Mutant Allele Frequency Research Articles

342.3: Outcome Analysis of Patients With Genetic Mutations of Complement Regulatory Proteins in Relation to Incidence of Post Renal Transplant Thrombotic Microangiopathy

Introduction: Thrombotic Microangiopathy is an occlusive disorder of microvasculature characterized by thrombocytopenia, systemic or intrarenal platelet aggregation, thrombus formation and hemolytic anemia caused by fragmentation of erythrocytes due to the disturbed microvasculature. TMA developing after renal transplantation can be categorized into either De novo TMA, developing for the first time without any evidence of the disease before the transplant or recurrent TMA where native kidneys have failed due to TMA and recur after kidney transplantation. The contribution of complement regulatory gene (CRG) mutation to post-transplant Thrombotic Microangiopathy (TMA) in Indian kidney transplant recipients (KTR) is unknown. This is a study to determine the association of CRG on post-transplant TMA and graft failure. Methods: A Single Centre, Prospective Observational Cohort Study was undertaken in renal transplant recipients. All live and Deceased donor renal allograft recipients who underwent transplantation from 1st December 2019 onwards till 31st December 2020 were recruited and were followed up for 1 year beyond the last enrolment. Genetic testing was performed with MLPA and Clinical exome sequencing. Results: 78 KTRs with a mean age of 37.09 ± 13.23 years were enrolled. The Native cause of kidney disease in 55 (70.5%) patients was undetermined and 23 (29.5%) had established cause of chronic kidney disease. 58 (74.4%) were males and 20 (25.6%) were females. CRG mutation was seen in 48/78 (61.5%) KTRs. The allele frequency of the CRG mutation was seen in 48/78 (61.53%) of which duplication of CFHR gene was predominantly seen in 40/78 (51.2%) patients. CRG mutation in this cohort was seen more in the younger age group patients. There was no gender predominance for patients with CRG mutation. None of the patients had a mutation for CFH, CFI, CFB, C3, THBD, DGKE genes. One patient had a mutation for the PLG gene. The incidence of post-transplant TMA was seen in 4/78 (5.12%) patients. Mutation related to aHUS was seen in 2 patients. There was no significant difference in serum creatinine levels of patients with or without CRG mutation at the end of 1 year. Conclusion: The prevalence of CRG mutation in our KTR cohort was high and is associated with post-transplant TMA in 50% of patients with de novo TMA. The graft outcome at the end of 1 year was similar in patients with mutation and those without mutation. This study suggests that the presence of CRG mutation should not preclude renal transplantation in patients with ESRD.

1145P Atezolizumab and stereotactic body radiotherapy in patients with advanced non-small cell lung cancer: The ComIT-1 trial

The emerging use of immune checkpoint inhibitors has transformed the treatment landscape of metastatic non-small cell lung cancer (NSCLC). However, challenges remain to increase the number of patients achieving durable clinical responses to these drugs, and to help monitor the treatment effect. In this phase II trial, we investigated the toxicity, systemic responses and circulating tumor DNA (ctDNA) responses in patients with advanced NSCLC treated with atezolizumab and stereotactic body radiotherapy (SBRT). Patients with metastatic NSCLC previously treated with a platinum doublet were included. SBRT (3 doses of 6 Gy) was given with concurrent atezolizumab 1200 mg every three weeks until no clinical benefit, intolerable toxicity, or a maximum of two years. Safety and clinical responses were registered. Plasma collected at baseline, during treatment and at progression was analyzed by next-generation sequencing using a 52-gene panel. We examined the relationship between longitudinal changes in ctDNA levels quantified using the allele frequency (AF) of lung cancer-associated somatic mutations, and radiographic responses for each patient. 21 patients were enrolled, of whom 15 with programmed death-ligand 1 (PD-L1) negative tumors. Grade 3 treatment-related adverse events were reported in three patients, leading to permanent discontinuation of treatment in one. No patients experienced grade 4–5 toxicity. Median overall survival time was still not reached at data cut-off. As best overall response, four patients had a partial response (PR), eight had stable disease (SD) and five had progressive disease (PD). Eight patients, including six with PD-L1 negative tumors, were on treatment for more than 12 months. ctDNA was detectable at baseline in 11 plasma samples. A rapid decline in ctDNA to <30% of baseline levels was seen for three patients, two of them radiographic responders and one considered clinically benefiting from therapy for almost one year. Atezolizumab was safely administered concomitant with SBRT, but further trials are needed to assess the efficacy of this combination. ctDNA holds potential as a dynamic biomarker in lung cancer patients receiving immune checkpoint inhibitors.

