Study population and design
Data were retrospectively collected from all consecutive eligible patients with advanced NSCLC who were treated with ICBs between January 2014 and May 2018, at five St. Mary’s Hospitals in Seoul, Suwon, Uijeongbu, Bucheon, and Yeouido, Korea. This study was approved by the Institutional Review Board of Catholic Medical Center [KC18SESI0440]. ICBs including nivolumab, pembrolizumab, avelumab, atezolizumab, or durvalumab, and were prescribed under coverage by health insurance or an early access program. We excluded patients who were lost to follow-up while showing a favorable response to ICBs or who did not have information available regarding the previous treatment.
Clinical data included age at diagnosis, sex, primary tumor location, TNM stage at diagnosis, number of prior systemic treatments, best tumor response during immunotherapy, baseline and post-immunotherapy imaging, patterns of recurrence, and location of distant metastases. Patients were divided into four groups: HPD, non-HPD progressive disease (non-HPD PD), stable disease (SD), and partial/complete response (PR/CR) displaying different tumor response to immunotherapy. We recorded time-series laboratory data including serum C-reactive protein, erythrocyte sedimentation rate, albumin, lactate dehydrogenase (LDH), and white blood cell count immediately before starting treatment, at the beginning of immunotherapy, and at the first tumor response assessment, i.e., 6-8 weeks after initiation of immunotherapy. The neutrophil-to-lymphocyte ratio (NLR) was defined as the absolute neutrophil count divided by absolute lymphocyte count, and the platelet-to-lymphocyte ratio (PLR) was defined as thrombocyte count divided by the lymphocyte counts. The C-reactive protein-to-albumin ratio (CAR) was calculated by dividing the C-reactive proteins level by the albumin level.
Tumor growth kinetics
Radiological changes were evaluated based on the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST ver. 1.1)  and irRECIST . We defined HPD as having (1) a tumor growth kinetics ratio (TGKr) exceeding the tumor growth rate by two-fold between the reference period (before immunotherapy) and the experimental periods during anti–PD-1/PD-L1 therapy and (2) a time-to-treatment failure (TTF) less than 2 months [7, 13, 25, 26]. We reviewed all pre- and post-immunotherapy images and determined the two points for determining tumor growth kinetics (i.e., before starting immunotherapy [TGKPRE] and after immunotherapy [TGKPOST]) [12, 13, 21]. TPRE, T0, and TPOST denote the time of the reference period’s baseline, experimental period’s baseline, and the experimental period’s first-post imaging, respectively. SPRE, S0, and SPOST denote the sum of the largest diameter of target lesions at the reference period’s baseline, experimental period’s baseline, and first follow-up image of the experimental periods, respectively. TGKPRE was defined as the difference in the sum of the largest diameters of the target lesions per unit of time between the reference period and experimental baseline imaging: (S0SPRE)/(T0TPRE). Similarly, TGKPOST was defined as (S0SPOST)/(T0TPOST). TGKr was defined as the ratio of TGKPOST to TGKPRE. TGKr >1 indicated tumor growth acceleration, whereas 0 < TGKr ≤ 1 and TGKr ≤0 indicated tumor deceleration and tumor shrinkage, respectively [13, 25-27].
Assessment of PD-L1 expression level using immunohistochemistry
We used archival tumor tissues obtained by core needle biopsy or excisional biopsy at the initial diagnosis. PD-L1 expression is widely used as a key predictive biomarker for PD-1/PD-L1 blockade and has been approved as a companion diagnostic test for pembrolizumab (Kytruda®; Merck, Kenilworth, NJ, USA). PD-L1 expression was assessed using immunohistochemistry (IHC) in formalin-fixed paraffin-embedded tumor tissue using the PD-L1 IHC 22C3 pharmDx assay (Dako, Santa Clara, CA, USA) at a hospital pathology laboratory. These data were determined by means of a Combined Positive Score, which includes the number of PD-L1 positive cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100.
Analysis of immune cell composition using multiplex IHC
To examine the TME, we used a quantitative multispectral imaging method using the Opal Multiplex IHC kit (Perkin-Elmer, Waltham, MA, USA) and Vectra automated quantitative pathology imaging system (Perkin-Elmer). Multiplex IHC staining for immune cells and antagonists of the PD-1/PD-L1 pathway was performed using a Leica Bond Rx™ Automated Stainer (Leica Biosystems, Newcastle, UK). We analyzed scanned images using inForm image analysis software (Perkin-Elmer) and TIBCO Spotfire software (TIBCO, Palo Alto, CA, USA).
We analyzed differences in the immune composition of the TME using multiplex IHC. T cell markers, including CD4, CD8, FOXP3, CD45RO, and CD3 were placed on panel 1, and co-inhibitory signal markers including TIM3, LAG3, PD-1, and PD-L1 were placed on panel 2. We also examined the degree of penetration of CD14, CD68, CD163, and CD206 as macrophage markers on panel 3 as well as CD11c as a myeloid-derived cell marker, CD16, CD56, CD86, and CD103 as NK cell and dendritic cell markers on panel 4.
The correlation of HPD with the clinicopathologic factors was analyzed using Pearson's chi-squared test and Student’s t-test. The quantified multiplex IHC data were analyzed using the Mann Whitney U test and compared using the Kruskal Wallis test. Overall survival and progression-free survival were calculated from the start of ICB administration until the date of death and disease progression, respectively. For survival analysis, living patients or those with no disease progression were censored from the last follow-up date. Multivariate analyses were identified by cox regression and overall survival and progression-free survival were performed using logistic regression and the log-rank test. All statistical analyses were performed using SPSS software (version 21; IBM Corp., Armonk, NY, USA) and R-studio package (version 1.8; https://rstudio.com/ Boston, MA, USA). A two-sided P value of <0.05 was considered statistically significant. Spider plots, scatterplots, and Kaplan-Meier survival curve were generated using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA).