Plasma EBV DNA: A Promising Diagnostic Marker for Endemic Burkitt Lymphoma

Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in regions of equatorial Africa where P. falciparum malaria is holoendemic. The tumor is consistently associated with Epstein-Barr virus (EBV). Screening for EBV DNA in plasma in a high-risk population in Hong Kong has been shown to be useful in facilitating the early diagnosis of nasopharyngeal carcinoma, another EBV-associated tumor. Here, we investigate plasma EBV as a diagnostic marker for eBL in children in Uganda. We studied plasma specimens from 25 children with eBL and 25 controls matched for age (<3-16 years), gender and geography, including many with asymptomatic P. falciparum infection. These specimens were previously collected under the auspices of the EMBLEM (Epidemiology of Burkitt lymphoma in East African children and minors) study. After cell-free DNA isolation, plasma EBV DNA was measured using a quantitative PCR assay that amplifies the large internal repeats of the EBV genome. All children with eBL had measurable plasma EBV, as compared to 84% of control children. The median plasma EBV DNA level was 5.23 log10 copies/mL (interquartile range 3.54-6.08 log10 copies/mL) in children with eBL. In contrast, the median plasma EBV DNA level was 0.37 log10 copies/mL (interquartile range 0.18-1.05 log10 copies/mL) in children without lymphoma. An EBV threshold of 2.52 log10 copies/mL yielded a sensitivity of.88 and a specificity of 1. The estimated AUC was 0.936 (95% CI: 0.8496 – 1.00) for the corresponding ROC curve. Plasma EBV copy number did not depend on age, gender, or malaria screening status. However, two control children with asymptomatic P. falciparum infection and parasitemia also had high plasma EBV copy number. Our analysis suggests that measurements of EBV copy number in plasma may be useful in identifying children with eBL versus control children. A promising area for future research is the differentiation of high copy number associated with tumor versus high copy number associated with asymptomatic parasitemia.

MET Gene High Copy Number (Amplification/Polysomy) Identified in Melanoma for Potential Targeted Therapy

Objectives Aberrant expression of the mesenchymal epithelial transition factor (MET) gene has been observed in several malignancies, and drugs targeting the MET gene have been implicated in clinical trials with promising results. Hence, MET is a potentially targetable oncogenic driver. We explored the frequency of MET gene high copy number in melanomas and carcinomas. Methods The study group included 135 patients. Tissue microarrays were constructed with 19 melanomas and 116 carcinomas diagnosed from 2010 to 2012. We screened MET gene copy number by fluorescence in situ hybridization analysis using probes for MET gene and CEP7 as control. Results We found MET gene amplification in 2 (11%) of 19 melanoma cases, whereas 5 (26%) of 19 melanoma cases showed polysomy. For carcinomas, there was no MET gene amplification identified. However, 8 (7%) of 116 cases showed polysomy. Conclusions In our study, MET gene amplification was identified in 11% of melanomas and is relatively concordant with few reported studies. However, about 26% of the additional melanoma cases showed MET gene polysomy, which has not been reported as per our knowledge. If these results are validated with further orthogonal studies, more of the melanoma cases could potentially benefit from targeted therapy with MET tyrosine kinase inhibitors.

More from less: Genome skimming for nuclear markers for animal phylogenomics, a case study using decapod crustaceans

Low coverage genome sequencing is rapid and cost-effective for recovering complete mitochondrial genomes for crustacean phylogenomics. The recovery of high-copy-number nuclear genes, including histone H3, 18S and 28S ribosomal RNAs, is also possible using this approach based on our research with freshwater crayfishes (Astacidea). We explored the potential of genome skimming (GS) to recover additional nuclear genes from shallow sequencing projects using decapod crustaceans. Using an in silico-baited approach, we recovered three additional core histone genes (H2A, H2B, and H4) from our low-coverage decapod dataset (99 species, 69 genera, 38 families, 10 infraorders). Phylogenetic analyses using various combinations of mitochondrial and nuclear genes for the entire decapod dataset and a subset of 40 species of crayfishes showed that the evolutionary rates for different classes of genes varied widely. A very high level of congruence was nevertheless found between trees from the six nuclear genes and those derived from the mitogenome sequences for freshwater crayfish. These findings indicate that nuclear genes recovered from the same genome skimming datasets designed to obtain mitogenomes can be used to support more robust and comprehensive phylogenetic analyses. Further, a search for additional intron-less nuclear genes identified several high-copy-number genes across the decapod dataset, and recovery of NaK, PEPCK, and GAPDH gene fragments is possible at slightly elevated coverage, suggesting the potential and utility of GS in recovering even more nuclear genetic information for phylogenetic studies from these inexpensive and increasingly abundant datasets.

