L1 Expression Research Articles

Locus specific reduction of L1 expression in the cortices of individuals with amyotrophic lateral sclerosis

The activation and dysregulation of retrotransposons has been identified in the CNS of individuals with the fatal neurodegenerative disorder Amyotrophic lateral sclerosis (ALS). This includes elements from multiple different families and subfamilies of retrotransposons, however there is limited knowledge of the specific loci from which this expression occurs in ALS. The long interspersed element-1 (L1) is the only autonomous retrotransposon in the human genome and members of this family of elements maintain the ability to mobilise. Despite L1s contributing to 17% of the human genome only 80–100 L1s encode the required proteins for mobilisation and are retrotransposition competent. Identifying the specific loci from which L1 expression occurs will inform on the potential functional consequences of their expression, such as the potential for somatic retrotransposition or DNA damage caused by the endonuclease activity of the ORF2 protein of the L1. Here we characterised L1 loci expression using the L1EM tool (https://github.com/FenyoLab/L1EM) in RNA sequencing data from 518 samples across four tissues (motor cortex, frontal cortex, cerebellum and cervical spinal cord) in the Target ALS cohort obtained from the New York Genome Center. There was a significant reduction in total intact L1 expression (those that encode functional proteins) in two brain regions of individuals with ALS compared to controls and clustering of the ALS brain regions occurred based on their intact L1 expression profile. Although overall the levels of L1 expression were reduced in ALS/ALS with other neurological disorder (ND) there were individuals in which L1s were expressed at much higher levels than the rest of the ALS/ALSND cohort. Expressed L1 loci were more frequently located in introns compared to those not expressed and the level of L1 expression positively correlated with the expression of the gene in which it was located. Significant differences were observed in the expression profiles of L1s in ALS and specific features of these elements, such as location in the genome and whether or not they are intact, were significantly associated with those that were expressed in the cohort.

Serum exosomal miR-16-5p functions as a tumor inhibitor and a new biomarker for PD-L1 inhibitor-dependent immunotherapy in lung adenocarcinoma by regulating PD-L1 expression.

ObjectivesWe aimed at investigating whether serum exosomal miR‐16‐5p could be utilized as an immunotherapy biomarker in lung adenocarcinoma (LUAD) patients administered by programmed cell death ligand‐1 (PD‐L1) inhibitors, and to evaluate its functions in LUAD progression.MethodsSixty LUAD sufferers and 20 healthy controls (HCs) were covered in this work. We applied both IHC and WB to examine PD‐L1 level in clinical tissue samples and utilized WB to quantify PD‐L1 expression in LUAD cells and LUAD xenograft tissues, respectively. Transmission electron microscopy (TEM), WB, and nanoparticle tracking analysis (NTA) were executed to confirm the exosomes isolated from serum specimens and cell culture media. To quantify of exosomal miR‐16‐5p level from serum and culture medium of cultured cell, qRT‐PCR experiment was utilized. The connection between tissue PD‐L1 level and serum exosomal miR‐16‐5p expression in PD‐L1‐positive sufferers administered by PD‐L1 inhibitors was verified using Spearman correlation coefficient analysis. In addition, the overall survival (OS) and progression‐free survival (PFS) rates among PD‐L1 inhibitor managed sufferers were acquired through a follow‐up visit. Finally, we used a group of assays, including 5‐bromo‐2′‐dexoyuridine (BrdU) and colony formation test, wound healing experiment, flow cytometry, and nude mice xenograft experiment, to explore the functions of circulating exosomal miR‐16‐5p on LUAD cell proliferation, apoptosis, and migration, as well as tumor development, respectively.ResultsPD‐L1 expression was positively related to T stage (tumor size stage), and PD‐L1 inhibitor treatment reduced the PD‐L1 expression and mitigated T stage in PD‐L1‐positive LUAD sufferers. For PD‐L1‐positive LUAD sufferers, elevated PD‐L1 expression or reduced serum exosomal miR‐16‐5p level were linked to longer PFS and OS upon PD‐L1 inhibitor treatment. The number of exosomes in patient's serum was more than that in the serum of healthy individuals, and PD‐L1 inhibitor treatment decreased the number of serum‐derived exosomes in PD‐L1‐positive LUAD sufferers. Exosome‐derived miR‐16‐5p was downregulated in patient's serum and cell culture medium, and this was negatively linked to tumor stage and PD‐L1 expression. Meanwhile, PD‐L1 inhibitor treatment could increase the serum exosomal miR‐16‐5p expression, and the expression change of serum exosomal miR‐16‐5p was diametrically related to PD‐L1 after the treatment. Moreover, the overexpression of PD‐L1 accelerated tumor growth and decreased the exosomal miR‐16‐5p content in cell culture media, while exosomal miR‐16‐5p overexpression in cell culture media inhibited tumor development by decreasing the PD‐L1 expression. Exosomal miR‐16‐5p overexpression in cell culture media also depressed LUAD cell proliferation and migration, and stimulated cell apoptosis, especially in the cells which cultured in the mediums with PD‐L1 inhibitor in vitro.ConclusionsSerum exosomal miR‐16‐5p may be a latent tumor inhibitor and a new biomarker for PD‐L1 inhibitor‐dependent immunotherapy in LUAD by regulating the PD‐L1 expression.

