Expression of PD-L1 and PD-L2 in colorectal cancer (CRC): A post-hoc integrated analysis of SCRUM-Japan GI-SCREEN CRC.

120 Background: PD-1, PD-L1/L2 axis is responsible for cancer immune escape which facilitate disease progression. However, expression of these proteins in CRC and its significance in clinical prognosis are yet to be fully clarified. Methods: This was a post-hoc analysis using FFPE tumor samples from the Nationwide Cancer Genome Screening Project, SCRUM-Japan GI-SCREEN for metastatic CRC. Patients (Pts) with MSI-H or BRAF V600E mutant tumors were prioritized to be included. PD-L1 (22C3) and PD-L2 expressions were centrally assessed using immunohistochemical assays at QualTek and NeoGenomics, respectively. Tumor infiltrating lymphocytes (TILs) were morphologically evaluated by H&E staining. Clinical information including biomarker status and overall survival (OS) were extracted from SCRUM-Japan GI-SCREEN database. Results: In total, 200 pts were included in this study with a median age of 65 (range, 29–88) years; male/female (116/84); MSI status MSI-H/non-MSI-H/unknown (8/189/3); RAS wild-type/mutant (113/87); and BRAF V600E wild-type/mutant (173/27). Positivity rate of PD-L1 in tumor cells (TC), immune cells (IC), PD-L2 on TC and IC were 10.8%, 46.9%, 0% and 20.3%, respectively. Expression of PD-L1 on TC or IC and PD-L2 on IC were associated with TIL (p = 0.023, p = 0.009, respectively). PD-L1 on TC was highly seen in BRAF V600E-mutated tumors (p = 0.043), but not related to other factors including RAS and MSI status. The pts with PD-L1+ tumors on TC or IC had longer OS than those with PD-L1- (Median, 31.9 vs 23.5 months; HR = 0.67, 95% CI, 0.46–0.99; p = 0.040), while OS in pts with PD-L2+ tumors was similar to that with PD-L2- (Median, 21.4 vs 27.3 months; HR = 0.87, 95% CI, 0.53–1.44; p = 0.60). The PD-L1 expression was also associated with the longer OS in pts with BRAF wild-type or non-MSI-H tumors. Conclusions: Our study suggested different prognostic impact of PD-L1 and PD-L2 expression in CRC pts, which require further evaluations as potential therapeutic targets for CRC. Clinical trial information: UMIN000016343.

Aryl Hydrocarbon Receptor (AHR) Ligands as Selective AHR Modulators (SAhRMs)

The aryl hydrocarbon receptor (AhR) was first identified as the intracellular protein that bound and mediated the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and dioxin-like compounds (DLCs). Subsequent studies show that the AhR plays an important role in maintaining cellular homeostasis and in pathophysiology, and there is increasing evidence that the AhR is an important drug target. The AhR binds structurally diverse compounds, including pharmaceuticals, phytochemicals and endogenous biochemicals, some of which may serve as endogenous ligands. Classification of DLCs and non-DLCs based on their persistence (metabolism), toxicities, binding to wild-type/mutant AhR and structural similarities have been reported. This review provides data suggesting that ligands for the AhR are selective AhR modulators (SAhRMs) that exhibit tissue/cell-specific AhR agonist and antagonist activities, and that their functional diversity is similar to selective receptor modulators that target steroid hormone and other nuclear receptors.

Analysis of biosynthesis and composition of cuticular wax in wild type bilberry (Vaccinium myrtillus L.) and its glossy mutant

Cuticular wax plays an important role in fruits in protection against environmental stresses and desiccation. In this study, biosynthesis and chemical composition of cuticular wax in wild type (WT) bilberry fruit was studied during development and compared with its natural glossy type (GT) mutant. The cuticular wax load in GT fruit was comparable to WT fruit. In both fruits, triterpenoids were the dominant wax compounds with decreasing proportion during the fruit development accompanied with increasing proportion of aliphatic compounds. Gene expression studies supported the pattern of compound accumulation during fruit development. Genes CER26-like, FAR2, CER3-like, LTP, MIXTA, and BAS exhibited prevalent expression in fruit skin indicating role in cuticular wax biosynthesis and secretion. In GT fruit, higher proportion of triterpenoids in cuticular wax was accompanied by lower proportion of fatty acids and ketones compared to WT fruit as well as lower density of crystalloid structures on berry surface. Our results suggest that a marked reduction in ketones in cuticular wax may play a significant role in the formation of glossy phenotype leading to the loss of rod-like structures in epicuticular wax layer of GT fruit.HighlightChemical composition and morphology of cuticular wax along with gene expression for wax biosynthetic genes varied between glossy type mutant (GT) and wild type (WT) fruit.