More from less: Genome skimming for nuclear markers for animal phylogenomics, a case study using decapod crustaceans

Low coverage genome sequencing is rapid and cost-effective for recovering complete mitochondrial genomes for animal phylogenomics. The recovery of high copy number nuclear genes, including histone H3, 18S and 28S ribosomal RNAs, is also possible using this approach. In this study, we explore the potential of the genome skimming (GS) to recover additional nuclear genes from shallow sequencing projects. Using an in silico baited approach, we recover three additional core histone genes (H2A, H2B and H4) from our existing collection of low coverage decapod crustacean dataset (99 species, 69 genera, 38 families, 10 infraorders). Phylogenetic analyses based on various combinations of mitochondrial and nuclear genes for the entire decapod dataset and 40 species of crayfish (Infraorder Astacidea) found that the evolutionary rates for different classes of genes varied widely. The highlight being a very high level of congruence found between trees from the six nuclear genes and those derived from the mitogenome sequences for freshwater crayfish. These findings indicate that nuclear genes recovered from the same genome skimming datasets designed to obtain mitogenomes can be used to support more robust and comprehensive phylogenetic analyses. Further, a search for additional intron-less nuclear genes identified several high copy number genes across the decapod dataset and recovery of NaK, PEPCK and GAPDH gene fragments is possible at slightly elevated coverage, suggesting the potential and utility of GS in recovering even more nuclear genetic information for phylogenetic studies from these inexpensive and increasingly abundant datasets.

Clinical and Molecular Description of a High-Copy IncQ1 KPC-2 Plasmid Harbored by the International ST15 Klebsiella pneumoniae Clone

ABSTRACT This study provides the genomic characterization and clinical description of bloodstream infections (BSI) cases due to ST15 KPC-2 producer Klebsiella pneumoniae. Six KPC-K. pneumoniae isolates were recovered in 2015 in a tertiary Brazilian hospital and were analyzed by whole-genome sequencing (WGS) (Illumina MiSeq short reads). Of these, two isolates were further analyzed by Nanopore MinION sequencing, allowing complete chromosome and plasmid circularization (hybrid assembly), using Unicycler software. The clinical analysis showed that the 30-day overall mortality for these BSI cases was high (83%). The isolates exhibited meropenem resistance (MICs, 32 to 128 mg/liter), with 3/6 isolates resistant to polymyxin B. The conjugative properties of the blaKPC-2 plasmid and its copy number were assessed by standard conjugation experiments and sequence copy number analysis. We identified in all six isolates a small (8.3-kb), high-copy-number (20 copies/cell) non-self-conjugative IncQ plasmid harboring blaKPC-2 in a non-Tn4401 transposon. This plasmid backbone was previously reported to harbor blaKPC-2 only in Brazil, and it could be comobilized at a high frequency (10−4) into Escherichia coli J53 and into several high-risk K. pneumoniae clones (ST258, ST15, and ST101) by a common IncL/M helper plasmid, suggesting the potential of international spread. This study thus identified the international K. pneumoniae ST15 clone as a carrier of blaKPC-2 in a high-copy-number IncQ1 plasmid that is easily transmissible among other common Klebsiella strains. This finding is of concern since IncQ1 plasmids are efficient antimicrobial resistance determinant carriers across Gram-negative species. The spread of such carbapenemase-encoding IncQ1 plasmids should therefore be closely monitored. IMPORTANCE In many parts of the world, carbapenem resistance is a serious public health concern. In Brazil, carbapenem resistance in Enterobacterales is mostly driven by the dissemination of KPC-2-producing K. pneumoniae clones. Despite being endemic in this country, only a few reports providing both clinical and genomic data are available in Brazil, which limit the understanding of the real clinical impact caused by the dissemination of different clones carrying blaKPC-2 in Brazilian hospitals. Although several of these KPC-2-producer K. pneumoniae isolates belong to the clonal complex 258 and carry Tn4401 transposons located on large plasmids, a concomitant emergence and silent dissemination of small high-copy-number blaKPC-2 plasmids are of importance, as described in this study. Our data identify a small high-copy-number IncQ1 KPC plasmid, its clinical relevance, and the potential for conjugative transfer into several K. pneumoniae isolates, belonging to different international lineages, such as ST258, ST101, and ST15.