1706P Artificial intelligence-powered tumor purity assessment from H&E whole slide images associates with variant allele frequency of somatic mutations across 23 cancer types in TCGA cohorts

Precise tumor purity assessment is a crucial preparation step to determine target sequencing depth to detect oncogenic variants from tissue samples. Moreover, knowing tumor purity improves somatic variant calling during post-sequencing analysis. We developed an artificial intelligence (AI)-powered model to quantify tumor purity from whole slide images (AI-P), named Lunit SCOPE TP, to address these challenges. In this study, we analyzed the correlation between AI-P, variant allele frequency (VAF), and average depth of sequencing across 23 cancer types.

Multicentre single-arm phase II trial evaluating the safety and effiCacy of Panitumumab and iRinOtecan in NeoRAS Wild-type mEtaStatic colorectal cancer patientS (C-PROWESS trial): study protocol

IntroductionA new concept of ‘NeoRAS wild-type (WT)’, which means conversion of RAS status from RAS mutant to RAS WT after treatment, has been reported. Previous observational and proof-of-concept studies have...

Association of KISS1 Gene SNPs (Rs35431622) with Metabolic Parameters in Iraqi Women Polycystic Ovary Syndrome

Background: Polycystic ovarian syndrome (PCOS) is among the most common types of gynecological hormonal abnormalities and metabolic problems that have an impact females throughout adolescence , with a prevalence of 6-10%.Hyperandrogenism, unreliable menstrual periods, and polycystic ovaries are the hallmarks of PCOS. Objectives: The goal of this find out about used to be to analyze the affiliation of kiss1 Gene SNPs (rs35431622) with metabolic Parameters in PCOS . Methodology: a case –control find out about enrolled one hundred sixty members of which eighty female have PCOS and eighty symbolize the manage crew who had-been interestingly healthful. Patients with PCOS had been chosen patients from our health center and the teaching hospital's gynecology and obstetrics department in Tikrit. Assessment of serum (fasting blood glucose , cholesreol .HDL and triglyceride) was performed using smart 150 full automated clinical chemistry machine .and insulin by ELISA KIT ,HOMA-IR By calculation and the maker company's provided methodology was followed to extract genomic DNA from whole blood. KISS1 Gene SNPs rs35431622 T&gt;A gene polymorphisms was once detected by using (T-ARMS-PCR) method . Results: The findings indicate, the frequency of the kiss1 gene rs35431622 AT and TT genotypes in PCOS-affected women increased 3.524-fold and 9.633- in comparison to the group of healthy women which have odds ratio; 3.524and 9.633, 95% confidence interval [CI], 1.622-7.657 and 2.916-31.816; P-value ≤0.01**and ≤0.01**) respectively. and The levels of FBS ,Insulin ,HOMA-IR, cholesterol, HDL and triglyceride showed significant difference among PCOS against control group (P≤ 0.0001) . Conclusions: Our find out about suggests a greater awareness of serum (FBS ,Insulin , cholesterol, HDL and triglyceride) seemed in PCOS companies as in contrast with control and also significant difference in the value of HOMA-IR as well, Cases and controls had statistically different frequencies of the identified polymorphisms, and Control 34 had a higher frequency of the homozygous allele (CC) than Cases 12 ., while the frequency of heterozygous allele (AT) were significantly different in cases 51 higher than the controls 41 as well the frequency of mutant allele (tt) were significantly different in cases 17 higher than the controls 5 .The allelic frequency of SNP rs35431622.C/T used.to,be detected extra regularly with significant affiliation infemale with PCOS than in control. Keywords: PCOS, kiss1,(IR)

UMIErrorCorrect and UMIAnalyzer: Software for Consensus Read Generation, Error Correction, and Visualization Using Unique Molecular Identifiers.

