The First Eight Mitogenomes of Leaf-Mining Dactylispa Beetles (Coleoptera: Chrysomelidae: Cassidinae) Shed New Light on Subgenus Relationships

The taxonomic classification of Dactylispa, a large genus of leaf-mining beetles, is problematic because it is currently based on morphology alone. Here, the first eight mitochondrial genomes of Dactylispa species, which were used to construct the first molecular phylogenies of this genus, are reported. The lengths of the eight mitogenomes range from 17,189 bp to 20,363 bp. All of the mitochondrial genomes include 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 1 A + T-rich region. According to the nonsynonymous/synonymous mutation ratio (Ka/Ks) of all PCGs, the highest and the lowest evolutionary rates were found for atp8 and cox1, respectively, which is a common phenomenon among animals. According to relative synonymous codon usage, UUA(L) has the highest frequency. With two Gonophorini species as the outgroup, mitogenome-based phylogenetic trees of the eight Dactylispa species were constructed using maximum likelihood (ML) and Bayesian inference (BI) methods based on the PCGs, tRNAs, and rRNAs. Two DNA-based phylogenomic inferences and one protein-based phylogenomic inference support the delimitation of the subgenera Dactylispa s. str. and Platypriella as proposed in the system of Chen et al. (1986). However, the subgenus Triplispa is not recovered as monophyletic. The placement of Triplispa species requires further verification and testing with more species. We also found that both adult body shape and host plant relationship might explain the subgeneric relationships among Dactylispa beetles to a certain degree.

PATH-33. MOLECULAR TYPING AND MUTATION SPECTRUM OF CHINESE CHILDREN WITH MEDULLOBLASTOMA WERE IDENTIFIED BY NEXT-GENERATION SEQUENCING

There are few reports on molecular typing and mutation spectrum of Chinese children with medulloblastoma. Here, we aimed to update the clinical manifestations and MB transcriptional mutation spectrum in Chinese Mb patients. Medical records of 59 Mb patients were reviewed. Genomic DNA was extracted from pathological tissues of children. Mutation analysis of whole coding regions, promoter regions and flanking splice sites in whole genome sequencing was performed. Nine cases (15%)with WNT subtype: CTNNB1 mutation was found in all specimens, and TP53 mutation was found in 33.3%. DDX3X mutation occurred in 33.3%. 33.3% had SMARCA4 mutations. Chr6 - associated large fragment deletion occurred in 88.9s. Comparison results between 19 cases (32%) with SHH data and previous studies: The proportion of SHH-TP53 mutants was11% ( 2/19).The proportion of SHH-TP53 wild-type was 89%.The prognosis of the mutated SHH-TP53 was worse than that of the wild-type SHH-TP53, and patient follow-up information could be integrated for comparison. Secondly, PTCH1 is more prevalent in patients with SHH (16%). The DDX3X mutation proportion was 11%. The mutation ratio of Gli2 amplification5%.The mutation ratio of MYCN amplification was 5%.Comparison results of 3 cases with G3 subtype data and previous studies: Variations mainly occur at the chromosomal level. Only one G3 patient had a genetic mutation (TP53 mutation). The proportion of Chr17q+ was 33.3%. CHR7 + proportion was 33.3%. The incidence rates of CHR10Q and CHR11Q were 66.7%.No MYC amplification was found. For 28 cases with G4 subtype: Chr17q +, Chr7 +, Chr18 +, and Chr8 - are the major chromosomal mutations. The incidence rate of CHR17Q + was 61%, and CHR7 + was 61%. CHR8- occurred in 11%. The above results are consistent with previous reports. The Chinese population and other population have similar molecular genotyping gene variation map on medulloblastoma.

Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.

Relationship between DICER1 mutations and immunotherapy biomarkers in solid tumors.