Loss of heterochromatin and retrotransposon silencing as determinants in oocyte aging.

Mammalian oocyte quality reduces with age. We show that prior to the occurrence of significant aneuploidy (9M in mouse), heterochromatin histone marks are lost, and oocyte maturation is impaired. This loss occurs in both constitutive and facultative heterochromatin marks but not in euchromatic active marks. We show that heterochromatin loss with age also occurs in human prophase I‐arrested oocytes. Moreover, heterochromatin loss is accompanied in mouse oocytes by an increase in RNA processing and associated with an elevation in L1 and IAP retrotransposon expression and in DNA damage and DNA repair proteins nuclear localization. Artificial inhibition of the heterochromatin machinery in young oocytes causes an elevation in retrotransposon expression and oocyte maturation defects. Inhibiting retrotransposon reverse‐transcriptase through azidothymidine (AZT) treatment in older oocytes partially rescues their maturation defects and activity of the DNA repair machinery. Moreover, activating the heterochromatin machinery via treatment with the SIRT1 activating molecule SRT‐1720, or overexpression of Sirt1 or Ezh2 via plasmid electroporation into older oocytes causes an upregulation in constitutive heterochromatin, downregulation of retrotransposon expression, and elevated maturation rates. Collectively, our work demonstrates a significant process in oocyte aging, characterized by the loss of heterochromatin‐associated chromatin marks and activation of specific retrotransposons, which cause DNA damage and impair oocyte maturation.

Analysis of epigenetic features characteristic of L1 loci expressed in human cells.

Only a select few L1 loci in the human genome are expressed in any given cell line or organ, likely to minimize damage done to the genome. The epigenetic features and requirements of expressed L1 loci are currently unknown. Using human cells and comprehensive epigenetic analysis of individual expressed and unexpressed L1 loci, we determined that endogenous L1 transcription depends on a combination of epigenetic factors, including open chromatin, activating histone modifications, and hypomethylation at the L1 promoter. We demonstrate that the L1 promoter seems to require interaction with enhancer elements for optimal function. We utilize epigenetic context to predict the expression status of L1Hs loci that are poorly mappable with RNA-Seq. Our analysis identified a population of ‘transitional’ L1 loci that likely have greater potential to be activated during the epigenetic dysregulation seen in tumors and during aging because they are the most responsive to targeted CRISPR-mediated delivery of trans-activating domains. We demonstrate that an engineered increase in endogenous L1 mRNA expression increases Alu mobilization. Overall, our findings present the first global and comprehensive analysis of epigenetic status of individual L1 loci based on their expression status and demonstrate the importance of epigenetic context for L1 expression heterogeneity.

Cytoplasmic L1 Levels in Cancer

In addition to shaping genome diversification over evolutionary time, L1 retrotransposition alters gene expression as well. The most notable gene altering process involves insertional mutagenesis. The aim of the study was the examination of both nuclear L1 expression levels and cellular localization in cancer cell lines, PBMCs from healthy volunteers and PBMCs from cancer patients. L1 was detected by FISH in chromosome preparations. L1 probe was custom-made using end-point PCR against L1-ORF2 and conjugated with FITC. It was found that cancer cell lines and clinical samples from cancer patients contained significantly elevated levels of L1 per nucleus compared to healthy volunteers. Cytoplasmic L1 was also increased in the above mentioned samples denoting that cancer could be associated with increased L1 activation and mobility. Our results may provide a novel cancer diagnostic marker and highlight the possibility of cytoplasmic L1 inhibition as a therapeutic intervention for cancer.

CgRab1 regulates Cgcathepsin L1 expression and participates in the phagocytosis of haemocytes in oyster Crassostrea gigas

