Mean Copy Number Research Articles

Multiple routes to fungicide resistance: Interaction of Cyp51 gene sequences, copy number and expression.

We examined the molecular basis of triazole resistance in Blumeria graminis f. sp. tritici (wheat mildew, Bgt), a model organism among powdery mildews. Four genetic models for responses to triazole fungicides were identified among US and UK isolates, involving multiple genetic mechanisms. Firstly, only two amino acid substitutions in CYP51B lanosterol demethylase, the target of triazoles, were associated with resistance, Y136F and S509T (homologous to Y137F and S524T in the reference fungus Zymoseptoria tritici). As sequence variation did not explain the wide range of resistance, we also investigated Cyp51B copy number and expression, the latter using both reverse transcription-quantitative PCR and RNA-seq. The second model for resistance involved higher copy number and expression in isolates with a resistance allele; thirdly, however, moderate resistance was associated with higher copy number of wild-type Cyp51B in some US isolates. A fourth mechanism was heteroallelism with multiple alleles of Cyp51B. UK isolates, with significantly higher mean resistance than their US counterparts, had higher mean copy number, a high frequency of the S509T substitution, which was absent from the United States, and in the most resistant isolates, heteroallelism involving both sensitivity residues Y136+S509 and resistance residues F136+T509. Some US isolates were heteroallelic for Y136+S509 and F136+S509, but this was not associated with higher resistance. The obligate biotrophy of Bgt may constrain the tertiary structure and thus the sequence of CYP51B, so other variation that increases resistance may have a selective advantage. We describe a process by which heteroallelism may be adaptive when Bgt is intermittently exposed to triazoles.

Needles in haystacks: monitoring the potential escape of bioaerosolised antibacterial resistance genes from wastewater treatment plants with air and phyllosphere sampling.

Wastewater treatment plants are well-known point sources of emissions of antibacterial resistance genes (ARGs) into the environment. Although most work to date has focused on ARG dispersal via effluent, aerial dispersal in bioaerosols is a poorly understood, but likely important vector for ARG dispersal. Recent evidence suggests that ARG profiles of the conifer needle phyllosphere could be used to measure bioaerosol dispersal from anthropogenic sources. Here, we assessed airborne dispersal of ARGs from wastewater treatment plants in Wales, UK and Quebec, Canada, using conifer needles as passive bioaerosol monitors. ARG profiles of wastewater were compared to those of conifer phyllosphere using high-throughput qPCR. ARG richness was significantly lower in conifer phyllosphere samples than wastewater samples, though no differences were observed across the dispersal gradients. Mean copy number of ARGs followed a similar trend. ARG profiles showed limited, but consistent patterns with increasing distance from wastewater treatment plants, but these did not align with those of wastewater samples. For example, proportional abundance of aminoglycosides decreased over the dispersal gradient in Wales, whereas mobile genetic elements showed the inverse relationship. In summary, while distinct ARG profiles exist along dispersal gradients, links to those of wastewater were not apparent.

Abstract 3040: Tumor-specific super-enhancer drives overexpression of EVI1 in ovarian cancer

Abstract Epithelial ovarian cancer (OC) is typically diagnosed at a late stage and, despite treatment with cytoreductive surgery and chemotherapy, 70-80% of patients relapse and eventually die due to the disease. Major challenges to improve patient outcomes are to overcome therapy resistance and to individually tailor therapy. Uncovering the molecular drivers of OC, especially in the most common subgroup high-grade serous (HGS), will help develop novel therapeutic strategies to meet these challenges. Using the Cancer Genome Atlas (TCGA) data, we observed that a locus in 3q26.2, near the EVI1 oncogene, was the most significantly amplified region in HGS OC, occurring in more than 80% of those tumors. We validated these findings in an OC dataset from the Hartwig Medical Foundation and in OC cell lines from the DepMap database. Furthermore, HGS OC had the highest mean copy number of the 3q26.2 locus co-occurring with high mean EVI1 expression among all tumor entities in the pan-cancer TCGA dataset. These EVI1 expression levels are significantly higher than in normal fallopian tube, from which HGS OC most likely arises. EVI1 overexpression promotes leukemogenesis in 3q26-rearranged acute myeloid leukemia (AML), as a result of chromosomal translocations that enable hijacking of hyperactive enhancers. Here, we hypothesize that EVI1 also plays a crucial role in OC development, but that its aberrant expression in this disease is driven by the amplification of yet undiscovered regulatory elements near EVI1. We showed that EVI1-positive OC cell lines, of which the majority are HGS, are EVI1-dependent and harbor a ~200 kb amplified region near EVI1. This region interacts with the EVI1 promoter (4C-Seq), is characterized by open chromatin (ATAC-Seq), and shows high levels of H3K27 acetylation (H3K27ac ChIP-Seq), suggesting the presence of an active enhancer. Furthermore, based upon H3K27ac signal, we identified within this region a super-enhancer of approximately 65 kb. In OC cell lines without detectable EVI1 levels or cell lines from other cancer subtypes, such as breast and colon cancer, this location had no active enhancer and did not loop to the EVI1 promoter. Altogether, these data point to the generation of a hyperactive super-enhancer driving EVI1 overexpression in OC. These findings emphasize the importance of epigenetic control in the regulation of EVI1 expression, as previously shown in 3q26-rearranged AML. Since OC tumor cell lines are strongly dependent on EVI1, interference with this super-enhancer, leading to loss of EVI1 expression, could be exploited to inhibit tumor growth. For this purpose, we generated GFP-tagged EVI1 cell line models (e.g. SKOV3), which will be used to discover small molecules to target the activity of this super-enhancer. In conclusion, we discovered a new mechanism of enhancer-mediated overexpression of the EVI1 oncogene as a molecular driver in ovarian cancer, with possible implications for therapy. Citation Format: Leonie Smeenk, Sabrina Kruse, Roger Mulet-Lazaro, Ingrid Boere, John Martens, Stefan Gröschel, Claudia Scholl, Stefan Fröhling, Ruud Delwel. Tumor-specific super-enhancer drives overexpression of EVI1 in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3040.