2603 Background: Dicer1 functions as a tumor suppressor in mouse models. In humans, somatic mutations are associated with many cancers in adults, and patients with DICER1 syndrome with DICER1 germline mutations are susceptible to childhood cancers. DICER1 is the core caner-intrinsic CTL-evasion gene, especially positive correlate with innate anti-PD-1 resistance signature or IPRES signature and hERV expression which involved in sensitivity and resistance to ICIs. Nevertheless, the association between mutations in DICER1 and the Chinese patients, the relationship between DICER1 mutations with immunotherapy biomarkers are unknown. Methods: NGS and clinical data were collected from 10953 Western pan-cancer patients (TCGA cohort). A 539-gene panel targeted sequencing assay was performed on FFPE tumor samples from 3514 Chinese pan-cancer patients (Chinese cohort). Both DICER1 mutation ratio and TMB were calculated on the two cohorts following the same criteria.DNA NGS testing (MSI-high vs low/stable (MSS)) in Chinese cohort were included. NGS data of 3514 patients who also detected PD-L1 expression from Chinese clinical dataset were analyzed to explore the association with mutation and PD-L1. The survival information was collected from 1661 pan-cancer patients to analyze the association between DICER1 mutation and efficacy of immunotherapy (MSKCC cohort). Results: In total, 2.91% (319/10953) patients in TCGA harbored DICER1 mutation; in the Chinese cohort, the DICER1 mutation ratio (2.67%, 94/3514) was similar to TCGA. The top 5 mutant DICER1-associated cancer types in Chinese cohort were lung cancer, colon adenocarcinoma, liver cancer, uterine corpus endometrial carcinoma, melanoma. In both cohorts, TMB level of mutation group was significantly higher than wild-type group (p < 0.001). The ration of mutation group in MSI-H (50%) and MSI-L (23.53%) was significantly higher than wild-type group in Chinese cohort (2.17%) (p < 0.001). In addition, the ratio of PD-L1 positive expression (≥1%) in mutation group (48.94%, 46/48) was significantly higher than wild-type in Chinese cohort (38.48%,1316/2104) (p < 0.05). The survival probability of mutation group was significantly longer than wild-type group in immunotherapy. Conclusions: The results indicated that DICER1 mutation was associated with a higher TMB, MSI-H and PD-L1 expression level in Chinese patients. Patients with DICER1 mutations may benefit more from ICIs.

Automated Generation of EQ-Algebras through Genetic Algorithms

This article introduces an approach to the automated generation of special algebras through genetic algorithms. These algorithms can be also used for a broader variety of applications in mathematics. We describe the results of research aiming at automated production of such algebras with the help of evolutionary techniques. Standard approach is not relevant due to the time complexity of the task, which is superexponential. Our research concerning the usage of genetic algorithms enabled the problem to be solvable in reasonable time and we were able to produce finite algebras with special properties called EQ-algebras. EQ-algebras form an alternate truth–value structure for new fuzzy logics. We present the algorithms and special versions of genetic operators suitable for this task. Then we performed experiments with application EQ-Creator are discussed with proper statistical analysis through ANOVA. The genetic approach enables to automatically generate algebras of sufficient extent without superexponential complexity. Our main results include: that elitism is necessary at least for several parent members, a high mutation ratio must be set, optional axioms fulfilment increases computing time significantly, optional properties negatively affect convergence, and colorfulness was defined to prevent trivial solutions (evolution tends to the simplest way of achieving results).

Revisiting neoantigen depletion signal in the untreated cancer genome

This study is arising from Van den Eynden J. et al. Nature Genetics. Lack of detectable neoantigen depletion signals in the untreated cancer genome. Van den Eynden J. et al. tried to address a very important scientific question: could the immune system eliminate cancer cells with immunogenic mutations in untreated situation? Van den Eynden J. et al. first annotated the human exome into “HLA-binding regions” and “non HLA-binding regions” based on the predicted binding affinity of nonapeptides translated from the un-mutated reference coding genome with type I HLA alleles. They hypothesized that if neoantigen depletion signal exist, the nonsynonymous mutations in “HLA-binding regions” will be negatively selected during cancer evolution, while nonsynonymous mutation in “non HLA-binding regions” will not be negatively selected. This will lead to decreased nonsynonymous vs synonymous mutation ratio (n/s) in “HLA-binding regions” compared with “non HLA-binding regions”. They defined HLA-binding mutation ratio (HBMR) as the ratio of n/s in “HLA-binding regions” to “non HLA-binding regions”, and reported that HBMRs are close to 1 in different types of cancer after background corrections, meaning neoantigen depletion signals are not detectable in different types of cancer. The fundamental problem of their hypothesis lies in that the actual neoantigens with immunogenicity do not overlap with their defined “HLA-binding regions”. Actually, most neoantigens with immunogenicity are not located in “HLA-binding regions”, when dissimilarity between mutant and wild type peptide are considered. It is the neoantigen with immunogenicity, but not nonsynonymous mutation in their defined “HLA-binding regions” undergo immunoediting based negative selection. Thus the results reported in that study are fundamentally flawed, and at this current stage we could not draw a solid conclusion as to whether the neoantigen depletion signal exists or not.

