Multivariate Analysis of Associations between Clinical Sequencing and Outcome in Glioblastoma

Background Many factors impact survival in patients with glioblastoma, including age, Karnofsky Performance Status, post-operative chemoradiation, IDH1/2 mutation status, MGMT promoter methylation status, and extent of resection. High-throughput next generation sequencing is a widely available diagnostic tool, but the independent impact of tumors harboring specific mutant genes on survival and the efficacy of extent of resection are not clear. Methods We utilized a widely available diagnostic platform (FoundationOne CDx) to perform high-throughput next generation sequencing on 185 patients with newly diagnosed glioblastoma in our tertiary care center. We performed multivariate analysis to control for clinical parameters with known impact on survival to elucidate the independent prognostic value of prevalent mutant genes and the independent impact of gross total resection. Results When controlling for factors with known prognostic significance including IDH1/2 mutation and after multiple comparisons analysis, CDKN2B and EGFR mutations were associated with reduced overall survival while PTEN mutation was associated with improved overall survival. Gross total resection, compared to other extent of resection, was associated with improved overall survival in patients with tumors harboring mutations in CDKN2A, CDKN2B, EGFR, PTEN, TERT promoter, and TP53. All patients possessed at least one of these six mutant genes. Conclusions This study verifies the independent prognostic value of several mutant genes in glioblastoma. Six commonly found mutant genes were associated with improved survival when gross total resection was achieved. Thus, even when accounting for known predictors of survival and multiple mutant gene comparisons, extent of resection continues to be strongly associated with survival.

Monitoring of RAS mutant clones in plasma of patients with RAS mutant metastatic colorectal cancer

Purpose Some patients with histologically confirmed primary mCRC and mutated RAS reported undetectable RAS mutant clones in plasma after receiving anti-VEGF treatment. The aim was to prospectively assess it with its potential therapeutic implications. Methods RAS mutant genes in solid biopsy (before first-line treatment: FOLFOX/CAPOX + bevacizumab) were compared in liquid biopsy (before second-line treatment: panitumumab + FOLFIRI), using Idylla™ system. Discordant results between solid/liquid biopsies were assessed by the next-generation sequencing (NGS) test (solid/liquid biopsies). Results Twenty-three patients were assessed (seven had RAS mutant discrepancies between solid/liquid biopsies). The NGS test confirmed that 3/23 (13%) patients had undetectable RAS mutant clones in liquid biopsy and 3/23 (13%) presented discrepancies in solid biopsy (Idylla™ system vs. NGS test). Conclusion Thirteen percentage of patients had undetectable RAS mutant clones in liquid biopsy after first-line treatment. However, some discrepancies between solid and liquid biopsies have been observed. These results suggest a need to improve accuracy of RAS analyses, especially in solid biopsies.

Molecular docking studies of potential anticancer agents from Thunbergia Fragrans against Colorectal cancer mutant genes through In silico study

Colorectal cancer (CRC) is one of the deadly diseases which incidence rate will increase every year due to people lifestyle and food habit etc., Moreover, people’s required a new therapeutic molecule to resolve this problem. Therefore plant-based chemical constituents are the best option due to the low side effects, easy availability and cost-effective manner. The flowering plant of Thunbergia fragrans Roxb belongs to the Acanthaceae family has a vast range of medicinal properties, anticancer activity is one among them. Thunbergia fragrans has reported to had chemical constituents of Palmitic acid, Cis-9-Hexadecenal and Campesterol which possess anticancer activity. For the beginning of TF chemical constituents were studied against the Colorectal cancer (CRC) mutant genes such as NRAS (PDB ID: 6ZIZ), Beta-Catenin (PDB ID: 6M93) – Oncogenes; APC (PDB ID: 3NMX), Smad2 (PDB ID: 1KHU) – Tumor Suppressor genes through insilico docking studies. AutoDock 4.2 tool was used to predict the interaction between ligand and receptor, Binding energy and Bond specification in a 3D space. Finally, the results revealed TF chemical constituents showed excellent binding energy against CRC mutant genes such as Palmitic acid against Beta-Catenin (-4.75) and APC (-4.01), Cis-9-Hexadecenal against NRAS (-1.92), Beta-Catenin (-3.96) and APC (-4.41), Campesterol against Beta-Catenin (-8.55) and APC (-8.85) respectively.