Abstract 4002: Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model

Abstract Introduction: Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as a promising approach for treating hematological malignancies, particularly B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to delve into the activation dynamics and therapeutic efficacy of CD19 CAR-T cells in a disseminated NALM6-luc human B-ALL murine model. Experimental design: NALM6-Luc-mCh-Puro cells were implanted intravenously in female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Animals were treated with CD19 CAR-T cells alone and in combination with ibrutinib. The anti-tumor activity was assessed, and the persistence and activation of CAR-T cells in the blood were evaluated using flow cytometry. Additionally, ddPCR analysis was employed to quantify CAR copy numbers, providing insights into the T-cell persistence over time. Results: Treatment with CD19 CAR-T cells exhibited notable anti-cancer activity and that activity was enhanced in combination with ibrutinib, in line with other reports. The increased time to progression was noted as 69.2% for the CAR-T group and 138.4% for the combination of CAR-T and ibrutinib compared to control. Flow cytometry analysis showed that CAR-T cells persisted in the blood throughout the study. The mean CAR-T absolute cell count for both CAR-T and CAR-T plus ibrutinib treated groups increased from 2 cell/µL blood on Day 0 to 11 and 51 cells/µL of blood, respectively, by Day 39. The progressive increase of PD-1+ and LAG-3+ percentage cells correlates positively with tumor burden, indicating a dynamic link between biomarker upregulation and the presence of tumor cells in mice. ddPCR analysis revealed an initial CAR copy decrease in all groups, followed by gradual increase in tumor-bearing mice treated with CD19 CAR-T cells, with a dramatic increase observed by Day 39. Results from CAR-T treated and CAR-T plus ibrutinib treated groups were comparable, showing mean copy numbers per ng of DNA increasing from less than 1 on day 4 to 17.9 and 23.3, respectively, on day 39. Importantly, no changes were noted in non-tumor implanted mice, emphasizing the tumor dependency for CAR-T cell expansion. Conclusion: This study emphasizes the marked anti-cancer activity of CD19 CAR-T cells in the NALM6-Luc-mCh-Puro B-ALL murine model. Comprehensive analysis of CAR-T cell persistence and activation, including insights from ddPCR, provides a robust understanding of therapeutic response dynamics. The observed in vivo anti-cancer efficacy aligns with the persistence of CAR-T cells revealed by flow cytometry and the dynamic expansion captured through ddPCR analysis. Our results indicate ddPCR and flow cytometry as complementary and independent methods for assessing the systemic CAR-T cell persistence, contributing to optimizing therapy for B-cell acute lymphoblastic leukemia. Citation Format: Prashant Pandey, David Draper, Sheri Barnes, Yewei Xing, Derrik Germain, Lauren Kucharczyk, Roys Stacey. Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4002.

