Hyperprogressive Disease During PD-1/PD-L1 Blockade in Patients With Non-Small-Cell Lung Cancer

C. G. Kim; K. H. Kim; K.-H. Pyo; C.-F. Xin; M. H. Hong; B.-C. Ahn; Y. Kim; S. J. Choi; H. I. Yoon; J. G. Lee; C. Y. Lee; S. Y. Park; S.-H. Park; B. C. Cho; H. S. Shim; E.-C. Shin; H. R. Kim


Ann Oncol. 2019;30(7):1104-1113. 

In This Article


Prognostic Score Systems

Royal Marsden Hospital (RMH) score [1], Gustave Roussy Immune Score (GRIM) score [2], and lung immune prognostic index (LIPI) score [3] were calculated between each patients as previously suggested. RMH score was composed of lactate dehydrogenase (LDH) level, albumin level, and sites of metastasis. GRIM score was composed of LDH level, albumin level, and neutrophil-to-lymphocyte ratio. LIPI score was composed of LDH level and neutrophil-to-lymphocyte ratio.

Exploratory Biomarker Analysis With Peripheral Blood

Peripheral blood mononuclear cells were isolated in 144 patients as previously described [4]. To characterize CD8+ T lymphocytes, single-cell suspensions were incubated with LIVE/DEAD Fixable Red Dead Cell Stain kit (Thermo Fisher Scientific) and then stained with fluorochrome-conjugated antibodies for 15 min at room temperature. For intracellular staining, cells were permeabilized and fixed with the FoxP3 staining buffer kit (Thermo Fisher Scientific) and further stained with fluorochrome-conjugated antibodies. All flow cytometry analysis was conducted using an LSR II system (BD Biosciences) and FlowJo software version 10.4.0 (Tree Star Inc.) according to the predefined gating strategies (supplementary Figure S16). The reagents used for flow cytometry are described in supplementary Table S5. There were no differences in baseline characteristics between the blood-based biomarker subcohort and the cohort of entire subjects (supplementary Table S6).

Supplementary Figure S16.

Gating strategies for flow cytometry analysis.

Cell Sorting, Assessment of Activation-induced Cell Death and Functionality Upon Stimulation

Peripheral blood CD8+ T cells from NSCLC patients were negatively isolated using magnetic bead separation kit (Miltenyi Biotec). Isolated CD8+ T cells were stained with 7-aminoactinomycin D (BD Biosciences), anti-CD8, anti-CCR7, anti-CD45RA, anti-PD-1, and anti-TIGIT antibodies. They were further sorted using FACSAria II cell sorter (BD Biosciences). Then cells were stimulated with plate-bound anti-CD3 microbead (Miltenyie Biotec) in the presence of anti-PD-1 blocking antibody. Twenty-four hours after stimulation, cells were harvested and proportion of apoptotic cells were evaluated by PE Annexin V Apoptosis Detection Kit I (BD Biosciences) according to manufacturer's instructions. Culture supernatants were collected and concentration of soluble factors were measured by LEGNEDplex™ bead-based immunoassays according to manufacturer's instructions (BioLegend).

Analysis of CD8+ T Cell Infiltration to Tumor and Major Histocompatibility Complex Class I Expression With Formalin-fixed, Paraffin-embedded Tumor Tissue

Sections from tumor tissue were stained using the automated staining platform (Ventana Benchmark XT automated staining system, Ventana Medical Systems) according to the manufacturer's instructions. Sections from tissue blocks (4 μm) were deparaffinized and rehydrated with xylene and alcohol solutions. Cell conditioning solution (CC1; Ventana Medical Systems) was used for antigen retrieval. Then, the tissue sections were subsequently incubated with primary antibody for CD8. After a chromogenic visualization step using the ultraView Universal DAB Detection Kit (Ventana Medical Systems), slides were counterstained with hematoxylin and cover-slipped. CD8+ T cell infiltration were semiquantitatively analyzed on a scale of 0 to 3 based on the extent of positive lymphocytes infiltrating within tumor cells. Each score was defined on the basis of the fraction of CD8+ T lymphocytes in tumor bed: score 0, non or rare; score 1, <5%; score 2, ≥5% and < 25%; and score 3, ≥25% based on the previous report [5]. For statistical comparison, the scores were dichotomized in to negative (score 0–1) positive (score 2–3). Expression of major histocompatibility complex class I was evaluated based on the previous report [6].


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