Implementasi Honey Bee Mating Optimization Pada Vehicle Routing Problem With Time Windows Dalam Perencanaan Jalur Wisata Malang

Abstrak Vahicle Routing Problem adalah suatu masalah pencaian jalur yang akan dilalui dengan tujuan mencari rute yang paling cepat atau pendek. Vahicle Routing Problem with Time Windows (VRPTW) yang merupakan sebutan bagi VRP dengan kendala tambahan berupa adanya time windows pada masing-masing pelanggan yang dalam hal ini berupa destinasi wisata. Dalam penelitian ini diterapkan Honey Bee Mating Optimization (HBMO) dalam menyelesaikan VRPTW. HBMO sendiri terinspirasi oleh perilaku koloni lebah ketika bereproduksi. Algoritma tersebut bertujuan untuk mengevaluasi pencarian individu atau solusi terbaik. Tujuan dari penelitian ini adalah bagaimana mengimplementasikan Honey Bee Mating Optimization dalam menyelesaikan VRPTW pada perencanaan jalur wisata di Malang. Sehingga dapat meminimumkan waktu dan jarak tempuh perjalanan. Berdasarkan hasil pengujian, parameter yang optimal untuk optimasi VRPTW menggunakan HBMO pada kasus perencannan jalur wisata Malang yaitu dengan menggunakan 800 generasi, populasi lebah jantan sebesar 300, batas kapasitas spermatheca sejumlah 100, nilai mutation ratio (Pm) dan royal jelly masing-masing bernilai 0.5.Abstract Vahicle Routing Problem is a problem of finding the best route that will be passed with the purpose to finding the fastest or shortest route. Vahicle Routing Problem with Time Windows (VRPTW) is a part of VRP with additional obstacles in the form of time windows in each customer. In this research, Honey Bee Mating Optimization (HBMO) was applied to completing VRPTW. HBMO itself was inspired by the behavior of bee colonies when reproducing. The purpose of this algorithm is to evaluate the best individual or the best solutions. The purpose of this research is how to implement Honey Bee Mating Optimization to completing VRPTW in Malang tourism route planning. So that it can minimize travel time and distance. Based on the results of the testing, the optimal parameters for VRPTW optimization using HBMO in Malang tourism route planning case are using 800 generations, the male bee population is 300, the capacity limit of spermatheca is 100, the mutation ratio (Pm) and royal jelly are respectively 0.5.

Ex-Vivo Drug Response Profiling for Tailoring Treatment in Hematologic Malignancies: The Prospective Non-Interventional SMART-Trial

Introduction Ex-vivo drug response profiling (DRP) represents a promising strategy to better understand the tumor's pathway dependencies, genotype-phenotype associations and the underlying molecular networks. However, little is known about the predictive value of drug response profiling and its utility for individualized treatment recommendations. Methods We report the first analysis of our ongoing prospective non-interventional SMARTrial (Systematic and Mechanism-based Approach to Rational Treatment Trial of Blood Cancer; ClinicalTrials.gov: NCT03488641). This trial aims to demonstrate the feasibility of high-throughput DRP in the clinical routine setting and the ability to predict in-vivo outcome by DRP. Ex-vivo responses to 107 different drugs including chemotherapeutic agents and FDA-approved or clinically investigated small molecules were assessed by an ATP-based assay after 48 hours of incubation. Results were processed by our customized analysis pipeline, enabling immediate reporting of DRP results. Patients with hematological malignancies and scheduled treatment were eligible if sufficient tumor cells were available from either lymph node biopsies, the bone marrow or peripheral blood. Results 58 patients out of the target study population of 80 patients have been recruited (AML, n = 24; B-cell lymphomas, n = 28; T-cell-lymphoma, n = 5; ALL, n = 1) as of July 24th, 2019. The median follow-up was 6 months. The primary endpoint, defined as proportion of successfully completed DRP tests within 7 days, is used to evaluate the feasibility of DRP in the clinical routine. In our study cohort, 93% of all recruited patients met this primary endpoint. We will describe our optimized workflow for drug testing and data analysis, which enables us to provide DRP reports in the clinical routine. First, we analyzed if the DRP reflected expected genotype-drug response associations and systematically regressed mutational data on DRP phenotypes. This analysis exemplarily revealed that FLT3-ITD-mutated AML cells with a mutation ratio &gt; 0.8 respond significantly stronger to FLT3 inhibitors than FLT3-ITD-mutated AML cells with a mutation ratio &lt; 0.8 or wildtype AML cells (Quizartinib, t-test, p = 0.022; Gilteritinib, t-test, p &lt; 0.001), confirming that the assay can uncover clinically meaningful vulnerabilities of cancer cells. 89.3% of all evaluable patients received treatments covered by our ex-vivo DRP assay, which facilitates the correlation of in-vivo and ex-vivo drug response. The majority of patients were scheduled for chemotherapy treatment (65%) or had received chemotherapy before (32%). In-vivo responses after scheduled treatment were categorized as response (Resp), incomplete response (IResp) or progressive disease (PD). We compared ex-vivo drug responses between these different in-vivo response categories. Cancer cells of patients with PD were significantly less sensitive to ex-vivo treatment with chemotherapy (mean of all ex-vivo tested chemotherapeutics) than patients with Resp (t-test, PD vs. Resp, p = 0.007; PD vs. IResp, p = 0.054). While the anthracycline daunorubicin (t-test, p = 0.034) and the purin analogon fludarabine (t-test, p = 0.004) were significantly more active in samples from patients with a clinical Resp than in samples from patients with PD, we did not observe this trend for vinca alkaloids (e.g. vincristine, vindesine) or antifolates (e.g. pralatrexate). Interestingly, individual patients exhibited an exceptional ex-vivo sensitivity to anti-folates, despite an almost complete insensitivity to all other chemotherapeutic drugs. For a patient with refractory Burkitt Lymphoma, we observed such a strong ex-vivo sensitivity to pralatrexate despite a general ex-vivo insensitivity to most drugs. This patient had progressed on treatment with DA-EPOCH and DHAP, but responded well to methotrexate, enabling him to undergo consolidating allogeneic stem cell transplantation. Conclusion Ex-vivo drug response profiling prior to treatment can be successfully implemented in the clinical routine. Our preliminary results suggest that DRP can predict in-vivo treatment resistance and support treatment guidance for individualized treatment. Disclosures Dreger: AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy.