Single-tube genotyping for small insertion/deletion mutations: simultaneous identification of wild type, mutant and heterozygous alleles

Current methods of genotyping small insertion/deletion (indel) mutations are costly, laborious, and can be unreliable. To address this, we have developed a method for small indel genotyping in a single polymerase chain reaction, with wild-type, heterozygous and mutant alleles distinguishable by band pattern in routine agarose gel electrophoresis. We demonstrate this method with multiple genes to distinguish 10 bp, 4 bp and even 1 bp deletions from the wild type. Through systematic testing of numerous primer designs, we also propose guidelines for genotyping small indel mutations. Our method provides a convenient approach to genotyping small indels derived from clustered regularly interspaced short palindromic repeats-mediated gene editing, N-ethyl-N-nitrosourea induced mutagenesis or diagnosis of naturally occurring polymorphisms/mutations.

Insights into the Effects of Cancer Associated Mutations at the UPF2 and ATP-Binding Sites of NMD Master Regulator: UPF1

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that recognizes post-transcriptionally abnormal transcripts and mediates their degradation. The master regulator of NMD is UPF1, an enzyme with intrinsic ATPase and helicase activities. The cancer genomic sequencing data has identified frequently mutated residues in the CH-domain and ATP-binding site of UPF1. In silico screening of UPF1 stability change as a function over 41 cancer mutations has identified five variants with significant effects: K164R, R253W, T499M, E637K, and E833K. To explore the effects of these mutations on the associated energy landscape of UPF1, molecular dynamics simulations (MDS) were performed. MDS identified stable H-bonds between residues S152, S203, S205, Q230/R703, and UPF2/AMPPNP, and suggest that phosphorylation of Serine residues may control UPF1-UPF2 binding. Moreover, the alleles K164R and R253W in the CH-domain improved UPF1-UPF2 binding. In addition, E637K and E833K alleles exhibited improved UPF1-AMPPNP binding compared to the T499M variant; the lower binding is predicted from hindrance caused by the side-chain of T499M to the docking of the tri-phosphate moiety (AMPPNP) into the substrate site. The dynamics of wild-type/mutant systems highlights the flexible nature of the ATP-binding region in UPF1. These insights can facilitate the development of drug discovery strategies for manipulating NMD signaling in cell systems using chemical tools.

beta-Glucan Increases IFN-gamma and IL-12 Production of Peripheral Blood Mononuclear Cells with/without Induction of Mycobacterium tuberculosis Wild-type/Mutant DNA

BACKGROUND: In tuberculosis infections, the immune system is weakened and cannot produce enough cytokines to against the infection. b-glucan is a potent immunomodulator that induces cytokine production in various bacterial infections. This study aimed to determine the effects of b-glucan on the production of interferon (IFN)-γ and interleukin (IL)-12 in peripheral blood mononuclear cells (PBMCs) induced by Mycobacterium tuberculosis DNA.METHODS: PBMCs were isolated from 11 healthy subjects. PBMCs were treated with/without 5 μg/mL b-glucan and M. tuberculosis rpoB wild-type or mutant DNA. The production of IFN-γ and IL-12 in the supernatant was performed with enzyme-linked immune-sorbent assay (ELISA).RESULTS: b-glucan increased significantly (p<0.05) IFN-γ of M. tuberculosis mutant DNA-induced PBMCs, M. tuberculosis wild-type DNA-induced PBMCs, and non-induced PBMCs. b-glucan also increased significantly (p<0.05) IL-12 of M. tuberculosis mutant DNA-induced PBMCs, M. tuberculosis wild-type DNA-induced PBMCs, and non-induced PBMCs. There were not any significant difference between male and female groups for IL-12 and IFN-γ in all treatment groups (p>0.05, ANOVA test).CONCLUSION: This in vitro study indicates that b-glucan increases the performance of PBMCs to produce IFN-γ and IL-12, with/without induction of M. tuberculosis wild-type/ mutant DNA.KEYWORDS: b-glucan, IFN-γ, IL-12, M. tuberculosis, rpoB