Targeted sequencing using unique molecular identifiers (UMIs) enables detection of rare variant alleles in challenging applications, such as cell-free DNA analysis from liquid biopsies. Standard bioinformatics pipelines for data processing and variant calling are not adapted for deep-sequencing data containing UMIs, are inflexible, and require multistep workflows or dedicated computing resources. We developed a bioinformatics pipeline using Python and an R package for data analysis and visualization. To validate our pipeline, we analyzed cell-free DNA reference material with known mutant allele frequencies (0%, 0.125%, 0.25%, and 1%) and public data sets. We developed UMIErrorCorrect, a bioinformatics pipeline for analyzing sequencing data containing UMIs. UMIErrorCorrect only requires fastq files as inputs and performs alignment, UMI clustering, error correction, and variant calling. We also provide UMIAnalyzer, a graphical user interface, for data mining, visualization, variant interpretation, and report generation. UMIAnalyzer allows the user to adjust analysis parameters and study their effect on variant calling. We demonstrated the flexibility of UMIErrorCorrect by analyzing data from 4 different targeted sequencing protocols. We also show its ability to detect different mutant allele frequencies in standardized cell-free DNA reference material. UMIErrorCorrect outperformed existing pipelines for targeted UMI sequencing data in terms of variant detection sensitivity. UMIErrorCorrect and UMIAnalyzer are comprehensive and customizable bioinformatics tools that can be applied to any type of library preparation protocol and enrichment chemistry using UMIs. Access to simple, generic, and open-source bioinformatics tools will facilitate the implementation of UMI-based sequencing approaches in basic research and clinical applications.

Bile is a reliable and valuable source to study cfDNA in biliary tract cancers.

ObjectiveThe aim of this study is to determine the clinical efficacy of bile-derived liquid biopsy compared with plasma and tumor tissue biopsy in patients with biliary tract carcinoma (BTC).MethodsA total of 13 patients with BTC were enrolled in this cohort. Tumor tissue, bile, and plasma samples were obtained and analyzed using next-generation sequencing for genomic profiling.ResultsBile and plasma samples were collected from all 13 patients, and 11 patients also had matched tumor tissues available. The cell-free DNA (cfDNA) concentration was significantly higher in the bile supernatant than in plasma (median: 1918 vs. 63.1 ng/ml, p = 0.0017). The bile supernatant and pellet had a significantly higher mean mutation allele frequency (MF) than plasma (median: 3.84% vs. 4.22% vs. 0.16%; p < 0.001). Genomic alterations were predominantly missense. Both bile supernatant and pellet had significantly more genomic alterations than plasma (average: 9.3 vs. 7.2 vs. 2.3 alterations per sample; p < 0.01). Among the top 10 most frequent genomic alterations, the consistency between bile supernatant and tumor tissue was 90.00% (18/20), that between bile pellet and tumor tissue was 85.00% (17/20), and that between the plasma and tissue was only 35.00% (7/20). MAF of both bile supernatant and pellet was positively correlated with that in tissue samples (ρ < 0.0001, spearman r = 0.777, and ρ < 0.0001, spearman r = 0.787, respectively), but no significant correlation with tissue was found in the plasma (ρ = 0.966, spearman r = 0.008). Furthermore, additional genomic alterations could be detected in bile supernatant and pellet than in tissue. Potential targets for targeted therapy were identified in bile supernatant and pellet. Regarding copy number variation (CNV) and chromosomal instability (CIN) detection, four additional CNVs from two patients were detected in the bile supernatant that was not detected in tissues (i.e., amplification of TERC, IL7R, RICTOR, and TERT). CIN was significantly higher in tumor tissue than in plasma. The CIN of the bile was also significantly higher than that of plasma. There was no significant difference in CIN between the tissue and the bile supernatant.ConclusionThe consistency of all genomic alterations and tumor tissue-determined genomic alteration in the bile supernatant/pellet was significantly higher than in plasma. Bile supernatants/pellets are better for genetic sequencing and may also have potential clinical value to guide targeted therapy and evaluate prognosis. Bile cfDNA may be a feasible substitute for tumor tissue in the genetic testing of patients with BTC.

Evaluation of circulating tumor DNA as a prognostic biomarker for metastatic pancreatic adenocarcinoma.