Rab protein plays an important role in a variety of cellular activities, especially the fusion process of the inner membrane during endocytosis. In the present study, a Rab1 protein (CgRab1) was identified from the Pacific oyster Crassostrea gigas. The full-length cDNA sequence of CgRab1 was of 2248 bp with an open reading frame of 618 bp, encoding a polypeptide of 205 amino acids containing a Rab domain. The deduced amino acid sequence of CgRab1 shared 94.2% and 89.3% identity with Rab1 from Pomacea canaliculata and Homo sapiens respectively. In the phylogenetic tree, CgRab1 was firstly clustered with the Rab1s from Aplysia californica and Pomacea canaliculata to form a sister group with Rab1 from invertebrates. The recombinant CgRab1 protein (rCgRab1) was able to bind GTP. The mRNA transcripts of CgRab1 were highly expressed in oyster haemocytes, and its expression level in oyster haemocytes was significantly up-regulated at 24 h after the stimulations with Vibro splendidus, which was 2.43-fold (p < 0.01) of that in the control group. After the expression of CgRab1 was knocked down (0.38-fold of that in EGFP-RNAi experimental group) by RNAi，the protein expression of Cgcathepsin L1 were reduced (0.63-fold, p < 0.01) compared with that in EGFP-RNAi experimental group. The phagocytic rate and phagocytic index of haemocytes in CgRab1-RNAi oysters decreased after V. splendidus stimulation, which was 0.50-fold (p < 0.01) and 0.58-fold (p < 0.01) of that in EGFP-RNAi experimental group. These results indicated that CgRab1 was involved in the process of haemocytes phagocytosis by regulating the expression of Cgcathepsin L1 in oyster C. gigas.

Investigation of H2S Donor Treatment on Neutrophil Extracellular Traps in Experimental Colitis.

Inflammatory bowel diseases (IBD) are chronic, immune-mediated disorders, which affect the gastrointestinal tract with intermittent ulceration. It is increasingly clear that neutrophil extracellular traps (NETs) seem to have a role in IBD; however, the associated pathogenesis is still not known. Furthermore, several conventional therapies are available against IBD, although these might have side effects. Our current study aimed to investigate the effects of hydrogen sulfide (H2S) treatment on NETs formation and on the expression of inflammatory mediators in experimental rat colitis. To model IBD, 2,4,6-trinitrobenzenesulfonic acid (TNBS) was administered intracolonically (i.c.) to Wistar–Harlan male rats. Animals were treated (2 times/day) with H2S donor Lawesson’s reagent per os. Our results showed that H2S treatment significantly decreased the extent of colonic lesions. Furthermore, the expression of members of NETs formation: peptidyl arginine deiminase 4 (PAD4), citrullinated histone H3 (citH3), myeloperoxidase (MPO) and inflammatory regulators, such as nuclear transcription factor-kappa B (NF-κB) and high-mobility group box 1 (HMGB1) were reduced in H2S treated group compared to TNBS. Additionally, H2S donor administration elevated the expression of ubiquitin C-terminal hydroxylase L1 (UCHL-1), a potential anti-inflammatory mediator. Taken together, our results showed that H2S may exert anti-inflammatory effect through the inhibition of NETs formation, which suggests a new therapeutic approach against IBD.

HCV Activates Somatic L1 Retrotransposition-A Potential Hepatocarcinogenesis Pathway.

Simple SummaryChronic hepatitis C virus (HCV) infection is a common cause of liver cancer in the developed world. Although anti-viral treatment can cure HCV infection, the risk of cancer development remains in individuals with consequent advanced fibrosis or cirrhosis. In this study, we show that HCV can influence transposable DNA elements called L1 retrotransposons. These are mobile genetic elements that can negatively alter the host genome, potentially promoting cancer development. L1 elements are known to be activated in several cancer types, including liver cancer. Hence, we suggest a novel pathway involved in liver cancer development in patients with chronic HCV, including those with active infection as well as after viral clearance.Hepatitis C virus (HCV) is a common cause of hepatocellular carcinoma (HCC). The activation and mutagenic consequences of L1 retrotransposons in virus-associated-HCC have been documented. However, the direct influence of HCV upon L1 elements is unclear, and is the focus of the present study. L1 transcript expression was evaluated in a publicly available liver tissue RNA-seq dataset from patients with chronic HCV hepatitis (CHC), as well as healthy controls. L1 transcript expression was significantly higher in CHC than in controls. L1orf1p (a L1 encoded protein) expression was observed in six out of 11 CHC livers by immunohistochemistry. To evaluate the influence of HCV on retrotransposition efficiency, in vitro engineered-L1 retrotransposition assays were employed in Huh7 cells in the presence and absence of an HCV replicon. An increased retrotransposition rate was observed in the presence of replicating HCV RNA, and persisted in cells after viral clearance due to sofosbuvir (PSI7977) treatment. Increased retrotransposition could be due to dysregulation of the DNA-damage repair response, including homologous recombination, due to HCV infection. Altogether these data suggest that L1 expression can be activated before oncogenic transformation in CHC patients, with HCV-upregulated retrotransposition potentially contributing to HCC genomic instability and a risk of transformation that persists post-viral clearance.

A narrative review of combination therapy of PD-1/PD-L1 blockade with standard approaches for the treatment of breast cancer: clinical application and immune mechanism.

