Tonsillar control tissue for CTLA-4 (10x objective).

<p>Photomicrograph of a portion of the tonsillar tissue used as control in this study. The upper prat of the photograph depicts the epithelial lining of a crypt with light brownish staining for CTLA-4. The rest of the tissue is formed of lymphoid cells in which the reactive cells show strong (3+ intensity) cytoplasmic and cell membrane reactions for CTLA-4 (presumably a subset of activated T-cells) scattered throughout, mostly in the germinal center (right lower quadrant of the picture). A few of these reactive cells are viewed at a high magnification in the right inset (60x objective). The arrows point to the squamous epithelial lining of a crypt showing a light granular reaction in the cytoplasm. A high magnification of the epithelial cells is shown in the left inset (60x objective). In the control tissue, the characteristic positive granules (light staining and lower in number) of the epithelial cells can be established as 1+ intensity.</p

Group I, summary of the breast tissue cores with ductal carcinoma in situ showing the CTLA-4 staining reactions, scores, interpretations, and percentages of stained lymphocytes.

<p>Table is primarily arranged based on the tumor grades and intensities.</p

Scores 1b & 2b in IDC (20x objective).

<p>Examples of invasive ductal carcinomas are photomicrographed showing the neoplastic cells invading the interstitial tissues. <b>B</b> CTLA-4 IHC stain with 2+ intensity showing cytoplasmic reaction in about 40% of the neoplastic cells scored as <b>1b</b> (from #2; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>). <b>D</b> shows 3+ intensity of CTLA-4 cytoplasmic granular reaction in 100% of the ductal carcinoma cells scored as <b>2b</b> (from #56; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>). The photomicrographs of the counterpart hematoxylin & eosin stains are displayed to the left of each respective CTLA-4 IHC stain (panels <b>A</b> & <b>C</b>).</p

Intensities of CTLA-4 reaction (60x objective).

<p>The immunohistochemical staining for CTLA-4 depicting different reaction intensities. Photomicrographs are obtained from the respective tissue cores in this study. <b>A</b> Intensity 0, showing no cytoplasmic reaction (from #8; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>). <b>B</b> Intensity 1+, showing a low number of reactive cytoplasmic granules (from #18; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>). <b>C</b> Intensity 2+, showing a moderate number of reactive cytoplasmic granules (from #31; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>). <b>D</b> Intensity 3+, showing a high number of reactive granules packed in the cytoplasm (from #7; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t002" target="_blank">Table 2</a>).</p

Score 2b in DCIS (10x objective).

<p>An example of ductal carcinoma in-situ is shown where the neoplastic lesion is confined within the ductal structures. <b>B</b> CTLA-4 with 3+ intensity showing a uniform strong cytoplasmic stain in 100% of the neoplastic cells. A counterpart hematoxylin & eosin stain of the same tumor is shown in panel <b>A</b> (from #7; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t002" target="_blank">Table 2</a>).</p

Expression of immune checkpoint regulators, cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death-ligand 1 (PD-L1), in female breast carcinomas

<div><p>Background</p><p>Immune checkpoint regulators, <i>cytotoxic T lymphocyte antigen 4</i> (CTLA-4) and the <i>programmed cell death protein-1/programmed death-ligand 1</i> (PD-1/PD-L1) have emerged as promising new targets for cancer therapeutics. While tumor expression of PD-L1 has been shown to have objective responses to anti-PD-L1 immunotherapies, the clinical implications of CTLA-4 expression in tumor cells or immune cells in the tumor microenvironment is still controversial. We investigated the expression of CTLA-4 and PD-L1 in human breast tumors and provided a scoring system for the systematic evaluation of CTLA-4 staining.</p><p>Methods</p><p>Immunohistochemical staining for PD-L1 and CTLA-4 expression was performed on a tissue microarray of 102 cores, which included normal and neoplastic breast tissues. Neoplastic cores were divided into four groups: <i>Ductal carcinoma in situ</i> (DCIS), <i>invasive ductal carcinoma</i> (IDC), <i>invasive lobular carcinoma</i> (ILC) and <i>invasive tubular carcinoma</i> (ITC). PD-L1 and CTLA-4 expressions were scored based on a system which accounted for the percentage and intensity of positivity and results provided in conjunction with available clinical and demographic data.</p><p>Results</p><p>Overall, CTLA-4 was over-expressed in 49 of 93 (52.7%) breast tumors. Subcategorically, CTLA-4 was positive in 3 of 8 (37.5%) ductal carcinoma in situ, 40 of 73 (55%) of invasive ductal carcinomas, 4 of 10 (40%) of invasive lobular carcinomas and 2 of 2 (100%) of invasive tubular carcinomas. All 6 normal breast tissues were interpreted as negative for CTLA-4 staining. Only 4.1% of the invasive ductal carcinomas were positive for PD-L1 reactivity and the remaining carcinomas stained negative.</p><p>Conclusions</p><p>This study shows a significant overexpression of CTLA-4 in >50% of breast carcinomas with no such overexpression of CTLA-4 in benign breast tissues. PDL-1 staining is seen in only a small number of invasive ductal carcinomas (4.1%). These findings suggest the need for further investigation of anti-CTLA-4 and anti-PD-L1 immunotherapies and their efficacy in the treatment of breast carcinomas with overexpression of these immune modulators. In addition, the proposed scoring system will facilitate a more systematic correlation between tumor reactivity and clinical outcome which can be applied to all intracytoplasmic tumor markers.</p></div

Summary of the <i>normal</i> breast tissue cores with the CTLA-4 reaction, score, and the interpretation.

<p>Summary of the <i>normal</i> breast tissue cores with the CTLA-4 reaction, score, and the interpretation.</p

Score 2a in benign breast tissue (20x objective).

<p>Photomicrographs of a normal (or benign) breast tissue showing the normal ductal structures with the adjacent interstitial tissue. <b>B</b> CTLA-4 stain with 1+ intensity showing a uniform light staining cytoplasmic granules in 100% of ductal cells. The luminal contents of the glands had a negative reaction. The inset is a portion of a ductal structure showing the sparsity of the cytoplasmic granules at a higher magnification (60x objective). A counterpart hematoxylin & eosin stain of the same tissue is shown in panel <b>A</b> (from #5; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t001" target="_blank">Table 1</a>).</p

Group II, summary of the breast invasive ductal carcinomas showing the CTLA-4 staining reactions, scores, interpretations, and percentages of stained lymphocytes.

<p>Table is primarily arranged based on the tumor grades and intensities.</p

PD-L1 in IDC (40x objective).

<p>Photomicrographs of an invasive ductal carcinoma are displayed. <b>B</b> shows PD-L1 reaction involving cell membranes of the tumor cells with large open nuclear chromatin. The reaction was observed in 20% of the tumor cells in this core. Small mononuclear cells are also present in between the tumor cells. A hematoxylin & eosin stain photomicrograph of the same tumor is shown in panel <b>A</b> (#37; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>).</p