PurposePancreatic cancer is an aggressive solid tumor with a severe prognosis. Although tumor biomarkers are often used to identify advanced pancreatic cancer, this is not accurate, and the currently used biomarkers are not indicative of prognosis. The present study evaluated circulating tumor DNA (ctDNA) as a biomarker for prognosis prediction and disease monitoring in metastatic pancreatic adenocarcinoma (PAC).MethodsFrom 2017 to 2018, 40 patients with metastatic PAC were enrolled, and tumor tissue and blood samples were collected from 40 and 35 patients, respectively. CtDNA was sequenced by next-generation sequencing (NGS) with a 425-gene capture panel. The association of clinical characteristics, laboratory indicators, and dynamic ctDNA with patient outcomes was analyzed.ResultsMutations in KRAS (87.5%, N = 35) and TP53 (77.5%, N = 31) were most common in 40 tumor tissue. Patients’ ECOG score, CA19-9, CEA, neutrophil-lymphocyte ratio (NLR), platelet- lymphocyte ratio (PLR) levels and mutations in ≥ 3 driver genes were strongly correlated with patients’ overall survival (OS). Patients’ gender, ECOG score, CA19-9, and CEA levels were associated with progression-free survival (PFS) (P<0.05). In 35 blood samples, univariate analysis showed a significant association between ECOG score, CA19-9, KRAS or CDKN2A mutation in ctDNA and OS and between CA19-9, CDKN2A or SMAD4 mutation in ctDNA and PFS. Cox hazard proportion model showed that patients’ CDKN2A mutation in ctDNA (HR=16.1, 95% CI=4.4-59.1, P<0.001), ECOG score (HR=6.2, 95% CI=2.4-15.7, P<0.001) and tumor location (HR=0.4, 95% CI=0.1-0.9, P=0.027) were significantly associated with OS. Patients’ CDKN2A mutation in ctDNA (HR=6.8, 95% CI=2.3-19.9, P=0.001), SMAD4 mutation in ctDNA (HR=3.0, 95% CI=1.1-7.9, P=0.031) and metastatic organ (HR=0.4, 95% CI=0.2-1.0, P=0.046) were significantly associated with PFS. Longitudinal changes in gene mutation allelic frequency (MAF) value were evaluated in 24 patients. Detection of progression disease (PD) by ctDNA was 0.9 months earlier than by radiological imaging (mean PFS: 4.6m vs 5.5m, P=0.004, paired t-test).ConclusionsThe ctDNA has the potential as a specific survival predictive marker for metastatic PAC patients. Longitudinal ctDNA tracking could potentially help identify disease progression and be a valuable complement for routine clinical markers and imaging.

Clinical sequencing uncovers the genomic characteristics and mutation spectrum of the 2018 African swine fever virus in Guangdong, China.

African swine fever (ASF) outbreak have caused tremendous economic loss to the pig industry in China since its emergence in August 2018. Previous studies revealed that many published sequences are not suitable for detailed analyses due to the lack of data regarding quality parameters and methodology, and outdated annotations. Thus, high-quality genomes of highly pathogenic strains that can be used as references for early Chinese ASF outbreaks are still lacking, and little is known about the features of intra-host variants of ASF virus (ASFV). In this study, a full genome sequencing of clinical samples from the first ASF outbreak in Guangdong in 2018 was performed using MGI (MGI Tech Co., Ltd., Shenzhen, China) and Nanopore sequencing platforms, followed by Sanger sequencing to verify the variations. With 22 sequencing corrections, we obtained a high-quality genome of one of the earliest virulent isolates, GZ201801_2. After proofreading, we improved (add or modify) the annotations of this isolate using the whole genome alignment with Georgia 2007/1. Based on the complete genome sequence, we constructed the methylation profiles of early ASFV strains in China and predicted the potential 5mC and 6mA methylation sites, which are likely involved in metabolism, transcription, and replication. Additionally, the intra-host single nucleotide variant distribution and mutant allele frequency in the clinical samples of early strain were determined for the first time and found a strong preference for A and T substitution mutation, non-synonymous mutations, and mutations that resulted in amino acid substitutions into Lysine. In conclusion, this study provides a high-quality genome sequence, updated genome annotation, methylation profile, and mutation spectrum of early ASFV strains in China, thereby providing a reference basis for further studies on the evolution, transmission, and virulence of ASFV.