In this review, we aimed to discuss the efficacy of immunotherapy of anti-programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) and potential immune mechanism combination with various standard systemic therapies for breast cancer (BC) such as chemotherapy, targeted therapy, endocrine therapy, and radiotherapy. Single-drug therapy of antibodies against PD-1 and its ligand, PD-L1, have only presented modest responses in patients with BC, partly due to the deficiency of tumor-infiltrating lymphocytes (TILs) and low mutation burden. Thus, the combinations of PD-1/PD-L1 blockade with other approaches which may increase the immune therapy effect are being studied. Moreover, an understanding of the immune mechanism of PD-1/PD-L1 blockade with other approaches will contribute to better application of clinical therapy. We searched the studies that focus on PD-1/PD-L1 therapy with or without other systemic therapy and relative immune mechanisms indicated between 2000 and 2020. Anti PD-1/L1 blockade combined with therapeutic approaches is safe and effective in BC, in particular for PD-L1 antibody atezolizumab plus nab-paclitaxel by inducing PD-1/L1 expression and the number of cytotoxic T cells. Otherwise, the toxicity also exists during clinical treatment. Future researches should be evaluated to explore the immune mechanism and vast clinical trials need to be conducted for evidential support for combination therapy of BC.

The expression and clinical value of ubiquitin carboxyl-terminal hydrolase L1 in the blood of neonates with hypoxic ischemic encephalopathy.

BackgroundNeonatal hypoxic ischemic encephalopathy (HIE) can result in mental retardation due to the associated brain damage. Early identification of brain injury is vital for the prevention and treatment of brain damage in neonates. This study investigated the expression levels of serum ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in neonates with HIE and its correlation with brain damage.MethodsFrom January 2019 to December 2020, 56 cases of neonatal patients with HIE were selected as the observation group, and 60 cases of healthy newborns delivered in our hospital during the same period were selected as the control group. Blood samples were obtained from neonates and the serum expression of UCH-L1 was detected by enzyme-linked immunosorbent assays (ELISAs). The relationship between UCH-L1 and neonatal prognosis and clinical features was analyzed.ResultsCompared with the healthy control group, the serum levels of UCH-L1 in the observation group was significantly higher (2.28±1.21 vs. 0.81±0.39 ng/mL, P=0.000). Furthermore, at 6 hours after birth, the serum levels of UCH-L1 were significantly higher in neonates with moderate to severe HIE compared to patients with mild HIE (2.92±0.80 and 1.76±0.72 ng/mL, respectively, P=0.000). Pearson correlation analysis showed that the expression levels of UCH-L1 were negatively correlated with the development quotient (DQ), intelligence index (MI), and the Neonatal Behavioral Neurological Assessment (NBNA) score of HIE newborns (P<0.05).ConclusionsThe level of UCH-L1 protein expression is elevated in the serum of newborns with HIE, and this may have a certain clinical value in predicting the intelligence of children.

Abstract 603: Viable circulating tumor cells predict occult metastatic disease and prognosis, and aberrant expression of PD-L1 on viable CTC in pancreatic cancer

Abstract [Introduction] CTCs as liquid biopsy are an emerging minimally invasive tool for cancer diagnosis, surveillance, and treatment. We tested novel methods for viable CTCs (v-CTC) isolation in the patients (pts) with pancreatic cancer (PC), and investigated the clinical potential of v-CTCs in prognosis. Furthermore, we analyzed the PD-L1(L1) expression in primary PC tumors, metastatic lymph nodes (LN) and v-CTCs. [Pts and Methods] 7.5 ml of venous blood was collected prospectively from 39 PC pts, either upfront surgery (Up group) or pre-treatment, consisted of Gem:800 mg/m2; and S-1:80 mg/m2 given concurrently with IMRT to 60 Gy (NACRT:N group). To detect v-CTCs, we employed a telomerase-specific replication-selective adenovirous expressing GFP. For Up group, samples were obtained before/after resection. For N group, samples were obtained before/after NACRT and after resection. To distinguish between leucocyte and cells with either epithelial or mesenchymal origin, cells were stained by anti-CD45, anti-Cytokeratin and anti-Vimentin Abs. GFP-positive and CD45-negative cells were counted as v-CTC. To assess L1 expression in PC tissues (PC tumors and LN) and v-CTCs, L1 IHC kit (22C3, for tissues) and anti-human L1 mAb (MIH1,for CTCs) were employed. [Results] Up group: 24 pts aged 53~85 years (male/female=12/12) were enrolled. 24 pts underwent curative resection. No v-CTCs were detected in 6 pts at both before and after resection, and 5 of 6 pts survived without recurrence. V-CTCs were identified in 18 of 24 pts, and 13 of 18 pts developed liver metastasis. Marked decrease of CTC counts were seen after resection in 10 of 18 pts, but 9 pts developed recurrence. N group: 15 PC pts aged 44~77 years (male/female=4/11) were enrolled. 15 pts underwent curative resection. No v-CTCs were detected in 5 pts, and all 5 pts survived without recurrence. V-CTCs was identified in 10 of 15 pts, and only 3 out of 10 pts developed disease recurrence. Marked increase in CTC counts was observed after NACRT in 5 of 6 CTC-positive pts before NACRT, and 3 of 5 pts developed liver metastasis and died. RT may induce tumor cell dissemination into the blood circulation for CTC-positive pts. PD-L1 expression: L1 expression were assessed for 21 pts (Up group:18, N group: 3). For PC tumors, L1 molecules were expressed in 12 pts and expression level of these pts were all low (57%, ≥50% [high]=0 pts, 1-49% [low]=12 pts {10%=6, 20%=4, 40%=2}, &amp;lt;1% [negative]=9 pts). For metastatic LNs, metastatic LNs were observed in 14 pts and L1-positive expression in these LNs were detected in only 4 pts (28%, all low expression). On the contrary, the majority of detected v-CTCs clearly expressed L1 molecule (92 CTCs out of 103 detected CTCs were L1-positive=89 %). [Conclusions] Viable CTC detection appears as a promising prognostic marker. Immunotherapy with anti-PD-1/PD-L1 Abs may effectively target v-CTCs to evade recurrence. Citation Format: Masahiro Tanemura, Kenta Furukawa, Manabu Mikamori, Yusuke Matsuura, Kenichi Matsumoto, Tadafumi Asaoka, Yasuo Urata. Viable circulating tumor cells predict occult metastatic disease and prognosis, and aberrant expression of PD-L1 on viable CTC in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 603.