Abstract 132: Proteomic Profiles Of Human Arterioles Isolated From Fresh Adipose Tissue Or Following Overnight Storage

Resistance arteries, or arterioles, are key determinants of the total peripheral vascular resistance, which, in turn, is a key determinant of arterial blood pressure (BP). However, the amount of protein available from one isolated human arteriole may be less than 5 μg, making proteomic analysis challenging. In addition, obtaining human arterioles requires manual dissection of unfrozen clinical specimens. This limits its feasibility, especially for powerful multi-center clinical studies in which clinical specimens need to be shipped overnight to a research lab for arteriole isolation. We performed a study to address low input, test overnight tissue storage, and develop a reference human arteriolar proteomic profile. We found that, in tandem mass tag proteomic analysis, the use of a booster channel consisting of endothelial and vascular smooth muscle cells (1:5 ratio) increased the number of proteins detected in a human arteriole segment with FDR < 0.01 from 1,051 to more than 3,000. We collected adipose tissues from three human subjects and isolated two arterioles from each fresh aliquot of the tissue or after 24h of cold storage of unfrozen aliquots in MACS tissue solution. The correlation coefficient of proteomic profile was similar (p=0.6) between replicate arterioles isolated freshly, following the cold storage, or before and after the cold storage. We built a human arteriolar proteomic profile consisting of 3,836 proteins based on the analysis of 12 arteriole samples from the three subjects. The average arteriolar protein had a mean copy number of 2.76х10 6 per cell using the histone proteomic ruler, which is based on the fact that the mass of DNA per cell is approximately equal to the protein mass of histones. Transgelin was the most abundant protein detected. We curated a set of BP-relevant human genes, which encode 1,945 proteins. Of these BP-relevant proteins, 476 (12.5%) were detected in the arteriolar proteome, which was a significant overrepresentation (p<0.05, Chi squared test). These findings demonstrate that proteomic analysis is feasible with arterioles isolated from human adipose tissue following cold overnight storage and provide a reference human arteriolar proteome profile highly valuable for studies of arteriole-related traits.

Development and validation of a nanoplate-based digital PCR assay for absolute MPXV quantification

Quantification of mpox virus (MPXV) across different human body anatomical sites can provide insights about the most likely transmission routes, so methods able to release absolute and exact quantitative values of MPXV DNA are crucial. Here, we optimized a new QIAcuity digital PCR (dPCR) protocol for the detection and quantification of MPXV DNA in clinical samples and assessed the performance of the assay by comparing the results obtained in 144 biological samples with those resulting from the use of an in-house real-time PCR (qPCR). Overall, the concordance between the two assays was 95%, with samples identified concordantly as MPXV DNA positive and having a mean number of copies per μl of 1708 (95% CI: 107–2830 copies/μl). The remaining samples gave discordant results, with 5 out of 7 detected with the QIAcuity dPCR assay but not with the in-house qPCR. MPXV DNA levels measured by QIAcuity dPCR were strongly correlated with the Ct values detected by in-house qPCR and with those detected by another dPCR assay previously developed in our laboratories. The QIAcuity dPCR assay may be a robust and easy-to-perform method for MPXV DNA quantification in several biological samples.

Digital droplet PCR-based quantification of ccfHPV-DNA as liquid biopsy in HPV-driven cervical and vulvar cancer.

More than 99% of cervical cancers and up to 40% of vulvar cancers are human papillomavirus (HPV) related. HPV 16 and 18 are the most relevant subtypes. Novel technologies allow the detection of minimal amounts of circulating cell-free HPV DNA (ccfHPV-DNA). The aim of this study was to evaluate ccfHPV-DNA assessed by droplet digital PCR (ddPCR) as a biomarker for molecular therapy monitoring in early, advanced, relapsed and metastatic HPV-driven cervical and vulvar cancer. Inclusion criteria of the study were histologically proven HPV 16/18-driven cervical and vulvar cancer with first diagnosed disease, newly diagnosed recurrence, or progression of disease. Blood samples were taken pre- and post-therapeutically. Circulating cell-free HPV DNA was quantified using ddPCR and the results were correlated with clinical data. The mean copy number of ccfHPV-DNA was 838.6 (± 3089.1) in pretreatment and 2.3 (± 6.4) in post-treatment samples (p < 0.05). The copy number of ccfHPV-DNA increased with higher FIGO stages (p < 0.05), which are commonly used for clinical staging/assessment. Furthermore, we compared the distribution of copy numbers between T-stage 1 versus T-stage 2/3. We could show higher copy number level of ccfHPV-DNA in T-stage 2/3 (p < 0.05). Therapy monitoring with determination of ccfHPV-DNA by ddPCR with a small amount of plasma reflects response to therapy and appears feasible for patients in advanced cancer stages of cervical and vulvar cancer. This promising tool should be examined as marker of therapy monitoring in particular in novel HPV-directed therapies.