Circulating Tumor DNA is Unreliable to Detect Somatic Gene Alterations in Gastrointestinal Peritoneal Carcinomatosis

IntroductionTumor agnostic circulating tumor DNA (ctDNA) is routinely used to guide treatment decisions in gastrointestinal (GI) cancers, especially metastatic cancers. The amount of ctDNA detected in plasma is affected by stage, tumor burden, and tumor vascularization. We hypothesized that peritoneal carcinomatosis (PC) is associated with lower ctDNA levels than other metastatic sites in GI cancers due to the plasma–peritoneal barrier.MethodsWe conducted a retrospective analysis of patients with stage II–IV GI cancers treated at our institution between 2015 and 2020 with available panel-based ctDNA results (Guardant 360TM). ctDNA analysis was performed on early and pretreatment samples. We compared the reported maximum variant allele frequency (mVAF) of somatic mutations across metastatic sites.ResultsOf the 279 patients with GI cancers (colorectal, upper GI, pancreaticobiliary), 212 had stage IV disease (PC: n = 61; visceral metastases: n = 138; other metastases: n = 13). Mean mVAF increased with increasing stages of disease (stage II: 3.6 ± 7; stage III: 6.4 ± 10; stage IV: 28.0 ± 51; p < 0.01). Among patients with stage IV disease, PC was associated with lower ctDNA levels independent of primary tumor site (PC only: 12.1%; PC+ visceral metastases: 26.8%; and visceral metastases only: 35.0%; p < 0.01). In a subset of patients (n = 27, matched pair analysis of genomic alterations (GAs) showed fewer GAs were detected in plasma compared with tissue.ConclusionsPC of GI origin is associated with significantly lower ctDNA levels compared with visceral metastasis. Caution is warranted when interpreting ctDNA results from patients with PC due to lower sensitivity for detecting actionable mutations.

A Prospective Validation Study of Lung Cancer Gene Panel Testing Using Cytological Specimens.

Simple SummaryThe gold standard for genetic panel test samples is considered to be tissue specimens. However, in clinical practice, tissue specimens are often unavailable. Therefore, using an amplicon-based high-sensitivity next-generation sequencing panel test capable of measuring eight druggable genes, we enrolled patients who underwent diagnostic procedures to evaluate analysis accuracy, nucleic acid yield, and quality using cytological specimens. Cytological specimens were suitable for both nucleic acid yield and specimen quality due to the ease of collection and processing. Cytological sample analysis detected gene mutations in 68.7% of lung adenocarcinomas, and these samples were consistent with companion diagnostic tests 99.5% of the time. Moreover, the allele frequency of gene mutations in cytological specimens showed a high correlation with tissue specimens. This is the first study to prospectively evaluate the feasibility of a lung cancer gene panel test using cytological specimens.Background: Genetic panel tests require sufficient tissue samples, and therefore, cannot always be performed. Although collecting cytological specimens is easier than tissue collection, there are no validation studies on the diagnostic accuracy of lung cancer gene panel tests using cytology samples. Methods: Using an amplicon-based high-sensitivity next-generation sequencing panel test capable of measuring eight druggable genes, we prospectively enrolled consecutive patients who underwent diagnostic procedures. We evaluated the analysis accuracy rate, nucleic acid yield, and the quality of cytological specimens under brushing, needle aspiration, and pleural effusion. We then compared these specimens with collected tissue samples. Results: In 163 prospectively enrolled cases, nucleic acid extraction and analysis accuracy was 100% in cases diagnosed with adenocarcinoma. Gene mutations were found in 68.7% of cases with 99.5% (95% CI: 98.2–99.9) concordance to companion diagnostics. The median DNA/RNA yield and DNA/RNA integrity number were 475/321 ng and 7.9/5.7, respectively. The correlation coefficient of the gene allele ratio in 64 cases compared with tissue samples was 0.711. Conclusion: The success of gene analysis using cytological specimens was high, and the yield and quality of the extracted nucleic acid were sufficient for panel analysis. Moreover, the allele frequency of gene mutations in cytological specimens showed high correlations with tissue specimens.

Bias and inconsistency in the estimation of tumour mutation burden

BackgroundTumour mutation burden (TMB), defined as the number of somatic mutations per megabase within the sequenced region in the tumour sample, has been used as a biomarker for predicting response to immune therapy. Several studies have been conducted to assess the utility of TMB for various cancer types; however, methods to measure TMB have not been adequately evaluated. In this study, we identified two sources of bias in current methods to calculate TMB.MethodsWe used simulated data to quantify the two sources of bias and their effect on TMB calculation, we down-sampled sequencing reads from exome sequencing datasets from TCGA to evaluate the consistency in TMB estimation across different sequencing depths. We analyzed data from ten cancer cohorts to investigate the relationship between inferred TMB and sequencing depth.ResultsWe found that TMB, estimated by counting the number of somatic mutations above a threshold frequency (typically 0.05), is not robust to sequencing depth. Furthermore, we show that, because only mutations with an observed frequency greater than the threshold are considered, the observed mutant allele frequency provides a biased estimate of the true frequency. This can result in substantial over-estimation of the TMB, when the cancer sample includes a large number of somatic mutations at low frequencies, and exacerbates the lack of robustness of TMB to variation in sequencing depth and tumour purity.ConclusionOur results demonstrate that care needs to be taken in the estimation of TMB to ensure that results are unbiased and consistent across studies and we suggest that accurate and robust estimation of TMB could be achieved using statistical models that estimate the full mutant allele frequency spectrum.