Expression and functional analysis of cathepsin L1 in ovarian development of the oriental river prawn, Macrobrachium nipponense

Cathepsin L is an important member of the cysteine protease family and has a central role in the development of ovaries in insects and fish. In this study, we analyzed the characteristics of the cathepsin L1 gene in the female oriental river prawn, Macrobrachium nipponense. The cathepsin L1 cDNA was 1173 bp in length, including a 960 bp open reading frame that encoded 319 amino acids. The cathepsin L1 gene was classified into the cathepsin L group and named Mn-CTS L1 by phylogenetic analysis. qRT-PCR analysis indicated that Mn-CTS L1 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different stages of ovarian development, Mn-CTS L1 expression in the hepatopancreas and ovaries peaked before ovarian maturation. Mn-CTS L1 expression was higher at the post-larval stage of day 15 than at other stages of embryogenesis. In situ hybridization studies revealed that Mn-CTS L1 was localized in the oocyte of the ovary. Injection of Mn-CTS L1 dsRNA significantly reduced Mn-CTS L1 expression and significantly increased the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CTS L1 dsRNA. These results suggest that Mn-CTS L1 has a key role in ovarian development.

Inducible T-cell co-stimulatory (ICOS) receptor agonist, feladilimab (fela), alone and in combination (combo) with pembrolizumab (P): Results from INDUCE-1 urothelial carcinoma (UC) expansion cohorts (ECs).

4519 Background: INDUCE-1 is a first-in-human trial evaluating fela, an IgG4 ICOS agonist non-T-cell depleting mAb, as monotherapy (mono) and in combo with P. ECs include tumor types, such as UC, with high ICOS expression and immunotherapy-favorable features. Fela induced IFNγ, increased PD-1/L1 expression, and enhanced antitumor activity in combo with PD-1 blockade nonclinically. We report preliminary efficacy, safety, and biomarker data of fela ± P in INDUCE-1 UC ECs. Methods: Eligible patients (pts) had recurrent/metastatic (R/M) UC of the upper or lower urinary tract, ≤6 prior systemic therapy lines in the advanced setting, measurable disease, and no active autoimmune disease. Pts received 0.3 or 1 mg/kg fela (mono EC; anti-PD-1/L1–experienced [exp] pts) or 0.3 mg/kg fela + 200 mg P (combo EC; anti-PD-1/L1–naïve pts) every 3 wks, up to 35 cycles until disease progression or unacceptable toxicity. Disease was assessed every 9 wks through wk 54, then every 12 wks. Archival and/or fresh biopsy tumor tissue was collected for biomarker analyses and safety assessed. Results: By Nov 6 2020, 13 anti-PD-1/L1–exp and 32 anti-PD-1/L1–naïve pts were evaluable in the mono and combo ECs, respectively. In the mono EC, median age was 69 yrs (range: 47–82), 92% of pts were male, and 85% received ≥2 prior therapy lines in the metastatic setting. In the combo EC, median age was 70 yrs (range: 42–84), 75% of pts were male, and 72% received ≥1 prior therapy line in the metastatic setting. In the mono EC, median duration of follow-up (mDoF) was 10.6 mo (range: 1.1–22.8); overall response rate (ORR) was 8% (1 partial response [PR]; 95% CI: 0.2, 36.0) with a duration of response (DoR) of 6.1 mo; disease control rate (DCR [response or stable disease for ≥9 wks]) was 23% (95% CI: 5.0, 53.8), and median overall survival (mOS) was 14.5 mo (95% CI: 2.8, NR), with 74% of pts alive at 6 mo. In the combo EC, mDoF was 9.6 mo (range: 0.9–28.3); ORR was 22% (7 PRs; 95% CI: 9.3, 40.0) with a median DoR of 8.3 months (range: 3.5–23.3+); DCR was 63% (95% CI: 43.7, 78.9), and mOS was 10.7 mo (95% CI: 5.2, 18.1), with 64% of pts alive at 6 mo. Grade ≥3 treatment-related AEs were reported for 0% and 9% of pts in the mono (N = 16) and combo (N = 44) safety populations, respectively. PD-L1 expression and ICOS-specific biomarkers are being evaluated, with promising trends observed in enrichment of clinical activity in preliminary analyses. Conclusions: Fela is the first ICOS agonist with reported single-agent activity in anti-PD-1/L1–exp relapsed/refractory UC. Fela + P in combo shows promising clinical activity and manageable safety in PD-1/L1–naïve R/M UC. Further study is warranted. Updated data to be presented. Funding: Study 204691 (NCT02723955) funded by GlaxoSmithKline in collaboration with Merck Sharp &amp; Dohme Corp, a subsidiary of Merck &amp; Co, Inc, Kenilworth, NJ, USA. Clinical trial information: NCT02723955.