Clinicopathological and Molecular Characteristics of Atypical Pulmonary Sclerosing Pneumocytoma With Multiple Metastases

Background: Few patients with pulmonary sclerosing pneumocytoma (PSP) may suffer from recurrence and oligometastasis as it is a benign tumor or has a low malignancy potential. Herein, an in-depth study, based on an extremely rare PSP case with atypical features, was carried out to elaborate the potential mechanism underlying the rapid malignant progression. Methods: The clinicopathological data of this atypical PSP (AP) case were obtained. Formalin‑fixed and paraffin‑embedded tissues from all the lesions of AP and five classic PSP cases (as control) were used for whole-exome sequencing (WES) and immunohistochemistry. Results: A 23-year-old male showed a 6.5-cm pulmonary nodule in the right middle lobe (RML) and enlarged mediastinal lymph nodes (LNs). He underwent thoracoscopic RML lobectomy, systematic LNs dissection, and mediastinal lymphadenectomy. The metastases to the cervical LNs and liver were detected in a short period and then resected. Postoperative pathological examination confirmed the diagnosis of PSP in all the lesions, based on the typical histological characteristics and immunophenotypes. Furthermore, WES identified both AKT1 E17K somatic mutation and TP53 C176Y germline mutation in this AP case. The genomic evolution analysis showed different evolutionary branches in the metastatic tumors distinct from the primary lesion. Moreover, compared to the control group, the AP case showed high copy number variations (CNVs) and significantly high copy number instability (CNI). Conclusions: We speculated that the atypical histopathological features and malignant behaviors may be due to the co-mutations of somatic AKT1 E17K and germline TP53 C176Y, combined with the high CNVs and CNI.

Vector backbone integration in transgenic cassava is significantly correlated to T-DNA copy number

Multiple T-DNA integrations often occur with transgenic technology, resulting in complex integration patterns and transgene silencing. This study, investigates the correlation coefficient of T-DNA copy number on vector backbone (VBB) integration in transgenic cassava using Dot blot and PCR analysis. Thirty-nine, fifty-one and thirty-eight transgenic cassava plant lines recovered from transformations of cassava friable embryogenic callus with A. tumefaciens strain LBA4404 independently carrying p8016, p8052, and p900 were randomly selected and evaluated for VBB integration and T-DNA copy number. The occurrences of events with low (1-2) and high (≥ 3) T-DNA copy numbers were correlated with the presence and absence of VBB integration. Seventy-two to ninety-eight percent of VBB-free events were low copy number events while 2 to 28% of same where high copy number events. Correlation coefficient of the data revealed that the number of VBB-free events showed a significant positive correlation (r = 0.821, n = 9, p = 0.01) for events with low T-DNA copy number and a significant negative correlation (r = -0.739, n =9, p = 0.02) for high copy number events. This shows that the recovery of events with low T-DNA copy number increases the chances of recovering VBB-free events thereby enhancing the production of quality transgenic events. Key words: Copy number, DsRed, T-DNA, transformation, transgenic cassava, vector backbone integration.