MiR-31-5p as a Potential Circulating Biomarker and Tracer of Clinical Improvement for Chronic Inflammatory Demyelinating Polyneuropathy.

MicroRNAs are endogenous, small noncoding RNA molecules that play a pivotal role in the regulation of gene expression. MicroRNAs are involved in many biological processes such as proliferation, cell differentiation, neovascularization, and apoptosis. Studies on microRNA expression may contribute to a better understanding of the pathomechanism of chronic inflammatory demyelinating polyneuropathy (CIDP) and consequently enable the development of new therapeutic measures using antisense miRNAs (antagomirs). In this study, we evaluated the level of miR-31-5p in the serum of patients with CIDP and its correlation with the miR-31-5p level and clinical presentation and electrophysiological and biochemical parameters. The study group consisted of 48 patients, mean age 61.60 ± 11.76, who fulfilled the diagnostic criteria of a typical variant of CIDP. The expression of miR-31-5p in patient serum probes was investigated by droplet digital PCR. The results were correlated with neurophysiological findings and the patient's clinical and biochemical parameters. The mean copy number of miRNA-31 in 100 μl serum was 1288.64 ± 2001.02 in the CIDP group of patients, while in the control group, it was 3743.09 ± 4026.90. There was a significant positive correlation (0.426) between IgIV treatment duration and miR-31-5p expression. Patients without IgIV treatment showed significantly lower levels of miR-31 compared to the treated group (259.44 ± 304.02 vs. 1559.48 ± 2168.45; p = 0.002). The group of patients with body weight > 80 kg showed statistically significantly lower levels of miRNA-31-5p than the patients with lower body weight (934.37 ± 1739.66 vs. 1784.62 ± 2271.62, respectively; p = 0.014). Similarly, the patients with elevated cerebrospinal fluid (CSF) protein levels had significantly higher miRNA-31-5p expression than those with normal protein levels (1393.93 ± 1932.27 vs. 987.38 ± 2364.10, respectively; p = 0.044). The results may support the hypothesis that miR-31-5p is strongly involved in the autoimmune process in CIDP. The positive correlation between higher miR-31-5p levels and duration of IVIg treatment may be an additional factor explaining the efficacy of prolonged IVIg therapy in CIDP.

Two fragments of HBV DNA integrated into chrX: 11009033 and its genetic regulation in HepG2.2.15.

Hepatitis B virus (HBV) integration into human genome causes hepatocellular carcinoma (HCC). The present study used inverse nested PCR; the full sequence of HBV DNA fragments of the chrX: 111009033 integration site was detected (987bp), containing two fragments of double‑stranded linear DNA with the same orientation (1,744‑1,094 and 1,565‑1,228nt). By reverse transcription‑quantitative PCR, HBV‑cell fusion transcript was observed in HepG2.2.15 cells. The mean copy number of this site in cells with H2O2 treatment (8.73x10‑2±1.65x10‑2 copies/cell) was significantly higher than that in the cells without H2O2 treatment (3.02x10‑2±2.33x10‑2 copies/cell; P<0.0001). The mean levels of P21‑activated kinase3 (PAK3) were 15.67±5.65 copies/cell in HepG2.2.15 cells with H2O2 treatment, significantly higher than in the cells without H2O2 treatment (11.34±4.58 copies/cell, P=0.0076) and in HepG2 cells (5.92±1.54 copies/cell, P<0.0001). Significant difference of PAK3 levels was also found between HepG2.2.15 cells without H2O2 treatment and HepG2 cells (11.34±4.58 vs. 5.92±1.54 copies/cell, P<0.0001). The average copy numbers of the integration site chrX: 111009033 were positively correlated with the average levels of PAK3 (P=0.0013). The overall trend of PAK3 expression was significantly increased in HepG2.2.15 cells with H2O2 treatment compared with that in HepG2.2.15 cells without H2O2 treatment (37.63±8.16 and 31.38±7.94, P=0.008) and HepG2 cells (21.67±7.88, P<0.0001). In summary, the chrX: 11009033 integration site may originate from primary human hepatocytes, occurrence and clonal expansion of which may upregulate PAK3 expression, which may contribute to hepatocarcinogenesis.

