*2.1. Patients*

We retrospectively selected 52 advanced NSCLC patients who were administered PD-1 inhibitor from 2016 to 2019, and they previously received one or two lines of chemotherapy. This study was approved by the Institutional Review Board of Ajou University School of Medicine. Informed consent was waived due to the retrospective nature of the study (AJIRB-BMR-KSP-19-050 and 2019-03-26).

Patients treated with PD-1 inhibitor were assigned to either a responder group (complete response, partial response, or stable disease) or a nonresponder group (disease progression), according to the response evaluation criteria for solid tumors (RECIST) version 1.1 [13].

### *2.2. Immunohistochemical Staining and HIP1R Expression Scoring*

One board-certified pathologist (YWK) reviewed hematoxylin and eosin (H&E)-stained tissue samples to determine a definitive pathologic diagnosis according to the 2015 World Health Organization Classification of Lung Tumors [14]. All patients were pathologically staged according to the eighth edition of the TNM classification.

HIP1R immunohistochemical (IHC) staining was performed with a Benchmark XT automatic IHC staining device (Ventana Medical Systems, Tucson, AZ, USA). The samples were incubated with an anti-HIP1R antibody (dilution 1:1000, 16814-1-AP, polyclonal, Proteintech, Rosemont, IL, USA). We used a human placenta tissue as positive control according to the manufacturer's recommendations (Figure S1). We also evaluated the intensity of HIP1R staining on a four-point intensity scale: 0 (no staining), 1 (light yellow = faint staining), 2 (yellow-brown = moderate staining), and 3 (brown = strong staining) (Figure 1). We also evaluated the percentages (0–100%) of cytoplasmic versus membranous localization of HIP1R. We used H-scores to interpret HIP1R staining [15], where H-score = [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]. H-scores (0–300) were obtained by multiplying the percentage of cells by the intensity score.

**Figure 1.** Huntingtin-interacting protein 1-related protein (HIP1R) expression in nonsmall cell carcinoma. (**A**) No staining of HIP1R, x400. (**B**) Faint HIP1R staining, X400. (**C**) Moderate HIP1R staining, X400. (**D**) Strong HIP1R staining, X400.

### *2.3. Immunohistochemical Staining and PD-L1 Expression Scoring*

Two PD-L1 antibodies (clone name SP263 or 22C3) were used to detect PD-L1 expression. Sp263 was a companion diagnostic assay for OPDIVO® (nivolumab), and 22c3 was a companion diagnostic assay for KEYTRUDA® (pembrolizumab). We performed SP263 and/or 22C3 assays prior to PD-1 inhibitor treatment for all NSCLC patients. Thirteen (25%) of the 52 specimens were tested for both SP263 and 22C3, 27 (51.9%) for only SP263, and 12 (23.1%) for only 22C3. Two PD-L1 tests used prediluted antibody (ready to use) according to the protocol. The SP263 assay was performed using a VENTANA BenchMark ULTRA instrument (Roche, Basel, Switzerland), and the 22C3 assay was conducted using the Dako Link-48 platform (Dako, Carpinteria, California, US), as recommended by the manufacturers [16]. PD-L1 intensity was also evaluated on a four-point intensity scale (0, none; 1, faint; 2, moderate; and 3, strong), and the percentage of membranous expression of PD-L1 was determined (Figures S2 and S3). When both the 22C3 and SP263 tests were conducted, mean values were used. High PD-L1 expression was defined as ≥ 50% of definitive tumor cells exhibiting PD-L1 staining, because 50% was the cut-off used for NSCLC [17].