A Severe Case of Hypothyroidism: Immunotherapy Induced

Background: Immune check point inhibitors are playing a crucial role in the treatment of many cancers. Their primary role is to reactivate anti tumor cytotoxic T cells. Programmed cell death 1(PD 1) is a transmembrane protein expressed on T cells, B cells and Natural killer cells. PD 1 ligand is expressed in many tumor cells along with hematopoietic cells and their interaction directly inhibits apoptosis of the tumor cell. Pembrolizumab binds PD1. FDA approved pembrolizumab initially for nonsquamous cell lung cancer and metastatic melanoma that express PD1 ligand which showed improved survival compared to standard therapy. Additionally it has been used in several others tumors with more than 50% PD L1 expression. Although well tolerated, these agents can cause immune related adverse events. Thyroid dysfunction is reported to range between 3.2-10.1% patients receiving pembrolizumab. Clinical Case: 86 year old Caucasian female with past medical history of hypertension and osteoarthritis was diagnosed with stage III metastatic colon cancer in the hepatic flexure and underwent hemicolectomy. Pathology showed 6.5cm mass and 2/30 lymph nodes positive. Due to fear of side effects from chemotherapy, patient refused further treatment. Her follow up labs showed elevation of carcinoembyronic antigen (CEA). Her oncologist suggested Pembrolizumab, immunotherapy. She was started on 5 cycles every 3 weeks. She had fatigue and fell twice after first cycle, but her CEA levels were improving, so it was decided to continue treatment. Soon after fifth cycle she became extremely weak, lethargic, and unable to do any ADLS and was exhausted all the time. Her labs showed TSH 137, free T4 0.4. At that point decision was made to stop pembrolizumab. She was started on levothyroxine 25mcg by her oncologist and referred to me few months later. She complained of tiredness, low energy. On exam Pulse 78, Blood pressure 152/77. Her labs showed TSH 106 and Free T4 0.45. Her levothyroxine dose was increased to 75mcg and followed her 6 weeks later. Her energy levels improved significantly and she is back to her normal self. Her thyroid function was normal. At this point patient has decided not to undergo any treatments for colon cancer. Conclusion: Immune related thyroid events are increasingly noticed with wide use of pembrolizumab. There is insufficient evidence to suggest the cause of these events. Reversible destructive thyroiditis and overt hypothyroidism are the common clinical presentations. It is important to recognize patients at high risk for these adverse events so these highly efficacious agents can be safely used in patients. Thyroid has been a frequent target for anti PD 1 treatments, further research can be of benefit to use against thyroid cancers.

The Clinical Significance of HPV L1/PD-L1 Tests Combined with Colposcopy for Cervical Precancerous Lesions and Cervical Cancer