PRODUKSI LIPASE DARI ISOLAT KAPANG HASIL MUTASI UNTUK TRANSESTERIFIKASI

Lipase Production by Mutant Fungal Isolates for Transesterification ABSTRACTLipase is used amongst others in biodiesel production, namely in the transesterification reaction. Kernel B (KB) was a fungus isolated from the waste of palm kernel and seed. The fungus produced lipase that catalysed the transesterification reaction with a lower activity compared to that of AK Amano commercial lipase. The purpose of this study was to obtain mutant fungi with higher transesterification activities than the wild type (KB). The mutation process was carried out using ultraviolet (UV) light, ethyl methane sulfonate (EMS), and N-methyl-N’-nitro-N-nitrosoguanidine (NMNG) on KB fungus. The mutations using UV light produced 11 isolates, of which isolate m4.1KB1 produced a higher transesterification activity (0.172 U·mg-1) compared to the wild type. Mutant m5.7KB, which was generated from mutant m4.1KB1 treated using EMS, had its transesterification activity decreased to only 0.051 U·mg-1. Mutant m6.0,3KB2, which was resulted through NMNG treatment, experienced an increase in transesterification activity which was 91.2% higher than that of KB.Keywords: ethyl methane sulfonate, lipase, mutant fungi, N-methyl-N’-nitro-N-nitrosoguanidine, ultraviolet ABSTRAKLipase dimanfaatkan salah satunya dalam produksi biodiesel, yaitu dalam reaksi transesterifikasi. Kernel B (KB) merupakan kapang yang diisolasi dari limbah inti dan biji kelapa sawit, yang menghasilkan lipase sebagai katalis dalam reaksi transesterifikasi. Namun aktivitas transesterifikasi yang dihasilkan oleh lipase dari KB lebih rendah dibandingkan dengan lipase komersial AK Amano. Tujuan penelitian ini adalah mendapatkan mutan kapang dengan aktivitas transesterifikasi yang lebih tinggi dibandingkan tipe liarnya (KB). Proses mutasi dilakukan dengan menggunakan sinar ultraviolet (UV), ethyl methane sulfonate (EMS), dan N-methyl-N’-nitro-N-nitrosoguanidine (NMNG) terhadap kapang KB. Mutasi KB dengan menggunakan sinar UV menghasilkan 11 isolat, dimana isolat dengan kode m4.1KB1 menghasilkan aktivitas transesterifikasi yang lebih tinggi dibandingkan tipe liar, yaitu 0,172 U·mg-1. Mutan m5.7KB, yang dihasilkan dari mutan m4.1KB1 dengan perlakuan EMS, mengalami penurunan aktivitas transesterifikasi hingga hanya sebesar 0,051 U·mg-1. Mutan m6.0,3KB2 hasil perlakuan NMNG mengalami peningkatan aktivitas transesterifikasi sebesar 91,2% lebih tinggi dari KB.Kata Kunci: ethyl methane sulfonate, kapang mutan, lipase, N-methyl-N’-nitro-N-nitrosoguanidine, ultraviolet

Clinicopathological features, efficacy of anti-EGFR therapy, and survival outcomes in patients with BRAF non-V600 mutated metastatic colorectal cancer.

659 Background: BRAF non-V600 mutations occur in 2-3% of colorectal cancer. These mutants can be classified as RAS independent (class 2) or RAS dependent (class 3). We reported BRAF non-V600 mutations could be a negative predictive factor for anti-EGFR therapy in patients (pts) with pretreated metastatic colorectal cancer (mCRC), while mCRC pts with class 3 BRAF mutations could respond to anti-EGFR therapy because of its dependency on receptors and RAS. Methods: This study evaluates the efficacy to anti-EGFR therapy in a large cohort of pts with BRAF non-V600 mutated mCRC. Pts with mCRC referred to the participating centers from 2010 to 2017 were included. Clinicopathological features, efficacy of anti-EGFR therapy, and survival outcomes were stratified by BRAF mutational class. Results: One hundred seventeen pts with BRAF non-V600 mutated mCRC were identified. Median age was 58 years (range, 27-83), 68 pts (58%) were male, and 38 pts (33%) had right-sided tumors. Mucinous histology was seen in 11 cases (9%); concurrent RAS mutations occurred in 31 cases (27%), and 3 cases (3%) were MSI-H. Also, TP53 mutations were detected in 74 pts among 90 analyzed cases (82%). Regarding BRAF mutation subtype, 25/63/29 pts were classified as class 2/3/not reported (NR), respectively. Median OS in RAS wild-type/mutant were 44.8/34.6 months, respectively (p=0.082). The median OS in RAS wild-type pts with BRAF non-V600 mutations for class 2, 3, and NR were 25.7, 44.2, and 79.1 months, respectively (class 2 vs. 3, p=0.219). Among 40 pts treated with anti-EGFR therapy, response rates were 14%, 44%, and 40% for class 2, 3, and NR, respectively. Median PFS was 4.4, 8.3, 4.0 months for class 2, 3, and NR, respectively. Moreover, in 25 pts receiving anti-EGFR therapy as third or later line, response rate was 0%, 27%, and 50% in class 2, 3, and NR, and median PFS was 2.8, 3.7, and 4.0 months (p=0.762), respectively. Conclusions: Pts with class 2 BRAF mutations tend to have a poor prognosis compared to those with class 3 mutations. While almost half of pts with class 3 BRAF mutations responded to anti-EGFR therapy, response was rare for pts with class 2 BRAF mutations, and none achieved objective response in the third or later line.