexed. After phase separation, the upper aqueous phase was collected, and an equal volume of isopropanol was added. miRNA was purified using the NucleoSpin miRNA Plasma kit with DNA digestion treatment, according to the manufacturer's protocol, for subsequent miRNA microarray analysis.

Microarray analysis was performed using the GeneChip™ miRNA 4.0 Array (Thermo Fisher Scientific). RNA labeling was performed with the FlashTag™ Biotin HSR RNA Labeling Kit (Thermo Fisher Scientific) according to the manufacturer's protocol, with a minor modification: instead of adjusting the RNA input based on concentration, the maximum recommended sample volume (8 µl per sample) was used, due to variability in RNA yield from the upstream lectin-based procedure. Subsequent steps, including hybridization, washing, staining, and scanning, were performed according to the manufacturer's instructions.

miRNA microarray analysis was conducted as an exploratory discovery step. Only miRNAs classified as ‘T’ (true detection) by the microarray analysis software were included, whereas those flagged as ‘F’ (not reliably detected) were excluded from subsequent analyses. Candidate miRNAs were selected based on differential abundance between paired pre-treatment and post-treatment plasma samples from the same patients.

miRNAs exhibiting a fold change >2 and a nominal P-value <0.05 were considered eligible for further validation. To minimize potential sex-related bias, miRNAs encoded on sex chromosomes (X or Y) were excluded from candidate selection.

For miR-320a (assay ID 002277), miR-320b (assay ID 002844), and miR-3613-5p (assay ID 463197_mat; Thermo Fisher Scientific), RT-qPCR was performed, and cDNA was synthesized from total miRNA using the TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific) and specific stem-loop reverse transcription primers (Thermo Fisher Scientific) according to the manufacturer's protocol. The exact primer and probe sequences used in the TaqMan MicroRNA Assays are proprietary and were not disclosed by the manufacturer; therefore, the assay identification numbers are provided to ensure reproducibility. The 15 µl reaction volumes were incubated in 0.2 ml tubes, and the following temperature profile was used: 16°C for 30 min, followed by 42°C for 30 min, and 85°C for 5 min. PCR was performed using a QuantStudio™ 5 System (Thermo Fisher Scientific). The 10 µl PCR mix, including the TaqMan™ Fast Advanced Master Mix for RT-qPCR (Thermo Fisher Scientific), was incubated in a 384-well optical plate at 95°C for 20 s, followed by 40 cycles of 95°C for 1 sec and 60°C for 20 sec. Levels of miR-320a, miR-320b, and miR-3613-5p were normalized to that of miR-16-5p (used as an internal control; assay ID 000391) and analyzed using the 2^−∆Cq method. miR-16-5p was chosen as a suitable reference gene, as described previously (23–25). The fold change in target miRNA expression relative to that in healthy volunteers was determined using the 2^−ΔΔCq method (26,27).

Statistical analyses were performed using the Mann-Whitney U test for continuous variables and χ² test or Fisher's exact test for categorical variables. ROC curve analysis was performed to compare the prognostic utility of OAA1-captured plasma miRNAs with that of plasma miRNAs without OAA1 treatment. Kaplan-Meier curves were generated for overall survival, and statistical significance was examined using the log-rank test. Univariate analyses were performed using logistic regression or the Cox proportional hazards model. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using IBM SPSS Statistics, version 31 (IBM Corp., Armonk, NY) and GraphPad Prism version 10 (GraphPad Software, San Diego, CA). Figures were generated using GraphPad Prism version 10.

TCGA RNA-seq data were analyzed using the UALCAN web portal (http://ualcan.path.uab.edu). For lung adenocarcinoma and lung squamous cell carcinoma cohorts, mRNA expression levels (transcripts per million) of N-glycan biosynthesis and processing enzymes, including ALG3 α-1,3-mannosyltransferase (ALG3), mannosidase α class 1A member 1 (MAN1A1), mannosidase α class 1C member 1 (MAN1C1), α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1) and β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT3), were retrieved and compared between primary tumor and normal tissues using TCGA module of UALCAN. Statistical significance of expression differences was assessed using an unpaired two sample t-test implemented in UALCAN.

Fig. 1A shows the histochemical results for OAA1 in surgically resected lung cancer specimens. This analysis revealed that high-mannose glycan structures bound by OAA1 were more abundantly expressed in lung cancer cells than in adjacent non-tumorous tissues (Fig. 1A). Similarly, TCGA RNA-seq data analysis via UALCAN showed a higher expression of ALG3, with an early addition of mannose during N-glycan assembly in the endoplasmic reticulum, and lower expression of mannosidase MAN1A1 and MAN1C1, resulting in trimmed mannose residues in the Golgi to prepare glycans for further processing, as well as lower expression of MGAT1, which initiates complex N-glycan branching with the addition of GlcNAc, and MGAT3, resulting in a ‘bisecting’ GlcNAc that modulates later branching (28–30). These expression patterns were consistent with the attenuated trimming and maturation of N-glycans in tumors (Figs. S1 and S2).

Based on this finding, we used OAA1 to selectively capture glycan-containing molecules associated with tumors from the plasma of patients with lung cancer and conducted miRNA microarray analysis to identify circulating miRNAs that were elevated during treatment in patients who exhibited tumor progression to PD despite nivolumab therapy (Fig. 1B). As shown in Fig. 1C, several candidate miRNAs were identified. Among them, hsa-miR-320a, hsa-miR-320b, and hsa-miR-3613-5p were significantly upregulated in OAA1-captured plasma samples after treatment compared with their pre-treatment levels. Furthermore, we quantified these OAA1-captured miRNAs in pre-treatment plasma samples from patients with advanced or recurrent NSCLC (n=48) and healthy volunteers (n=11). As shown in Fig. 1D, the levels of these miRNAs were significantly higher in patients with NSCLC than in healthy controls.