To explore the expression of human papilloma virus (HPV) L1 protein and programmed cell death ligand-1 (PD-L1) protein in cervical precancerous lesions and cervical cancer, to analyze the correlation between HPV L1 and PD-L1 expression tests combined with colposcopy and the occurrence and development of of cervical lesions, and to determine the significance of the combined examination for auxiliary differential diagnosis. 260 patients with high-risk HPV (HR-HPV) infection who were treated at West China Second University Hospital, Sichuan University from January, 2018 to January, 2020 were included in the study. 260 cervical cytology specimens were collected, of which 218 cervical histology specimens were collected, of which 202 cases underwent colposcopy. Among the 260 cervical cytology specimens, 40 were of cervical inflammatory cells, 40 were of low-grade squamous intraepithilia lesions (LSIL) with mild atypical hyperplasia, 80 were of high-grade squamous intraepithilia lesions (HSIL) with moderate and severe atypical hyperplasia, and 100 were of cervical carcinoma cells (CCC). Among the 218 cervical histology specimens, 15 were of chronic cervicitis tissue, 20 were of cervical intraepithelial neoplasia (CIN) 1, 32 were of CIN 2, 51 were of CIN 3, and 100 were of cervical cancer (CC). Among the 260 patients, 202 underwent colposcopy. Immunocytochemistry and immunohistochemistry were used to assess the expression of HPV L1 protein and PD-L1 protein, and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the level of PD-L1 mRNA in chronic cervicitis tissues and CC. The sensitivity, specificity, positive predictive value and negative predictive value of HPV L1 and PD-L1 tests combined with colposcopy in the detection of cervical lesions were studied. ①In the cervical cytology group, the positive rate of HPV L1 expression was 82.50%, 57.50%, 11.25%, and 3.00% in cervical inflammatory cells, low-grade squamous intraepithilia lesions (LSIL), high-grade squamous intraepithilia lesions (HSIL) and CCC, respectively, showing decreasing levels of positive expression rate ( P<0.05). In the cervical histology group, the positive rate of HPV L1 expression was 86.67%, 65.00%, 34.38%, 11.76% and 4.00% in chronic cervicitis tissues, CIN 1 group, CIN 2 group, CIN 3 group and CC group, respectively, showing decreasing levels of positive expression ( P<0.05). ②In the cervical cytology group, the average positive expression scores of PD-L1 in the cervical inflammatory cells, LSIL group, HSIL group, and CCC group were 0.25±0.12, 1.05±0.67, 1.39±0.11 and 2.14±0.17, respectively, showing increasing levels of positive expression scores ( P<0.05). In the cervical histology group, the average positive expression scores of PD-L1 were 0.28±0.24, 1.21±0.79, 1.56±0.26, 1.80±0.24, and 2.10±0.19 in the chronic cervicitis tissue, CIN 1 group, CIN 2 group, CIN 3 group and CC group, respectively, showing increasing levels of positive expression scores ( P<0.05). The relative expression of PD-L1 mRNA in chronic cervicitis tissue and CC is 1.02±0.04 and 1.81±0.22, respectively ( P<0.05). ③The sensitivity and specificity of diagnosis of cervical tissue CIN2 and abovelesions, HPV L1 detection alone was 95.8%, 47.2%, PD-L1 detection alone was 96.5%, 32.8%, colposcopy alone was 77.5%, 70.8%, HPV L1/PD-L1 tests combined detection was 92.4%, 64.5%, HPV L1/PD-L1 detection combined colposcopy was 71.6% and 89.6%, respectively. The sensitivity and specificity of the HPV L1/PD-L1 combined test for the diagnosis of CIN3 and above cervical lesions were 71.9% and 86.1%, HPV L1/PD-L1 combined with colposcopy were 50.5% and 100.0%, respectively. The specificity of HPV L1/PD-L1 detection combined with colposcopy for CIN2 and above lesions is higher than that of HPV L1 detection alone, PD-L1 detection alone and colposcopy alone. HPV L1/PD-L1 detection combined with colposcopy detection for CIN3 and above lesions has an important auxiliary diagnostic value.

The Effect of Study-Abroad on Pragmatic Transfer

AbstractThis study aims to investigate the effect that studying abroad may have on pragmatic transfer in requests, refusals, and expressions of gratitude, produced by Japanese learners of English. Twenty-two Japanese college students completed a multimedia elicitation task (MET) before and after studying in the US for one semester, together with twenty-two L1 English speakers and twenty L1 Japanese speakers as baseline data. The MET is a computer-based instrument for eliciting oral data. Unlike previous studies on pragmatic transfer, which often lack statistical evidence, this study includes statistical analysis. The analysis revealed that negative pragmatic transfer occurs within a limited range. The identified transfer includes pragmalinguistic transfer, whereby, assuming that their politeness levels are equal, learners directly translate L1 expressions into L2; and sociopragmatic transfer, whereby learners transfer L1 discourse patterns and functions. Resistance to L2 norms and increased fluency can be influencing factors. The results indicated that the effect of study-abroad is limited because most of the negative transfer which was identified before studying abroad remained after studying abroad. Thus, the necessity of explicit pragmatic instruction was proposed.