Multiple traces of monkeypox detected in non-sewered wastewater with sparse sampling from a densely populated metropolitan area in Asia

The monkeypox virus is excreted in the feces of infected individuals. Therefore, there is an interest in using viral load detection in wastewater for sentinel early surveillance at a community level and as a complementary approach to syndromic surveillance. We collected wastewater from 63 sewered and non-sewered locations in Bangkok city center between May and August 2022. Monkeypox viral DNA copy numbers were quantified using real-time polymerase chain reaction (PCR) and confirmed positive by Sanger sequencing. Monkeypox viral DNA was first detected in wastewater from the second week of June 2022, with a mean copy number of 16.4 copies/ml (n = 3). From the first week of July, the number of viral DNA copies increased to a mean copy number of 45.92 copies/ml. Positive samples were Sanger sequenced and confirmed the presence of the monkeypox virus. Our study is the first to detect monkeypox viral DNA in wastewater from various locations within Thailand. Results suggest that this could be a complementary source for detecting viral DNA and predicting upcoming outbreaks.

Characteristics of MYC Amplification and Their Association with Clinicopathological and Molecular Factors in Patients with Breast Cancer.

MYC amplification is detected in ∼15% of breast tumors and is associated with poor prognosis by mediating acquired resistance to anticancer therapies. This study aimed to determine the prevalence of MYC amplifications in Chinese women with breast cancer (BRCA) and investigate the correlation between MYC amplification and clinicopathological and molecular characteristics and its clinical implications. We analyzed MYC alterations in tissue specimens from 410 women diagnosed with BRCA in our hospital from June 1, 2017 to September 27, 2018. We compared our results with publicly available data from The Cancer Genome Atlas (TCGA) BRCA cohort (n = 1079). MYC amplification was identified in 12.4% (51/410) of our cohort, with mean copy number (CN) of 4.42 (range: 2.84-11.27). In TCGA cohort, MYC amplification was identified in 21.2% (229/1079) and was associated with age, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 (HER2) status, and molecular subtype, whereas in our cohort, MYC amplification was associated with smaller tumor size (T1-2, p = 0.023) and higher Ki-67 levels (≥20%; p = 0.031). Analysis of molecular profiles revealed that MYC-amplified breast tumors had significantly more concurrent CN variations compared with MYC nonamplified BRCA in both Guangdong Provincial People's Hospital (GDPH) and TCGA cohorts (p < 0.001). Pathway mapping analysis demonstrated that MYC-amplified tumors had more mutations involved in 15 different but interrelated pathways critical in DNA repair, cell cycle, and cell proliferation. Patients in TCGA cohort with MYC-amplified hormone receptor (HR)-positive/HER2-positive BRCA (p = 0.038) and MYC nonamplified triple-negative BRCA (p = 0.027) had significantly shorter overall survival. In conclusion, this study contributes to a better understanding that MYC-amplified breast tumors had distinct clinicopathological and molecular features compared with MYC nonamplified breast tumors. Further research with a larger sample size is necessary to further elucidate the clinical and survival implications of MYC amplifications.

Sequence polymorphisms of PR39 cathelicidins and extensive copy variations in commercial pig breeds

Copy number polymorphisms (CNPs) of antimicrobial peptides (AMPs) in livestock can influence the innate immune response of individuals. We conducted a high-resolution analysis of the genomic variations of porcine cathelicidin PR39 using cloned PR39 amplicons corresponding to the 5′ untranslated region (UTR) to 3′ UTR from four individuals of three different pig breeds. We identified 15 different sequences corresponding to 9 different coding domain sequences (CDSs), encoding 7 different protein sequences consisting of 3 functional and 4 non-functional forms. Subsequently, we developed a PR39 CNP typing method using real-time polymerase chain reaction (PCR) and analyzed the PR39 copy numbers from 44 pigs of six breeds. Significant variations in PR39 copies ranging from 2 to 10 copies, with a mean copy number of 5, were observed among all commercial breeds, except the wild boar. Among the different breeds, the PR39 copy number was highest (10) in Korean native pigs. Gene expression analysis showed that PR39 expression was correlated with the copy number. Moreover, the comparative analysis of the cathelicidin cluster-containing region among eight mammalian species showed the complete evolutionary conservation of the region, except for differences in the degree of cathelicidin expansion in each species. Therefore, characterization of CNPs in AMP genes could aid in improving the genetic potential of innate immune responses in livestock animals.

Airway Fusobacterium is Associated with Poor Response to Immunotherapy in Lung Cancer.