Characterization of the mutational landscape of high-grade serous ovarian tumors before and after neo-adjuvant chemotherapy (267)

Characterization of the mutational landscape of high-grade serous ovarian tumors before and after neo-adjuvant chemotherapy (267)

CRISPR-mediated knockout of nicotinic acetylcholine receptor (nAChR) α6 subunit confers high levels of resistance to spinosyns in Spodoptera frugiperda

Spinosyn insecticides (spinosad and spinetoram) have been widely used to control a number of agricultural pests including the fall armyworm, Spodoptera frugiperda. Mutations of the nicotinic acetylcholine receptor α6 subunit (nAChRα6) have been reported to confer high levels of resistance to spinosyns in several insect pests. Here we used CRISPR-mediated gene knockout to determine the involvement of S. frugiperda nAChRα6 (Sfα6) in spinosyns susceptibility. A Sfα6 knockout strain of S. frugiperda (Sfα6-KO) was established using dual single guide RNA (sgRNA) directed large fragment deletion with the CRISPR/Cas9 system. Sfα6-KO showed high levels of resistance to spinosad (307-fold) and spinetoram (517-fold) compared with the progenitor strain YJ-19, while no resistance was observed to emamectin benzoate, indoxacarb, chlorfenapyr, chlorantraniliprole and broflanilide. Genetic analyses confirmed that spinosad resistance in Sfα6-KO was autosomal, incompletely recessive and tightly linked to the edited deletion mutation of Sfα6. Our results provided in vivo functional evidence for Sfα6 as the major target of spinosyns against S. frugiperda, and demonstrated that disruption of Sfα6 causes high level resistance to spinosyns. Although no mutations of Sfα6 have yet been reported in any field populations of S. frugiperda, it is critical to develop F1 screens and/or DNA-based methods to detect and monitor the mutant allele frequencies of Sfα6 across global populations of S. frugiperda.

Somatic GJA4 gain-of-function mutation in orbital cavernous venous malformations

Orbital cavernous venous malformation (OCVM) is a sporadic vascular anomaly of uncertain etiology characterized by abnormally dilated vascular channels. Here, we identify a somatic missense mutation, c.121G > T (p.Gly41Cys) in GJA4, which encodes a transmembrane protein that is a component of gap junctions and hemichannels in the vascular system, in OCVM tissues from 25/26 (96.2%) individuals with OCVM. GJA4 expression was detected in OCVM tissue including endothelial cells and the stroma, through immunohistochemistry. Within OCVM tissue, the mutation allele frequency was higher in endothelial cell-enriched fractions obtained using magnetic-activated cell sorting. Whole-cell voltage clamp analysis in Xenopus oocytes revealed that GJA4 c.121G > T (p.Gly41Cys) is a gain-of-function mutation that leads to the formation of a hyperactive hemichannel. Overexpression of the mutant protein in human umbilical vein endothelial cells led to a loss of cellular integrity, which was rescued by carbenoxolone, a non-specific gap junction/hemichannel inhibitor. Our data suggest that GJA4 c.121G > T (p.Gly41Cys) is a potential driver gene mutation for OCVM. We propose that hyperactive hemichannel plays a role in the development of this vascular phenotype.