Low curcumin concentration enhances the anticancer effect of 5-fluorouracil against colorectal cancer

BackgroundColon cancer treatments include surgery, radiotherapy, and chemotherapy. Chemotherapy using 5-fluorouracil (5-FU) has been widely applied to treat colorectal cancer (CRC). However, it is important to explore the use of chemotherapy drugs in combination with other agents to decrease severe adverse effects. PurposeThis study aimed to investigate the effects of curcumin in combination with 5-FU on the proliferation, migration, and apoptosis of CRC SW620 cell line both in vitro and in vivo. MethodsFlow cytometry was used to study the effect of curcumin on chemotherapy-induced apoptosis in CRC cells. The mechanism of curcumin's enhanced antitumor effect in vivo was investigated using gene knockdown, TUNEL, western blot, qRT-PCR and immunohistochemistry. ResultsThe results showed a synergistic effect of the two compounds on CRC cells. Considerable reduction in the proliferation and migration of SW620 cells was observed in the combination treatment group. Significantly increased apoptosis rate extended the survival of immunodeficient mice in the combination group as compared to that of the 5-FU group (p < 0.05). The results showed that curcumin significantly inhibited pERK signaling and downregulated L1 expression in SW620 cells. ConclusionsWe conclude that curcumin promotes chemosensitivity of CRC cells to 5-FU by downregulating L1 expression. Our findings provide experimental evidence for the synergism between curcumin and 5-FU, which can be utilized in clinical applications for reducing the toxicity and adverse effects of 5-FU.

ROLE OF PD-L1 ASSESSMENT IN THE ASPECT OF MOLECULAR-GENETIC CLASSIFICATION OF COLORECTAL CANCER. CURRENT STATE OF THE PROBLEM

The purpose of the study was to analyze and summarize data regarding a significance of PD -L1 expression in various molecular subtypes of colorectal cancer.Material and Methods. A systemic literature search was conducted in the electronic databases Medline, Cochrane Library, Elibrary, PubMed. Of identified and reviewed 201 full-text articles, we included data from 47 studies.Results. The literature review described the features of the molecular genetic classification of colorectal cancer and revealed the key characteristics of each of the molecular subtypes of this disease. Much attention was paid to the molecular mechanisms of anti-PD -1/PD -L1 therapy. The main problems associated with the standardization of methods for pathomorphological assessment of the expression of this marker and the difficulties of its interpretation in colorectal carcinomas were outlined.Conclusion. Analysis of the literature revealed problems associated with the assessment of PD -L1 expression in colorectal cancer, in particular, with the lack of generally accepted methods for interpreting research results and standardizing methods for pathomorphological diagnosis of malignant tumors of this localization. Further studies are needed for introducing the molecular genetic classification of colorectal carcinomas into a wide clinical practice and personalizing the approach to therapy of this disease.

39800 Immune Checkpoint Blockade during Periprosthetic Joint Infection

ABSTRACT IMPACT: If immune checkpoint blockade increases bacterial clearance with or without antibiotics in vitro, clinical application would be almost immediate and dramatic creating a seismic shift in the current therapeutic paradigm of periprosthetic joint infection. OBJECTIVES/GOALS: Periprosthetic joint infection (PJI) is a major cause of failure after joint replacement. Currently, the treatment of PJI relies on removing biofilm contaminated implants. Some of the bacteria within biofilm undergo a phenotypic shift becoming small colony variants (SCVs). SCVs induce local immunosuppression through PD-1/L1 signaling. METHODS/STUDY POPULATION: We will infect cultured human macrophages and bone marrow aspirate with stable Staphylococcus aureus SVCs and treat with anti-PD-1 or anti-PD-L1 monoclonal antibodies with and without antibiotics (e.g., gentamycin, cefazolin, vancomycin, rifampicin) and assess the residual bacterial viability. We will utilize multiplexed ion beam imaging to quantify PD-1/L1 expression in human tissue from patients with a chronic PJI and compare those to patients undergoing an aseptic revision. Patients with a chronic PJI are likely to have increased expression of PD-1/L1 as their tissue samples are prospectively screened. RESULTS/ANTICIPATED RESULTS: SCVs reduce the phagocytic activity of macrophages and can survive intracellularly. SCVs also induce anti-inflammatory M2-macrophage polarization and recruit a heterogeneous group of immature monocytes and granulocytes called myeloid-derived suppressor cells (MDSC) to the periprosthetic microenvironment. M2-macrophages and MDSCs then produce an immunosuppressive cytokine milieu characterized by increased IL-10 and decreased TNF-α. Clinically isolated SCVs up-regulate the expression of PD-L1 and PD-L2 on the surface of macrophages, representing a mechanism by which SCVs induce host immunosuppression and survive immune clearance. Our preliminary data show PD-L1 expression during septic PJI, but not in aseptic revisions. DISCUSSION/SIGNIFICANCE OF FINDINGS: If immune checkpoint blockade is shown to increase bacterial clearance with or without antibiotics, host immunomodulation would represent a novel class of therapeutic adjuvants to assist surgical debridement and antibiotic administration that could be superimposed on existing treatment algorithms to improve PJI related outcomes.

Amelioration of the abnormal phenotype of a new L1 syndrome mouse mutation with L1 mimetics.

L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.