PurposeThere is a major limitation in the immunotherapy for solid cancer is that it only benefited a minority of cancer patients. This study aims to investigate whether the differential composition of the lung microbiome could affect the sustained clinical responses in lung cancers treated with immunotherapy.MethodsTwenty-seven non-responders and 19 responders treated with anti-PD-1 therapy were included in the discovery set. Bacterial load in bronchoalveolar lavage from lung cancer patients was examined by quantitative PCR of 16S rRNA copies. Bacterial 16S rDNA was sequenced using the Illumina HiSeq on the 16S rDNA V3-V4 variable region. Operational taxonomic unit (OTU) analysis was performed using VSEARCH v2. The α-diversity and β-diversity were calculated using QIIME software.ResultsThe mean copy number of bacterial 16S DNA levels significantly decreased after anti-PD-1 treatment (after: 1.8 ± 0.6×104 copies per milliliter vs prior to treatment: 3.3 ± 1.1x104, p = 0.0036). In addition, longitudinal analysis revealed that microbial diversity was reduced taxonomically after treatment compared to those prior to the treatment (Shannon values: before: 3.291 ± 0.067 vs after: 2.668 ± 0.168, p < 0.01). Further, we observed a reduction of Fusobacterium nucleatum, including phylum Fusobacteria (p < 0.01), class Fusobacteria (p < 0.01), order Fusobacteria (p < 0.01), family Fusobacteria (p < 0.01), genus Fusobacteria (p = 0.025) in the responders post anti-PD-1 treatment. However, there was no significant difference of Fusobacterium in non-responders. An independent cohort was used to validate the levels of Fusobacterium, demonstrating that patients with higher abundance of Fusobacterium prior to treatment were significantly more likely to have poor response to anti-PD-1 therapy (p < 0.001).ConclusionAirway enriched Fusobacterium prior to anti-PD-1 therapy is associated with poor response in lung cancer, which indicated that potential resistance to immunotherapy can be attributed to lung microbiome.

Defence by duplication: The relation between phenotypic glyphosate resistance and EPSPS gene copy number variation in Amaranthus palmeri.

Gene copy number variation (CNV) has been increasingly associated with organismal responses to environmental stress, but we know little about the quantitative relation between CNV and phenotypic variation. In this study we quantify the relation between variation in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) copy number using digital drop PCR and variation in phenotypic glyphosate resistance in 22 populations of Amaranthus palmeri (Palmer Amaranth), a range-expanding agricultural weed. Overall, we detected a significant positive relation between population mean copy number and resistance. The majority of populations exhibited high glyphosate resistance yet maintained low-resistance individuals, resulting in bimodality in many populations. We also investigated threshold models for the relation between copy number and resistance, and found evidence for a threshold of ~15 EPSPS copies: there was a steep increase in resistance below the threshold, followed by a much shallower increase. Across 924 individuals, as copy number increased the range of variation in resistance decreased, yielding an increasing frequency of high phenotypic resistance individuals. Among populations we detected a decline in variation (s.d.) as mean phenotypic resistance increased from moderate to high, consistent with the prediction that as phenotypic resistance increases in populations, stabilizing selection decreases variation in the trait. Our study demonstrates that populations of A. palmeri can harbour wide variation in EPSPS copy number and phenotypic glyphosate resistance, reflecting the history of, and template for future, resistance evolution.

Comparison of MET gene amplification analysis by next-generation sequencing and fluorescence in situ hybridization.

MET gene alterations are known to be involved in acquired resistance to epidermal growth factor receptor inhibition. MET amplifications present a potential therapeutic target in non-small cell lung cancer. Although next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) are conventionally used to assess MET amplifications, there are currently no clinically defined cut-off values for NGS, with FISH still being the gold standard. A collective of 20 formalin-fixed paraffin-embedded lung cancer tissue samples (mean age 64 years) were selected based on increased MET gene copy number (CNV) status or the presence of mutations detected by NGS (GeneReader, QIAGEN) and were further assessed by FISH (MET/CEN7, Zytomed). Of these, 17 tumor samples were MET-amplified and one patient was found to have a MET rearrangement by NGS, while two samples had no MET gene alteration. In contrast to the NGS result, FISH analysis showed only one highly amplified sample and 19 negative samples. The single highly amplified case detected by FISH was also positive by NGS with a fold change (FC) of 3.18 and a mean copy number (CNMV 10−100%) of 20.5. Therefore, for the assessment of MET amplifications using the QIAGEN NGS workflow, we suggest detecting amplified cases with an FC value of ≥ 3.0 and a CNMV 10−100% value of ≥ 20.0 by FISH. In summary, NGS allows for DNA- and RNA-based analysis of specific MET gene amplifications, point mutations or rearrangements.