Heterogeneity of GNAQ/11 mutation inversely correlates with the metastatic rate in uveal melanoma

PurposeTo determine whether the GNAQ/11 mutation correlated with the outcome of patients with uveal melanoma (UM) when genetic heterogeneity was considered.MethodsWe performed a retrospective study of sixty-seven patients with UM. The heterogeneity of GNAQ/11 was examined by using droplet digital PCR. The correlation between metastasis and heterogeneity of the GNAQ/11 mutation was analysed. Disease free survival curves were constructed using the Kaplan-Meier method, and the Wilcoxon log-rank test was used to compare the curves.ResultsThe GNAQ/11 mutation ratio was varied between each case. Among these patients, 28.35% of them harboured homogeneous mutation of GNAQ/11, 62.69% present heterogeneous mutation and 8.96% didn’t present either GNAQ or GNA11 mutation. The tumour with heterogeneous mutation of GNAQ/11 has a higher metastatic rate than that with homogeneous mutation (13/29 vs 1/18, p=0.027). In Kaplan-Meier analysis, metastasis-free survival was not significantly associated with either homogeneous or heterogeneous mutation of GNAQ/11.ConclusionThe mutation ratio of GNAQ/11 in UM was quite variable. The tumour with heterogeneous mutation of GNAQ/11 is more likely to develop a poor prognosis than that with homogeneous mutation of GNAQ/11.

Mitochondrial Transfer from Wharton’s Jelly Mesenchymal Stem Cell to MERRF Cybrid Reduces Oxidative Stress and Improves Mitochondrial Bioenergetics

Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a maternally inherited mitochondrial disease affecting neuromuscular functions. Mt.8344A>G mutation in mitochondrial DNA (mtDNA) is the most common cause of MERRF syndrome and has been linked to an increase in reactive oxygen species (ROS) level and oxidative stress, as well as impaired mitochondrial bioenergetics. Here, we tested whether WJMSC has therapeutic potential for the treatment of MERRF syndrome through the transfer of mitochondria. The MERRF cybrid cells exhibited a high mt.8344A>G mutation ratio, enhanced ROS level and oxidative damage, impaired mitochondrial bioenergetics, defected mitochondria-dependent viability, exhibited an imbalance of mitochondrial dynamics, and are susceptible to apoptotic stress. Coculture experiments revealed that mitochondria were intercellularly conducted from the WJMSC to the MERRF cybrid. Furthermore, WJMSC transferred mitochondria exclusively to cells with defective mitochondria but not to cells with normal mitochondria. MERRF cybrid following WJMSC coculture (MF+WJ) demonstrated improvement of mt.8344A>G mutation ratio, ROS level, oxidative damage, mitochondrial bioenergetics, mitochondria-dependent viability, balance of mitochondrial dynamics, and resistance against apoptotic stress. WJMSC-derived mitochondrial transfer and its therapeutic effect were noted to be blocked by F-actin depolymerizing agent cytochalasin B. Collectively, the WJMSC ability to rescue cells with defective mitochondrial function through donating healthy mitochondria may lead to new insights into the development of more efficient strategies to treat diseases related to mitochondrial dysfunction.