Increased blood-based intratumor heterogeneity (bITH) is associated with unfavorable outcomes of immune checkpoint inhibitors plus chemotherapy in non-small cell lung cancer

BackgroundThe combination of immune checkpoint inhibitors (ICIs) and chemotherapy has been the standard first-line treatment for advanced non-small cell lung cancer (NSCLC) patients with driver-gene negative. However, efficacy biomarkers for ICIs-based combination therapy are lacking. We aimed to identify potential factors associated with outcomes of ICIs plus chemotherapy at baseline and dynamic changes in peripheral blood.MethodsWe collected plasma samples of 51 advanced NSCLC patients without EGFR/ALK/ROS1 alteration at baseline and/or after two treatment cycles of ICIs plus chemotherapy. A blood-based intratumor heterogeneity (bITH) score was calculated based on the allele frequencies of somatic mutations using a 520-gene panel. bITH-up was defined as a ≥ 10% increase in bITH score from baseline, with a second confirmatory measurement after treatment.ResultsAt baseline, the number of metastatic organs and lung immune prognostic index (LIPI) were significantly associated with shorter progression-free survival (PFS) of ICIs plus chemotherapy, while bITH and other common molecular biomarkers, including ctDNA level, blood-based tumor mutational burden (bTMB), and PD-L1 expression, had no effect on PFS. LRP1B mutation at baseline was significantly associated with favorable outcomes to ICIs plus chemotherapy. There were 37 patients who had paired samples at baseline and after two cycles of treatment, with the median interval of 53 days. Intriguingly, patients with bITH-up had significant shorter PFS (HR, 4.92; 95% CI, 1.72–14.07; P = 0.001) and a lower durable clinical benefit rate (0 vs 41.38%, P = 0.036) than those with bITH-stable or down. Case studies indicated that bITH was promising to predict disease progression.ConclusionsThe present study is the first to report that increased bITH is associated with unfavorable outcomes of ICIs plus chemotherapy in advanced NSCLC patients.

CMDB: the comprehensive population genome variation database of China.

A high-quality genome variation database derived from a large-scale population is one of the most important infrastructures for genomics, clinical and translational medicine research. Here, we developed the Chinese Millionome Database (CMDB), a database that contains 9.04 million single nucleotide variants (SNV) with allele frequency information derived from low-coverage (0.06×-0.1×) whole-genome sequencing (WGS) data of 141 431 unrelated healthy Chinese individuals. These individuals were recruited from 31 out of the 34 administrative divisions in China, covering Han and 36 other ethnic minorities. CMDB, housing the WGS data of a multi-ethnic Chinese population featuring wide geographical distribution, has become the most representative and comprehensive Chinese population genome database to date. Researchers can quickly search for variant, gene or genomic regions to obtain the variant information, including mutation basic information, allele frequency, genic annotation and overview of frequencies in global populations. Furthermore, the CMDB also provides information on the association of the variants with a range of phenotypes, including height, BMI, maternal age and twin pregnancy. Based on these data, researchers can conduct meta-analysis of related phenotypes. CMDB is freely available at https://db.cngb.org/cmdb/.

IDH1 p.R132H ctDNA and D-2-hydroxyglutarate as CSF biomarkers in patients with IDH-mutant gliomas.

We aimed to evaluate IDH1 p.R132H mutation and 2-hydroxyglutarate (2HG) in cerebrospinal fluid (CSF) as biomarkers for patients with IDH-mutant gliomas. CSF was collected from patients with infiltrating glioma, and 2HG levels were measured by liquid chromatography-mass spectrometry. IDH1 p.R132H mutant allele frequency (MAF) in CSF-ctDNA was measured by digital droplet PCR (ddPCR). Tumor volume was measured from standard-of-care magnetic resonance images. The study included 48 patients, 6 with IDH-mutant and 42 with IDH-wildtype gliomas, and 57 samples, 9 from the patients with IDH-mutant and 48 from the patients with IDH-wildtype gliomas. ctDNA was detected in 7 of the 9 samples from patients with IDH-mutant glioma, and IDH1 p.R132H mutation was detected in 5 of the 7 samples. The MAF ranged from 0.3 to 39.95%. Total 2HG level, D-2HG level, and D/L-2HG ratio in CSF were significantly higher in patients with IDH-mutant gliomas than in patients with IDH-wildtype gliomas. D-2HG level and D/L-2HG ratio correlated with total tumor volume in patients with IDH-mutant gliomas but not in patients with IDH-wildtype gliomas. Our results suggest that detection of IDH1 p.R132H mutation by ddPCR and increased D-2HG level in CSF may help identify IDH-mutant gliomas. Our results also suggest that D-2HG level and D/L-2HG ratio correlate with tumor volume in patients with IDH-mutant gliomas. Further prospective studies with larger cohorts are needed to validate these findings.