Broad spectrum of CRISPR-induced edits in an embryonic lethal gene

Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse. While CRISPR-based gene editing of multiple genes at once offers an attractive alternative strategy, subsequent breeding or establishment of permanent mouse lines will rapidly segregate the different mutant loci again. Direct phenotypic analysis of genomic edits in an embryonic lethal gene in F0 generation mice, or F0 mouse embryos, circumvents the need for breeding or establishment of mutant mouse lines. In the course of genotyping a large cohort of F0 CRISPants, where the embryonic lethal gene T/brachyury was targeted, we noted the presence of multiple CRISPR-induced modifications in individual embryos. Using long-read single-molecule Nanopore sequencing, we identified a wide variety of deletions, ranging up to 3 kb, that would not have been detected or scored as wildtype with commonly used genotyping methods that rely on subcloning and short-read or Sanger sequencing. Long-read sequencing results were crucial for accurate genotype–phenotype correlation in our F0 CRISPants. We thus demonstrate feasibility of screening manipulated F0 embryos for mid-gestation phenotypic consequences of CRISPR-induced mutations without requiring derivation of permanent mouse lines.

Sensitivity to tumor development by TALEN-mediated Trp53 mutant genes in the susceptible FVB/N mice and the resistance C57BL/6 mice

Background This study was undertaken to compare the sensitivities of mice strains during tumor induction by transcription activator-like effector nucleases (TALEN)-mediated Trp53 mutant gene. Alterations of their tumorigenic phenotypes including survival rate, tumor formation and tumor spectrum, were assessed in FVB/N-Trp53em2Hwl/Korl and C57BL/6-Trp53em1Hwl/Korl knockout (KO) mice over 16 weeks. Results Most of the physiological phenotypes factors were observed to be higher in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice, although there were significant differences in the body weight, immune organ weight, number of red blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total bilirubin (Bil-T) and glucose (Glu) levels in the KO mice relative to the wild type (WT) mice. Furthermore, numerous solid tumors were also observed in various regions of the surface skin of FVB/N-Trp53em2Hwl/Korl KO mice, but were not detected in C57BL/6-Trp53em1Hwl/Korl KO mice. The most frequently observed tumor in both the Trp53 KO mice was malignant lymphoma, while soft tissue teratomas and hemangiosarcomas were only detected in the FVB/N-Trp53em2Hwl/Korl KO mice. Conclusions Our results indicate that the spectrum and incidence of tumors induced by the TALEN-mediated Trp53 mutant gene is greater in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice over 16 weeks.

Distribution of Pfmdr 1 and Kelch 13 Genes Among the Children 5 Years and Below Attending a Secondary Health Facility, South-West, Nigeria.

