Programmed death-ligand 1 overexpression is a prognostic marker for aggressive papillary thyroid cancer and its variants

Abstract

Programmed death-ligand 1(PD-L1) expression on tumor cells is emerging as a potential predictive biomarker in anti-PD-L1 directed cancer immunotherapy. We analyzed PD-L1 expression in papillary thyroid carcinoma (PTC) and its variants and determined its prognostic potential to predict clinical outcome in these patients. This study was conducted at an academic oncology hospital which is a prime referral centre for thyroid diseases. Immunohistochemical subcellular localization (IHC) analyses of PD-L1 protein was retrospectively performed on 251 archived formalin fixed and paraffin embedded (FFPE) surgical tissues (66 benign thyroid nodules and 185 PTCs) using a rabbit monoclonal anti-PD-L1 antibody (E1L3N, Cell Signaling Technology) and detected using VECTASTAIN rapid protocol with diaminobenzidine (DAB) as the chromogen. The clinical-pathological factors and disease outcome over 190 months were assessed; immunohistochemical subcellular localization of PD-L1 was correlated with disease free survival (DFS) using Kaplan Meier survival and Cox multivariate regression analysis. Increased PD-L1 immunostaining was predominantly localized in cytoplasm and occasionally in plasma membrane of tumor cells. Among all combined stages of PTC, patients with increased PD-L1 membrane or cytoplasmic positivity had significantly shorter median DFS (36 months and 49 months respectively) as compared to those with PD-L1 negative tumors (DFS, both 186 months with p < 0.001 and p < 0.01 respectively). Comparison of PD-L1+ and PD-L1- patients with matched staging showed increased cytoplasmic positivity in all four stages of PTC that correlated with a greater risk of recurrence and a poor prognosis, but increased membrane positivity significantly correlated with a greater risk of metastasis or death only in Stage IV patients. In conclusion, PD-L1 positive expression in PTC correlates with a greater risk of recurrence and shortened disease free survival supporting its potential application as a prognostic marker for PTC.

Keywords: benign nodule; programmed death-ligand 1; protein biomarkers; subcellular localization; thyroid cancer.

Figure 1. Immunohistochemical analysis of PD-L1 in thyroid tissues: Paraffin embedded sections of different stages of PTC, benign thyroid nodules and concurrent thyroiditis were stained using anti-PD-L1 monoclonal antibody as described in the method sections

The representative photomicrographs show immunostaining of PD-L1 in thyroid carcinomas. Faint cytoplasmic staining was observed in early stage (Stage I) of thyroid carcinoma (A); Mild cytoplasmic staining was observed in Stage II (B); moderate to very strong cytoplasmic staining and mild to moderate membranous staining of PD-L1 were visible in advanced stages (Stage III, IV) of thyroid carcinomas (C), (D), (E). Benign tissues section showing no detectable PD-L1 staining (F); Human OSCC tissue as positive control (G); Thyroid tissue incubated with isotype specific IgG showing no detectable immunostaining for PD-L1 as Negative control (H). Lymphocytic Thyroiditis tissue section showing increased PD-L1 staining mostly cytoplasmic (arrow indicated as C) and occasionally in membrane (M) and presence of increased lymphocytes infiltration (I). Original magnification (A–F) and (H): ×200; (G, I): ×400.

Figure 2. Kaplan-Meier estimation of disease-free survival in PTC patients

Disease free survival curves showing PD-L1 expression among all the combined stages of PTCs in: (A) cytoplasm [PD-L1 positive median DFS = 49 months (blue lines) and PD-L1 negative DFS = 186 months (red lines); p < 0.001]; (B) plasma membrane [PD-L1 membrane positive tumors median disease free survival (DFS) = 36 months (blue lines) and PD-L1 negative DFS = 186 months (red lines); p < 0.01]; (C) Combined cytoplasmic and membrane PD-L1 positive patients [DFS = 42 months (blue lines) and PD-L1 negative [DFS = 186 months (red lines); p < 0.01].

Figure 3. Kaplan-Meier estimation of disease-free survival in stage I and II PTC patients

Disease free survival curves showing PD-L1 expression in (A) Stage I cytoplasm [PD-L1 positive median disease free survival (DFS) = 166 months (blue lines) and PD-L1 negative DFS = 185 months (red lines); p = 0.031]. (B) Stage I membrane [PD-L1 positive median disease free survival (DFS) = 124 months (blue lines) and PD-L1 negative DFS = 185 months (red lines); p = 0.164]. (C) Stage I combined cytoplasm and membrane [PD-L1 positive median disease free survival (DFS) = 168 months (blue lines) and PD-L1 negative DFS = 185 months (red lines); p = 0.063]. (D) Stage II cytoplasm [PD-L1 positive median disease free survival (DFS) = 62 months (blue lines) and PD-L1 negative DFS = 132 months (red lines); p = 0.027]. (E) Stage II membrane [PD-L1 positive median disease free survival (DFS) = 62 months (blue lines) and PD-L1 negative DFS = 132 months (red lines); p = 0.453]. (F) Disease free survival curves showing PD-L1 expression in Stage II combined cytoplasm and membrane [PD-L1 positive median disease free survival (DFS) = 62 months (blue lines) and PD-L1 negative DFS = 105 months (red lines); p = 0.267].

Figure 4. Kaplan-Meier estimation of disease-free survival in stage III and IV PTC patients

Disease free survival curves showing PD-L1 expression in (A) Stage III cytoplasm [PD-L1 positive median disease free survival (DFS) = 53 months (blue lines) and PD-L1 negative DFS = 128 months (red lines); p = 0.036]. (B) Stage III membrane [PD-L1 positive median disease free survival (DFS) = 43 months (blue lines) and PD-L1 negative DFS = 120 months (red lines); p = 0.025]. (C) Stage III combined cytoplasm and membrane [PD-L1 positive median disease free survival (DFS) = 45 months (blue lines) and PD-L1 negative DFS = 120 months (red lines); p = 0.046]. (D) Stage IV cytoplasm [PD-L1 positive median disease free survival (DFS) = 23 months (blue lines) and PD-L1 negative DFS = 140 months (red lines); p = 0.001]. (E) Stage IV membrane [PD-L1 positive median disease free survival (DFS) = 25 months (blue lines) and PD-L1 negative DFS = 81 months (red lines); p = 0.038]. (F) Disease free survival curves showing PD-L1 expression in Stage IV combined cytoplasm and membrane [PD-L1 positive median disease free survival (DFS) = 21 months (blue lines) and PD-L1 negative DFS = 140 months (red lines); p = 0.004].