Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Expression of PD-1 and Its Ligands, PD-L1 and PD-L2, in Smokers and Never Smokers with KRAS-Mutant Lung Cancer.

Author(s): Calles A, Liao X, Sholl LM, Rodig SJ, Freeman GJ, Butaney M, Lydon C, Dahlberg SE, Hodi FS, Oxnard GR, Jackman DM, Jänne PA

Publication: J Thorac Oncol, 2015, Vol. 10, Page 1726-35

PubMed ID: 26473645 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to determine if immunohistochemical staining of Programmed Cell Death Protein-1 (PD-1) and its ligands (Programmed Cell Death Ligand-1, PD-L1; Programmed Cell Death Ligand-2, PD-L2) in non-small cell lung cancer (NSCLC) patients with a KRAS mutation are affected by patient age, gender, or race; smoking history; tumor sampling method (core needle biopsy, CNB; transbronchial biopsy; fine needle aspiration, FNA); and the storage duration of formalin-fixed, paraffin-embedded (FFPE) blocks.  In total, 514 patients diagnosed with KRAS-mutant NSCLC that had previously undergone systematic genotyping were asked to complete a questionnaire that classified participants as a current smoker, a former smoker (ceased smoking 1 year prior), or a never smoker (smoked <100 cigarettes in their lifetime) and quantified the intensity of smoking by calculating “pack-years” [(mean number of cigarettes smoked per day/20) × years smoking].  Protein expression of PD-1, PD-L1, and PD-L2 were assayed by immunohistochemistry in tumor samples (85 surgical resections, 18 core needle or transbronchial biopsies, 11 FNA) collected from 114 KRAS-mutant patients (84 current/former smokers, 30 never smokers) using 4 µm-thick FFPE sections that underwent antigen retrieval by incubation in a steamer for 30 min in either citrate buffer, pH 6 (PD-1, PD-L2) or EDTA buffer, pH 8 (PD-L1) before incubation with a primary antibody, an horse radish peroxidase-conjugated secondary antibody, and diaminobenzidine labeling with a hematoxylin counterstain. Details regarding cold ischemia time; fixation and tissue processing conditions, duration, and temperatures; and FFPE block storage conditions were not provided. Staining was defined as positive for PD-L1 if ≥5% tumor cells displayed membrane staining, for PD-L2 if ≥10% of tumor cells were stained; staining intensity was also scored (0=negative, 1+ = weak, 2+ = moderate, 3+ = intense).  PD-1 staining was quantified as the number of stained tumor infiltrating lymphocytes (TILs) in 5 representative areas at a magnification of 20×.  Evaluation of all immunostaining was performed by a pathologist that was blinded to the associated clinical data. Of the 114 tumors that underwent IHC, 82% of patients had the biopsy at the time of diagnosis and had not received prior treatment.

   Summary of Findings:

   KRAS-mutant tumors from NSCLC patients that identified as smokers displayed a significantly higher incidence of positive PD-L1 expression than tumors from nonsmokers (44% current smokers, 20% of former smokers, 13% of never smokers; p=0.03) and a greater incidence of high intensity PD-L1 immunostaining than tumors from nonsmokers (3+; p=0.03). However, the median percentage of PD-L1-positive tumor cells did not differ between smokers (current and former) and never smokers (70 versus 55%).  PD-L1 staining intensity was also influenced by the smoking history of the patient, as patients with a PD-L1 staining intensity of 2+ and 3+ had a significantly higher number of pack-years (34 cigarette packs/year) than patients with weaker staining (0-1+: 20 cigarette packs/year) (p=0.015).  The percentage of patients with a PD-L1 positive/negative status did not differ based on patient age, race, histology, type of KRAS mutation (transversion or a transition), and method of tumor sampling.  When specimens were analyzed within 3 years of collection, there was a higher percentage of PD-L1 positive cases than when FFPE block storage was for longer than 3 years (stored 1-3 y: 39%, stored 4-6 y: 15%, stored >6 y: 15%; p=0.016).

   The percentage of patients with a PD-L2 positive/negative status did not differ based on patient age, gender, race; histology; type of KRAS mutation (transversion or a transition); tumor sampling method; smoking status of the patient; or storage duration of the FFPE block. There were no associations between the status or staining intensity of PD-L2 and PD-L1.  Nor was the number of PD-1-positive TILs associated with the intensity of PD-L1 or PD-L2 staining or the smoking status of the patient.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1-3 y 4-6 y >6 y
   Preaquisition	Diagnosis/ patient condition	Current smoker Former smoker (>1 y) Never Smoker (<100 cigarettes/life-time) Intensity of smoking history (3-115 pack years) Adenocarcinoma Squamous Adenosquamous Large cell lung cancer NSCLC Not Otherwise Specified (NOS) KRAS transversion KRAS transition
   Preaquisition	Patient race	Caucasian Asian African American Hispanic
   Preaquisition	Patient gender	Female Male
   Immunohistochemistry Specific	Targeted peptide/protein	PD-1 PD-L1 PD-L2
   Biospecimen Acquisition	Method of tissue acquisition	Surgical resection Core needle biopsy Fine needle aspiration Transbronchial biopsy
   Preaquisition	Patient age	20-84 y

   View more