Background: Malaria is a major public health concern in some part of the world especially in the tropical Africa where children are more vulnerable. The occurrence of resistant gene in Plasmodium falciparum to some antimalarial drugs could increase the malaria morbidity and mortality among the children. The study evaluates the distribution of P. falciparum resistant kelch protein gene on chromosome 13 (PfKelch 13) and multidrug resistant (Pfmdr1) mutant genes among children aged five years and below who attended Mother and Child Hospital, Akure, Nigeria. Methods: Thin and thick smears were prepared from the blood collected aseptically through venepuncture from five hundred (500) children (age 5years and below). Two hundred (200) malaria positive samples were randomly selected from the 500 samples for PCR analysis to detect Pfmdr1 and Kelch 13 mutant genes from the positive samples. Discussion: The results showed that of the 500 respondents who gave their consent to participate in the study, 288 (57.6%) were males while 212 (42.4%) were females. The distribution of Pfmdr1 are; mixed group (mutant/wild) 38.5% (77/200), mutant gene 35.5% (71/200), wild gene 20.5% (41/200) and the resistant genes were absent in 5.5% (11/200) of the infected children. The mixed group of Pfmdr1 gene was higher among infants (51.9%), male (44.3%), children with birth order 4 (60.0%) and children that have blood group B (51.3%), however, there is no significant difference in the distribution of Pfmdr1 between gender (χ2 = 0.634, df = 1, p>0.05). There was a point mutation in the codon position 557 where the amino acid Alanine was replaced by Serine in the PfK13. The research revealed high prevalence of Pfmdr1 mutant genes and point mutation in the PfK13 gene of P. falciparum among children which may be as a result of treatment of malaria with different antimalarial drugs which the parasite has developed resistance against. It is therefore important to administer other malaria drugs apart from the drugs the parasite has developed resistance against.

High Frequency Mutant Genes in Urothelial Carcinoma Based On Genomic Landscape

ObjectiveThe new generation of sequencing technology has been applied to the study of genomic genetic characteristics of urothelial carcinoma for 20 years. Researchers at home and abroad have done a lot of research work. Analyzing and summarizing the research results, we can clarify the genes with high-frequency mutations, which is of great significance for the screening of biomarkers and molecular targets of urothelial carcinoma.Method We will adopt the PICOS analysis method of evidence-based medicine; follow the principles of systematic evaluation and meta-analysis; formulate literature retrieval keywords and retrieval strategies; determine the inclusion criteria; and statistically analyze the name, mutation frequency, quantity, and the total number of times in repeated reports of significant mutant genes in the genomic landscape.Results A total of 6254 cases of urothelial carcinoma were sequenced in the 27 theses selected. Sequencing methods include whole genome sequencing, whole exome sequencing, and target exome sequencing. 27 genomic landscapes of urothelial carcinoma showed that the number of significant mutant genes was 5-58, with an average of 26 reported in each paper. There were 273 genes with significant mutations in urothelial carcinoma, 65.57% (179 / 273) of which were reported only once and 34.43% (94 / 273) were reported more than twice. The top 7 genes most frequently reported were TP53, PIK3CA, FGFR3, KDM6A, ARID1A , RB1 and STAG2.Conclusion There were 273 genes with significant mutations in the genome of urothelial carcinoma, and biomarkers may be selected from 94 genes with high-frequency of mutations.

Exploring Somatic Alteration Associating With Aggressive Behaviors of Papillary Thyroid Carcinomas by Targeted Sequencing

Wisely differentiating high-risk papillary thyroid carcinoma (PTC) patients from low-risk PTC patients preoperatively is necessary when comes to making a personalized treatment plan. It is not easy to stratify the risk of patients according to sonography or lab results before surgery. This study aims to seek out potential mutation gene markers that may be helpful in stratifying the risk of PTC. A custom panel of 439 PTC relevant and classic tumor metabolic pathway relevant genes was designed. Targeted capture sequencing was performed on 35 pairs of samples from 35 PTC tumors and 35 para-tumor thyroid tissues obtained during surgery. Variant calling and detection of cancer gene mutations were identified by bio-information analysis. Ingenuity Pathway Analysis (IPA) was performed to do functional enrichment analysis of high-frequency mutant genes. Immunohistochemistry (IHC) was performed on 6 PTC patients to explore the expression of protein associated with interested genes. Event-free survival (EFS) was calculated to determine which genes might affect the prognosis of patients. We have identified 32 high-frequency mutant genes in PTC including BRAF. RBL2 was found to be significantly correlated to event-free survival, FOXO1, MUC6, PCDHB9, NOTCH1, FIZ1, and RTN1 were significantly associated with EFS, while BRAF mutant was not correlated to any of the prognosis indicators. Our findings in this study might open more choices when designing thyroid gene panels used in FNA samples to diagnose PTC and predict the potentially aggressive behavior of PTC.