Detection of Epstein-Barr virus encoded small RNA genes in oral squamous cell carcinoma and non-cancerous oral cavity samples

BackgroundEpstein-Barr Virus (EBV) is a human oncogenic virus that can lead to cancer in lymphoid and epithelial cells and is one of the hypothesized causes of oral cavity lesions including oral squamous cell carcinoma (OSCC), but the etiological association remains undetermined. The present investigation aimed to explore the EBV presence, viral load, and EBV-encoded small RNA (EBER) sequence variation in tissue samples of patients with OSCC and other oral cavity lesions including oral lichen planus (OLP), and oral irritation fibroma (OIF).MethodsIn total, 88 oral cavity samples (23 with OSCC, 29 with OLP, and 36 with OIF diagnosis) were examined by Real-Time PCR technique and some of them were sequenced.ResultsViral EBER sequence was detected in 6 out of the 23 OSCC (31.4%), 6 out of the 29 OLP (20.7%), and 3 out of the 36 OIF cases (8.3%). The mean EBV copy number was higher in OSCC samples (1.2 × 10−2 ± 1.3 × 10−2 copies/cell) compared to OLP (2.2 × 10−3 ± 2.6 × 10−3 copies/cell) and OIF (2.4 × 10−4 ± 2.0 × 10−4 copies/cell) samples, although this difference was not statistically significant (P = 0.318). The EBER gene was amplified and sequenced in 5 OSCC, 3 OLP, and 2 OIF samples with high EBV viral load. One OSCC, two OLP, and two OIF isolates showed different nucleotide variations compared with EBV-WT and AG876 prototype sequences: C6834T, C6870T, C6981T, C7085T, C7085G, and C7094T.ConclusionIn our study the presence of more than one genome copies per tumor cell indicates the possible role of EBV infection in oral cancers. However, more studies should be conducted to clarify the role of EBV in OSCC carcinogenesis.

Comprehensive Genomic Profiling of Circulating Cell-Free DNA Distinguishes Focal MET Amplification from Aneuploidy in Diverse Advanced Cancers.

Amplification (amp) of MET can be observed in cases of focal gene copy number gain, such as MET-driven amp, or with a gain of chromosome 7, such as aneuploidy. Several studies have shown that only high-level focal MET amp (MET/CEP7 ratio ≥5) is oncogenic, with such tumors responding to targeted therapy. However, there are few reports on how to distinguish between focal amplification and aneuploidy using next-generation sequencing (NGS). A total of 1025 patients with advanced solid tumors (typically pre-treated) were tested with a non-invasive comprehensive cfDNA NGS panel (Guardant360) from July 2014 to June 2019. Since bioinformatics upgrades of Guardant360 were undergoing in September 2018, focal MET amp was determined by our independent algorithm using the cohorts tested before September 2018 (291 patients), and validation was performed in the remaining cohort (734 patients). MET alterations (alts) associated with aberrant signaling were found in 110 patients (10.7%) among nine different cancer types, most commonly in non-small cell (12.2%, 62/510) and small cell (33.3%, 3/9) lung cancers, gastroesophageal cancer (19.4%, 7/36), and prostate adenocarcinoma (15.6%; 5/32). Among 291 patients tested before September 2018, 37 (12.7%) had MET alts. Among these, 24 (64.9%) had amps, 5 (13.5%) had exon 14 skipping, and 13 (35.1%) had single nucleotide variants (SNVs). Co-alterations, such as amp + SNVs, were found in four samples (10.8%). Among 24 MET amps, 29.2% (7/24) were focal according to our algorithm. MET copy number was significantly higher with focal amp compared to non-focal amp (mean copy number 3.26 vs. 2.44, respectively, p = 0.00304). In 734 patients tested after September 2018, our definition of focal MET amp was detected in 4.2% (31/734). Overall, focal amplification based on our algorithm was 3.7% (=38/1025). This study describes an approach to distinguish focal and non-focal MET amplification using comprehensive genomic profiling of cfDNA in advanced cancer patients. Focal MET amp accounted for ~30% of all MET amp, which was found in 3.7% of patients with diverse cancers and was associated with a higher plasma copy number. Clinical studies are warranted to assess the clinical utility of targeted therapies for tumors with focal MET amplification detected by NGS of cfDNA.

Circulating Cell-Free Mitochondrial DNA in Cerebrospinal Fluid as a Biomarker for Mitochondrial Diseases.

Mitochondrial diseases (MD) are genetic metabolic disorders that impair normal mitochondrial structure or function. The aim of this study was to investigate the status of circulating cell-free mitochondrial DNA (ccfmtDNA) in cerebrospinal fluid (CSF), together with other biomarkers (growth differentiation factor-15 [GDF-15], alanine, and lactate), in a cohort of 25 patients with a molecular diagnosis of MD. Measurement of ccfmtDNA was performed by using droplet digital PCR. The mean copy number of ccfmtDNA was approximately 6 times higher in the MD cohort compared to the control group; patients with mitochondrial deletion and depletion syndromes (MDD) had the higher levels. We also detected the presence of both wild-type mtDNA and mtDNA deletions in CSF samples of patients with single deletions. Patients with MDD with single deletions had significantly higher concentrations of GDF-15 in CSF than controls, whereas patients with point mutations in mitochondrial DNA presented no statistically significant differences. Additionally, we found a significant positive correlation between ccfmtDNA levels and GDF-15 concentrations (r = 0.59, P = 0.016). CSF ccfmtDNA levels are significantly higher in patients with MD in comparison to controls and, thus, they can be used as a novel biomarker for MD research. Our results could also be valuable to support the clinical outcome assessment of MD patients.