Next, we measured the levels of miR-320a, miR-320b, and miR-3613-5p in plasma samples collected before nivolumab treatment from patients who exhibited PR (n=10) and SD + PD (n=38). This analysis was conducted under two conditions: With and without OAA1 enrichment. Without OAA1 enrichment, there was no significant difference in the levels of miR-320a between the PR and SD + PD groups. However, both miR-320b and miR-3613-5p were expressed at significantly higher levels in the SD + PD group than in the PR group (Fig. 2 A). In contrast, with OAA1 enrichment, the levels of all three miRNAs (miR-320a, miR-320b, and miR-3613-5p) were significantly higher in the SD + PD group than in the PR group (Fig. 2 B), suggesting that OAA1 enrichment enhances the sensitivity of circulating miRNA-based detection for predicting the response to nivolumab.

Univariate logistic regression analysis was performed to evaluate the association between circulating miRNA levels and response to nivolumab (SD or PD) in patients with NSCLC (Table I). Without OAA1 enrichment, the odds ratios for predicting SD/PD were 3.208 for miR-320a, 12.444 for miR-320b, and 19.5 for miR-3613-5p. In contrast, with OAA1 enrichment, the odds ratios increased to 12.889 for miR-320a, 27.222 for miR-320b, and 39.857 for miR-3613-5p, representing a substantial improvement compared with the values obtained without OAA1 enrichment (Table I). These results suggest that OAA1 enrichment improves the predictive utility of circulating miRNAs for identifying patients with poor response to nivolumab. This finding is consistent with an earlier comparison between the PR and SD + PD groups and supports the potential of OAA1-captured plasma miRNAs as predictive biomarkers in NSCLC.

To evaluate the prognostic value of circulating miR-320a, miR-320b, and miR-3613-5p in patients who responded to nivolumab, receiver operating characteristic (ROC) curve analyses were performed with and without OAA1 enrichment. Without OAA1 enrichment, the area under the curve (AUC) values for miR-320a, miR-320b, and miR-3613-5p were 0.671 [95% confidence interval (CI): 0.502–0.840], 0.779 (95% CI: 0.632–0.926) and 0.837 (95% CI: 0.722–0.952), respectively (Fig. 3A). In contrast, with OAA1 enrichment, the AUCs increased to 0.847 (95% CI: 0.723–0.967) for miR-320a, 0.853 (0.718–0.988) for miR-320b, and 0.897 (95% CI: 0.807–0.987) for miR-3613-5p, showing improved performance compared with the corresponding values obtained without OAA1 enrichment (Fig. 3B). These results suggest that OAA1 enrichment improves the prognostic performance of these circulating miRNAs for overall survival in patients with NSCLC treated with nivolumab.

The optimal cutoff values (2^−ΔCq) were determined using the ROC analysis. Without OAA1 enrichment, the cutoff values were 0.2335 for miR-320a, 0.01662 for miR-320b, and 6.947×10⁻⁴ for miR-3613-5p, whereas with OAA1 enrichment, the corresponding values were 0.2610, 0.01164, and 1.740×10⁻³, respectively.

Circulating miRNA profiles with and without OAA1 enrichment were categorized into high- and low-expression groups based on the cutoff values determined by the ROC curves for patients who responded to nivolumab, as shown in Fig. 3. Based on each ROC-based threshold, 48 patients with NSCLC were stratified into high or low miRNA expression groups. Table SI summarizes the association between the levels of the target miRNAs and clinicopathological features without OAA1 enrichment. In the group without OAA1, high levels of miR-320b and miR-3613-5p were significantly associated with low sensitivity to nivolumab (SD and PD); however, no other clinical features were significantly correlated (Table SI). In contrast, with OAA1 enrichment, a high expression of all three target miRNAs was significantly associated with SD and PD (Table SII).

Kaplan-Meier survival analysis with log-rank testing was performed to assess the prognostic relevance of circulating miR-320a, miR-320b, and miR-3613-5p in patients with NSCLC treated with nivolumab with and without OAA1 enrichment. High plasma levels of these miRNAs were significantly associated with poor overall survival, regardless of the OAA1 enrichment status (Fig. 4).

Cox regression analysis was used to evaluate the prognostic relevance of circulating miRNA levels for overall survival in patients with NSCLC based on univariate modeling (Table II). Without OAA1 enrichment, the hazard ratios (HRs) for predicting poor survival were 2.136 for miR-320a, 3.578 for miR-320b, and 3.386 for miR-3613-5p. In contrast, with OAA1 enrichment, the corresponding HRs increased to 2.389 for miR-320a, 7.922 for miR-320b, and 7.815 for miR-3613-5p (Table II). These results indicate that OAA1 enrichment enhances the prognostic utility of circulating miRNAs and improves their ability to stratify patients according to overall survival following nivolumab treatment. Multivariable Cox regression analysis, including CD8 expression and the miRNAs identified as significant in the univariate analysis, further supported these findings. In the analysis without OAA1 enrichment, CD8 expression (P=0.008, HR=0.149), miR-320b (P=0.024, HR=5.505) and miR-3613-5p (P=0.048, HR=3.152) were identified as independent prognostic factors. In contrast, in the analysis with OAA1 enrichment, only miR-3613-5p remained an independent prognostic factor (P=0.010, HR=7.462) (Tables SIII and SIV). Notably, the HR of miR-3613-5p was higher with OAA1 enrichment than without enrichment.