Representative images of PD-L1 expression in a case with heterogeneous PD-L1 expression.

<p>(<b>A</b>) The whole section (magnification, 3×), (<b>B</b>) the corresponding Spiral Array core (magnification, 15×), (<b>C</b>) magnified images from the regions delineated by the dotted lines in <b>A</b> (left) and <b>B</b> (right) (magnification, 200×), and (<b>D</b>) magnified images from the regions delineated by the solid lines in <b>A</b> (left) and <b>B</b> (right) (magnification, 200×). Images of the other 5 cases are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186192#pone.0186192.s001" target="_blank">S1 Fig</a>.</p

Representative images of the heterogeneous and homogeneous expression of PD-L1.

<p>(<b>A</b>) Heterogeneous and (<b>B</b>) homogeneous expression of PD-L1 (magnification, 150×).</p

Spiral Array construction.

<p>A representative block was selected from each case, and two continuous straight regions of interest (X and Y axes) were selected and marked by reviewing hematoxylin and eosin-stained slides. Two 120.0-μm-thick sections were sliced from each block and aligned with the X and Y axes. The two sections were rolled together into a cylindrical form and cut along the line reflecting the X and Y axes. Spiral Array cores were embedded vertically into a recipient block. Four-μm-thick sections were prepared from the Spiral Array blocks for further histopathological evaluation.</p

Patient characteristics.

<p>Patient characteristics.</p

Additional file 1: of Ciliated muconodular papillary tumors of the lung with KRAS/BRAF/AKT1 mutation

Antibodies used for immunohistochemistry. (XLSX 10 kb

Distributions of cases with negative, heterogeneous, and homogeneous PD-L1 expression.

<p>(<b>A</b>) Distributions of all 138 cases and (<b>B</b>) distributions according to histological type.</p

Schematic of the 8 segments of a Spiral Array core.

<p>Each core was divided into 8 segments for the scoring of PD-L1 expression.</p

Intratumoral heterogeneity of programmed cell death ligand-1 expression is common in lung cancer

<div><p>Programmed cell death ligand-1 (PD-L1) expression may predict the response to both programmed cell death-1 and PD-L1 inhibitors in lung cancer. However, the extent of intratumoral heterogeneity of PD-L1 expression, which may cause false negative results, is largely unexplored. We aimed to assess the intratumoral heterogeneity of PD-L1 expression in surgically resected lung cancer specimens by applying a novel method of tissue microarray, namely Spiral Arrays, which enables us to observe the heterogeneity in spiral-shaped tissue cores. Adenocarcinoma and squamous cell carcinoma specimens were obtained from consecutive patients with lung cancer who had undergone surgical resection at Nagasaki University Hospital (Nagasaki, Japan) since 2009. Small cell lung cancer and large cell carcinoma specimens were selected from patients in the same archive who had undergone resection since 1998. Spiral Arrays were constructed of spiral-shaped cores, prepared from representative blocks of each case, which were subjected to immunohistochemistry using an anti-PD-L1 antibody. Each core was divided into 8 segments and each segment was classified as either PD-L1-positive or PD-L1-negative using thresholds of 1.0%, 5.0%, 10.0%, and 50.0%, respectively. In total, 138 specimens were selected, including 60 adenocarcinomas, 59 squamous cell carcinomas, 12 small cell lung cancers, and 7 large cell carcinomas. The majority of specimens with PD-L1-positive segments exhibited heterogeneous expression (i.e., had a mixture of PD-L1-positive and PD-L1-negative segments within a core) irrespective of the threshold (1.0%, 66.7%; 5.0%, 74.4%; 10.0%, 75.8%; and 50.0%, 85.7%]. Large variations in the ratios of PD-L1-positive segments were observed. At least 50.0% of the segments within a core were negative in no fewer than 50.0% (range, 50.0–76.0%) of cases with heterogeneous PD-L1 expression. In conclusion, intratumoral heterogeneity of PD-L1 expression was frequently observed in cases of lung cancer. Thus, multiple tumor biopsy specimens may be needed to accurately determine the PD-L1 expression status.</p></div

Representative images of a region containing macrophages that resemble tumor cells.

<p>Immunohistochemical analysis of (<b>A</b>) PD-L1 and (<b>B</b>) CD68 expression, and (<b>C</b>) hematoxylin and eosin staining (magnification, 200×).</p

Illustration of the periods of inactive status and sirolimus treatment while on the transplantation waiting list.

<p>A. Eight of 21 LAM patients with a history of inactive status had received lung transplants. Five of eight patients had a period during which they had been on the sirolimus treatment (we could not confirm the period of time in case 3*). The reason why a patient had been inactive is indicated at the right end of each illustration by: A, anxiety and fear about transplantation; C, comorbidity (cases 6 and 8, psychiatric disorder and case 3, thyroid tumor); F, family matter; M, participated in the MILES trial; NA, not available; and S, on sirolimus treatment. Although the cases 1 and 7 participated in the MILES trial, they were in the placebo group; the case 7 took sirolimus after the MILES trial. The clinical courses of cases 2, 4, 5, and 7 who took sirolimus, but their clinical courses were not stabilized sufficiently. B. Of twelve patients who were in an inactive status as of March 2014, nine initiated sirolimus treatment before registration (cases 2 and 6) or while on a waiting list (cases 3, 4, 5, 8, 9, 11, and 12). The reason for being inactive is presented with the same manner as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146749#pone.0146749.g002" target="_blank">Fig 2A</a> (A and M); S(S) indicates that a patient has been on sirolimus treatment and respiratory condition has improved or been stabilized.</p