DataSheet_1_Anti-PD-1 antibodies, a novel treatment option for advanced chemoresistant pulmonary lymphoepithelioma carcinoma.docx

BackgroundPulmonary lymphoepithelioma-like carcinoma (LELC) exhibits a unique immune microenvironment, including high PD-L1 expression and abundant infiltrating-immune cells. However, the availability of PD-1/PD-L1 inhibitors in patients with LELC is still not determined.MethodsA total of 36 cases of pulmonary LELC treated with PD-1/PD-L1 inhibitors were reviewed, including 10 cases from our institute and 26 cases included from the literature. The Kaplan-Meier method and log-rank test were utilized to analyze the survival outcomes of LELC patients receiving immunotherapy, and the factors related to immunotherapy response were further examined.ResultsOf the 10 patients from our institute, the median age was 53.5 years, adrenal glands and distant lymph nodes were the most common metastatic sites, and 4 of 8 (50%) patients had a PD-L1 TPS ≥50%. The median progression-free survival and overall survival in patients from our institute and from the literature were 11.6 and 27.3 months, 17.2 months and not reached, respectively. In all 36 patients, the objective response rate was as high as 57.6%. Patients with higher PD-L1 expression were more likely to have a tumor response, but the association of PD-L1 expression with survival time remains to be determined.ConclusionsPD-1/PD-L1 inhibitors in patients with pulmonary LELC demonstrated a promising efficacy in retrospective cohorts, and deserve further validation in prospective studies administrating in front-line setting.</p

MiRNA-615-5p Functions as a Tumor Suppressor in Pancreatic Ductal Adenocarcinoma by Targeting AKT2

<div><p>Background</p><p>Aberrant microRNA (miRNA) expression is associated with tumor development. This study aimed to elucidate the role of miR-615-5p in the development of pancreatic ductal adenocarcinoma (PDAC).</p><p>Methods</p><p>Locked nucleic acid <i>in situ</i> hybridization (LNA-ISH) was performed to compare miR-615-5p expression in patients between PDAC and matched adjacent normal tissues. Effects of miR-615-5p overexpression on cell proliferation, apoptosis, colony formation, migration, and invasion were determined in the pancreatic cancer cell lines PANC-1 and MIA PaCa-2. Effects of miR-615-5p on <i>AKT2</i> were examined by dual-luciferase reporter assay. Lentivirus expressing miR-615 was used to create stable overexpression cell lines, which were subsequently used in mouse xenograft and metastasis models to assess tumor growth, apoptosis and metastasis.</p><p>Results</p><p>miR-615-5p expression was significantly lower in PDAC than in adjacent normal tissues. Low levels of miR-615-5p were independently associated with poor prognosis (HR: 2.243, 95% CI: 1.190-4.227, <i>P</i>=0.013). AKT2 protein expression was inversely correlated with miR-615-5p expression (<i>r</i>=-0.3, <i>P</i>=0.003). miR-615-5p directly targeted the 3’-untranslated region of <i>AKT2</i> mRNA and repressed its expression. miR-615-5p overexpression inhibited pancreatic cancer cell proliferation, migration, and invasion <i>in vitro</i>, and tumor growth and metastasis <i>in vivo</i>. Furthermore, miR-615-5p overexpression also induced pancreatic cancer cell apoptosis both <i>in vitro</i> and <i>in vivo</i>.</p><p>Conclusions</p><p>These results show that miR-615-5p inhibits pancreatic cancer cell proliferation, migration, and invasion by targeting AKT2. The data implicate miR-615-5p in the prognosis and treatment of PDAC.</p></div

MiR-615-5p overexpression inhibits tumor metastasis <i>in vivo</i>.

<p>LV-miR-615-5p-MIA or LV-control-MIA cells were injected into nude mice via tail vein. Mouse livers were harvested to evaluate tumor metastasis 4 weeks after injection. (A) Mouse liver tissue (left panel) and representative tumor nodules (right panel, H&E staining) (Magnification: ×2.5, upper; ×10, lower). (B) The number of tumor nodules in the liver was quantified and is presented as mean±SD (n = 6 in LV-control-MIA group; n = 6 in LV-miR-615-5p-MIA group). **<i>P</i><0.01 <i>vs</i>. LV-control-MIA group.</p

Kaplan-Meier analysis demonstrates that PDAC patients with low-miR-615-5p expression have a lower overall survival than those with high expression of miR-615-5p (log-rank test, <i>P</i><0.01).

<p>Kaplan-Meier analysis demonstrates that PDAC patients with low-miR-615-5p expression have a lower overall survival than those with high expression of miR-615-5p (log-rank test, <i>P</i><0.01).</p

Effects of miR-615-5p overexpression on cell proliferation, colony formation, and apoptosis in pancreatic PANC-1 and MIA PaCa-2 cancer cell lines.

<p>PANC-1 and MIA PaCa-2 cells were transiently transfected with the miR-615-5p miRNA mimic (miR-615-5p) or a control construct (NC). (A) Cell proliferation was measured by CCK-8 detection at 24, 48, 72, 96, and 120 hours after transfection both in MIA PaCa-2 and PANC-1 cells. (B) Cell proliferation was also determined by 5-ethynyl-2’-deoxyuridine (EdU) staining 48 hours after transfection (Scale bar: 200 μm). Red: EdU; Blue: DAPI. (C) Colony formation assay after 21 days in culture. (D) Cell apoptosis was determined by flow cytometry analysis using Annexin V/PI staining 48 hours after transfection. The data are shown as mean±SD. *<i>P</i><0.05 <i>vs</i>. untreated cells or NC group.</p

Multivariate Cox regression analysis of clinico-pathological variables and cumulative survival of patients with PDAC.