ASSOCIATION BETWEEN VASCULAR ENDOTHELIAL GROWTH FACTOR GENE POLYMORPHISMS AND THE RISK OF PREECLAMPSIA IN IRAQI PREGNANT WOMEN

Background: Vascular endothelial growth factor (VEGF) is an essential factor for angiogenesis and plays important role in placental development. Objective: To investigate the association between VEGF single nucleotide polymorphisms (SNPs) +936 Cytosine/Thymine (C/T) and -634 Guanine/Cytosine (G/C) and preeclampsia risk. Methods: A total of 50 cases of pregnant women with preeclampsia and 50 healthy pregnant women (as a control) were involved in this case control study. Blood samples were collected form each woman and the Deoxyribonucleic acid (DNA) was extracted. Amplification of VEGF gene was done by conventional polymerase chain reaction (PCR) and then detection of SNPs-634 G/C and +936 C/T were carried out by restriction fragment length polymorphism PCR (PCR-RFLP). Results: The frequency of different genotypes of VEGF SNPs +936 C/T &amp;-634 G/C are in accordance with Hardy Weinberg equilibrium. VEGF polymorphism +936 C/T appeared in 3 genotypes after digestion with restriction enzymes Cytosine Cytosine (CC), Cytosine Thymine (C/T) &amp; Thymine Thymine (TT). VEGF SNP -634 G/C appeared in 3 genotypes after digestion with restriction enzymes; those were Guanine Guanine (GG), Guanine Cytosine (GC) and CC. The heterozygous genotype CT of polymorphism +936 C/T was more frequent among preeclamptic patients than the controls (26% versus 16%). Likewise, TT genotype was more frequent among preeclamptic patients (8% versus 2%) with no significant differences. At the allelic level, the difference was more prominent. The frequency of mutant allele (T allele) was much more frequent in preeclamptic patients than controls (21% versus 10%) with a statistically significant difference (p=0.049). Although CC genotype of polymorphism -634 G/C was more frequent among preeclamptic patients than controls (14% versus 10%), the difference was not significant (p=0.704). Likewise, there were no significant differences in allele frequency. Conclusion: The study suggested that the mutant T allele of VEGF +936 C/T polymorphism was associated with increased preeclampsia risk and disease development. Keywords: Vascular endothelial growth factor, polymorphisms, preeclampsia Citation: Khaleel MF, Al-Moayad HA. Association between vascular endothelial growth factor gene polymorphisms and the risk of preeclampsia in Iraqi pregnant women. Iraqi JMS. 2022; 20(1): 106-112. doi: 10.22578/IJMS.20.1.14

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate.

Simple SummaryCanine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease caused by a DM-associated mutation (SOD1:c.118G>A, p.E40K) that commonly occurs in German Shepherd Dogs (GSDs). This study aimed to determine the mutant allele frequency in the Japanese GSD population and to analyze the clinical progression rate among GSDs with the homozygous mutant A/A genotype. The survey found 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%) among the 541 dogs examined, indicating that the mutant allele frequency was 0.220; the Japanese GSD population can be considered to be in Hardy–Weinberg equilibrium. Clinical analysis revealed that the clinical progression rate was particularly high (100%) among A/A dogs aged >10 years. Appropriate mating management is crucial for the management and prevention of DM in the Japanese GSD population.Canine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease reported in multiple canine breeds, including the German Shepherd Dog (GSD). Clinical signs include progressive motor neuron paralysis, which begins in the pelvic limbs and eventually leads to respiratory distress, which may necessitate euthanasia. A common DM-associated mutation is a single nucleotide substitution that causes an amino acid substitution (c.118G>A, p.E40K) in the canine SOD1 gene. This SOD1 mutation and the clinical progression rate of A/A risk genotype in the Japanese GSD population have not been analyzed before. Therefore, the aim of this study was to determine the frequency of the mutated allele and analyze the clinical progression rate in the Japanese GSD population. We studied 541 GSDs registered with the Japanese German Shepherd Dog Registration Society between 2000 and 2019. Genotyping was performed using real-time PCR with DNA extracted from the hair roots of each dog. The study revealed 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%), indicating a frequency of the mutant allele of 0.220, which are in Hardy–Weinberg equilibrium. We analyzed the clinical signs in A/A dogs with an age limit of 10 years based on information obtained from the dogs’ owners. Of the seven A/A dogs older than 10 years, owners reported DM-related clinical signs, indicating a clinical progression rate of 100%. These results, further genotyping, and thorough clinical examinations of SOD1 A/A risk genotype will help control and prevent DM in the Japanese GSD population.