Clinical usefulness of 16S ribosomal RNA real-time PCR for the diagnosis of scrub typhus

Scrub typhus is a major acute febrile disease in the Asia–Pacific region. The purpose of the present study is to investigate the clinical usefulness of real-time PCR (Q-PCR) of 16S rRNA for the diagnosis of scrub typhus. We examined blood specimens from 148 adult patients who were confirmed to have scrub typhus from September 2008 to December 2009. Among the 148 scrub typhus patients, 36 patients were treated with antibiotics before admission. To evaluate the clinical usefulness of 16S rRNA Q-PCR, we compared its diagnostic accuracy to the accuracy of the following methods: nested PCR (N-PCR) targeting the gene encoding the 56-kDa protein, Q-PCR targeting the gene encoding the 47-kDa protein, and conventional PCR (C-PCR), targeting the 16S rRNA gene. According to 16S rRNA Q-PCR and 47-kDa Q-PCR, the mild group had copy numbers of 234.4 ± 261.9 and 130.5 ± 128.3, whereas the severe group had copy numbers of 584.4 ± 911.4 and 244.7 ± 210.9, respectively. In both tests, the mean copy numbers were significantly greater in the severe group (P = 0.037 and P = 0.035). 16S rRNA Q-PCR detected Orientia tsutsugamushi infections with a sensitivity of 91.9% (95% CI 86.3–95.7), and 56-kDa N-PCR, 47-kDa Q-PCR, and 16S rRNA C-PCR exhibited lower sensitivities of 81.1% (95% CI 73.8–87.0), 74.3% (95% CI 66.5–81.1), and 87.8% (95% CI 81.5–92.6), respectively, for all 148 patients. In addition, 16S rRNA Q-PCR exhibited a sensitivity of 99.1% (95% CI 95.1–100.0) in the 112 patients who were not treated with antibiotics before admission. 16S rRNA Q-PCR is clinically useful for the rapid diagnosis of scrub typhus and is more accurate than the 56-kDa N-PCR, 47-kDa Q-PCR, and 16S C-PCR methods.

Molecular Peritoneal Staging for Pancreatic Ductal Adenocarcinoma Using Mutant KRAS Droplet-Digital Polymerase Chain Reaction: Results of a Prospective Clinical Trial

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with predilection for peritoneal dissemination. Accurate peritoneal staging is imperative for treatment recommendations, as one-third of patients develop peritoneal recurrence after resection. Because >90% of PDAC tumors harbor mutant KRAS (mKRAS), we sought to determine feasibility of mKRAS DNA detection in peritoneal lavage (PL) fluid using droplet-digital polymerase chain reaction (ddPCR) via a prospective trial. Patients with nonmetastatic PDAC undergoing staging laparoscopy with PL were included. PL fluid was sent for cytologic examination, CA19-9/CEA levels, and mKRAS ddPCR assay. Clinically positive laparoscopy was defined as gross metastases or positive cytology. PL mKRAS status was compared with gross findings, cytology, and CA19-9/CEA levels. There were 136 patients enrolled; 70 of 136 (51%) patients received neoadjuvant therapy before PL, and 32 of 136 (24%) patients had clinically positive laparoscopy. Cytology was positive in 17 of 136 (13%) patients, and 22 of 136 (16%) patients had gross metastases. Of patients with gross metastases, only 8 of 22 (36%) had positive cytology; 97 of 136 (71%) patients had mKRAS in PL. PL mKRAS was present in 27 of 32 (84%) clinically positive laparoscopies, with higher mean copy number in clinically positive patients (643 vs 10, p= 0.02). Peritoneal mKRAS was positive in an additional 70 clinically negative patients. This prospective study establishes the feasibility of PL mKRAS detection. Clinically positive disease was identified in 1 in 4 staging laparoscopies. Although PL mKRAS was highly associated with clinically positive findings, many clinically negative laparoscopies had detectable PL mKRAS, suggesting that standard staging may be inadequate. Longer follow-up will elucidate utility of this promising molecular assay.