<p><b><i>Note</i></b>: HR: hazard ratio; 95% CI: 95% confidence interval.</p><p>Multivariate Cox regression analysis of clinico-pathological variables and cumulative survival of patients with PDAC.</p

Locked nucleic acid <i>in situ</i> hybridization (LNA-ISH) shows differential expression of miR-615-5p in pancreatic ductal adenocarcinoma (PDAC) compared to normal pancreatic tissues. Bar = 100 μm.

<p>(A) Strong positive (2+) staining of miR-615-5p in normal pancreas; note the dark acinar nuclear staining with cytoplasmic stippling. (B) Weak positive (+) staining of miR-615-5p in PDAC tissue. (C) Positive control staining for the U6 probe in PDAC tissue; note the dark nuclear staining observed in cancer cells. (D) Negative control for the scrambled probe in normal pancreatic tissue; no positive staining is observed.</p

AKT2 is a direct target of miR-615-5p.

<p>(A) Schematic representation of the firefly luciferase reporter construct containing the 3’-UTR of AKT2 with either wild-type (WT) or mutant (MUT) miR-615-5p target site. The mutant construct contains mutations in 6 nucleotides within the seed region of the target site to disrupt binding of miR-615-5p. PANC-1 (B) and MIA PaCa-2 (C) cells (0.5 × 10⁶ cells per well) were transiently co-transfected with reporter plasmids (200 ng, WT or MUT) and 100 nM of either miR-615-5p miRNA mimic (miR-615-5p) or control mimic (NC) for 24 h. Protein lysates were then prepared and relative luciferase activity was determined using the dual-luciferase assay system. Renilla luciferase was used for normalization of transfection efficiency. Data are represented as normalized fold change of luciferase activity. The data are shown as mean±SD. *<i>P</i><0.05 <i>vs</i>. miR-615+AKT2WT. AKT2 protein expression in MIA PaCa-2 (D) and PANC-1 (E) cells transiently transfected with mir-615-5p miRNA mimic (miR-615-5p) or control mimic (NC) were determined by Western blot. (F) AKT2 protein expression in tumor tissues from nude mice stably overexpressing miR-615-5p was determined by Western blot. β-actin was used as a loading control. (G) Higher magnification after AKT2 detection in PDAC tissues by immunohistochemistry (IHC) (Magnification: ×200); negative, focally positive and diffusely positive cells can be observed. Two arrows point to two group of cancer cells, one with positive AKT2 expression (left) and the other AKT2 negative (right). (H) AKT2 expression is inversely correlated with miR-615-5p in PDAC tissues. AKT2 expression was determined by IHC (upper panel). miR-615-5p expression was determined by locked nucleic acid <i>in situ</i> hybridization (LNA-ISH) (lower panel). Magnification: ×200. Normal: normal pancreas; PC G1: well differentiated PDAC tissue; PC G3: poorly differentiated PDAC tissue. LNA-ISH and immunohistochemistry slides were counterstained with nuclear fast red and hematoxylin, respectively.</p

miR-615-5p expression in PDAC and normal pancreas tissues determined by locked nucleic acid <i>in situ</i> hybridization (LNA-ISH).

<p><b><i>Note</i>:</b> **<i>P</i> <0.01 PDAC tissue <i>vs</i>. normal pancreas tissues (Fisher’s exact test). PDAC: Pancreatic ductal adenocarcinoma.</p><p>miR-615-5p expression in PDAC and normal pancreas tissues determined by locked nucleic acid <i>in situ</i> hybridization (LNA-ISH).</p

miR-193b regulates the expression of KRAS by directly targeting its 3′-UTR.

<p>(A) The TargetScan online database (<a href="http://www.targetscan.org" target="_blank">http://www.targetscan.org</a>) predicted KRAS as an miR-193b target. The site between 1074 and 1080 (boxed region), one of the two predicted miR-193b binding sites in the 3′-UTR of KRAS, is shown, highlighting the evolutionarily conservation among humans (hsa), the chimpanzees (Ptr), rhesus monkey (Mml), bushbaby (Oga), and treeshrew (Tbe). (B) The two miR-193b binding sites at positions 303 to 309 and 1074–1080 in the KRAS 3′-UTR, identified using the TargetScan online database (<a href="http://www.targetscan.org" target="_blank">http://www.targetscan.org</a>), are shown. Seed sequences were mutated as shown in the boxed regions. The wild-type or mutant constructs were inserted into the pGL3 vector directly downstream of the luciferase gene. (C) 293A cells were co-transfected with miR-193b or scrambled oligonucleotide, wild-type or mutant firefly luciferase constructs of the KRAS 3′-UTR segment containing miR-193b binding sites, and Renilla luciferase (endogenous control). Luciferase activity was measured 24 hours after transfection using the Dual-Luciferase Reporter Assay Systems. Renilla-normalized luciferase activity is expressed relative to that obtained for the scrambled oligonucleotide under each condition. WT, wild-type; Mut, mutant. (D) KRAS and CCND1 protein levels were assessed by Western blotting 48 hours after transfection. (E) The protein levels of p-ERK and p-Akt were evaluated by Western blotting 48 hours after miR-193b transfection.</p