Liquidhope: methylome and genomic profiling from very limited quantities of plasma-derived DNA.

Analysis of the methylome of tumor cell-free deoxyribonucleic acid (DNA; cfDNA) has emerged as a powerful non-invasive technique for cancer subtyping and prognosis. However, its application is frequently hampered by the quality and total cfDNA yield. Here, we demonstrate the feasibility of very low-input cfDNA for whole-methylome and copy-number profiling studies using enzymatic conversion of unmethylated cysteines [enzymatic methyl-seq (EM-seq)] to better preserve DNA integrity. We created a model for predicting genomic subtyping and prognosis with high accuracy. We validated our tool by comparing whole-genome CpG sequencing with in situ cohorts generated with bisulfite conversion and array hybridization, demonstrating that, despite the different techniques and sample origins, information on cfDNA methylation is comparable with in situ cohorts. Our findings support use of liquid biopsy followed by EM-seq to assess methylome of cancer patients, enabling validation in external cohorts. This advance is particularly relevant for rare cancers like neuroblastomas where liquid-biopsy volume is restricted by ethical regulations in pediatric patients.

A Tar aspartate receptor and Rubisco-like protein substitute biotin in the growth of rhizobial strains.

Biotin is a key cofactor of metabolic carboxylases, although many rhizobial strains are biotin auxotrophs. When some of these strains were serially subcultured in minimal medium, they showed diminished growth and increased excretion of metabolites. The addition of biotin, or genetic complementation with biotin synthesis genes resulted in full growth of Rhizobium etli CFN42 and Rhizobium phaseoli CIAT652 strains. Half of rhizobial genomes did not show genes for biotin biosynthesis, but three-quarters had genes for biotin transport. Some strains had genes for an avidin homologue (rhizavidin), a protein with high affinity for biotin but an unknown role in bacteria. A CFN42-derived rhizavidin mutant showed a sharper growth decrease in subcultures, revealing a role in biotin storage. In the search of biotin-independent growth of subcultures, CFN42 and CIAT652 strains with excess aeration showed optimal growth, as they also did, unexpectedly, with the addition of aspartic acid analogues α- and N-methyl aspartate. Aspartate analogues can be sensed by the chemotaxis aspartate receptor Tar. A tar homologue was identified and its mutants showed no growth recovery with aspartate analogues, indicating requirement of the Tar receptor in such a phenotype. Additionally, tar mutants did not recover full growth with excess aeration. A Rubisco-like protein was found to be necessary for growth as the corresponding mutants showed no recovery either with high aeration or aspartate analogues; also, diminished carboxylation was observed. Taken together, our results indicate a route of biotin-independent growth in rhizobial strains that included oxygen, a Tar receptor and a previously uncharacterized Rubisco-like protein.

The Embryonic Key Pluripotent Factor NANOG Mediates Glioblastoma Cell Migration via the SDF1/CXCR4 Pathway.

NANOG is a key transcription factor required for maintaining pluripotency of embryonic stem cells. Elevated NANOG expression levels have been reported in many types of human cancers, including lung, oral, prostate, stomach, breast, and brain. Several studies reported the correlation between NANOG expression and tumor metastasis, revealing itself as a powerful biomarker of poor prognosis. However, how NANOG regulates tumor progression is still not known. We previously showed in medaka fish that Nanog regulates primordial germ cell migration through Cxcr4b, a chemokine receptor known for its ability to promote migration and metastasis in human cancers. Therefore, we investigated the role of human NANOG in CXCR4-mediated cancer cell migration. Of note, we found that NANOG regulatory elements in the CXCR4 promoter are functionally conserved in medaka fish and humans, suggesting an evolutionary conserved regulatory axis. Moreover, CXCR4 expression requires NANOG in human glioblastoma cells. In addition, transwell assays demonstrated that NANOG regulates cancer cell migration through the SDF1/CXCR4 pathway. Altogether, our results uncover NANOG-CXCR4 as a novel pathway controlling cellular migration and support Nanog as a potential therapeutic target in the treatment of Nanog-dependent tumor progression.

Phase 3 Trial of 177Lu-Dotatate for Midgut Neuroendocrine Tumors.

BACKGROUND: Patients with advanced midgut neuroendocrine tumors who have had disease progression during first-line somatostatin analogue therapy have limited therapeutic options. This randomized, controlled trial evaluated the efficacy and safety of lutetium-177 (177Lu)-Dotatate in patients with advanced, progressive, somatostatin-receptor-positive midgut neuroendocrine tumors. METHODS: We randomly assigned 229 patients who had well-differentiated, metastatic midgut neuroendocrine tumors to receive either 177Lu-Dotatate (116 patients) at a dose of 7.4 GBq every 8 weeks (four intravenous infusions, plus best supportive care including octreotide long-acting repeatable [LAR] administered intramuscularly at a dose of 30 mg) (177Lu-Dotatate group) or octreotide LAR alone (113 patients) administered intramuscularly at a dose of 60 mg every 4 weeks (control group). The primary end point was progression-free survival. Secondary end points included the objective response rate, overall survival, safety, and the side-effect profile. The final analysis of overall survival will be conducted in the future as specified in the protocol; a prespecified interim analysis of overall survival was conducted and is reported here. RESULTS: At the data-cutoff date for the primary analysis, the estimated rate of progression-free survival at month 20 was 65.2% (95% confidence interval [CI], 50.0 to 76.8) in the 177Lu-Dotatate group and 10.8% (95% CI, 3.5 to 23.0) in the control group. The response rate was 18% in the 177Lu-Dotatate group versus 3% in the control group (P<0.001). In the planned interim analysis of overall survival, 14 deaths occurred in the 177Lu-Dotatate group and 26 in the control group (P=0.004). Grade 3 or 4 neutropenia, thrombocytopenia, and lymphopenia occurred in 1%, 2%, and 9%, respectively, of patients in the 177Lu-Dotatate group as compared with no patients in the control group, with no evidence of renal toxic effects during the observed time frame. CONCLUSIONS: Treatment with 177Lu-Dotatate resulted in markedly longer progression-free survival and a significantly higher response rate than high-dose octreotide LAR among patients with advanced midgut neuroendocrine tumors. Preliminary evidence of an overall survival benefit was seen in an interim analysis; confirmation will be required in the planned final analysis. Clinically significant myelosuppression occurred in less than 10% of patients in the 177Lu-Dotatate group. (Funded by Advanced Accelerator Applications; NETTER-1 ClinicalTrials.gov number, NCT01578239 ; EudraCT number 2011-005049-11 .).

Robust association between vascular habitats and patient prognosis in glioblastoma: An international multicenter study.

BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor, characterized by a heterogeneous and abnormal vascularity. Subtypes of vascular habitats within the tumor and edema can be distinguished: high angiogenic tumor (HAT), low angiogenic tumor (LAT), infiltrated peripheral edema (IPE), and vasogenic peripheral edema (VPE). PURPOSE: To validate the association between hemodynamic markers from vascular habitats and overall survival (OS) in glioblastoma patients, considering the intercenter variability of acquisition protocols. STUDY TYPE: Multicenter retrospective study. POPULATION: In all, 184 glioblastoma patients from seven European centers participating in the NCT03439332 clinical study. FIELD STRENGTH/SEQUENCE: 1.5T (for 54 patients) or 3.0T (for 130 patients). Pregadolinium and postgadolinium-based contrast agent-enhanced T1 -weighted MRI, T2 - and FLAIR T2 -weighted, and dynamic susceptibility contrast (DSC) T2 * perfusion. ASSESSMENT: We analyzed preoperative MRIs to establish the association between the maximum relative cerebral blood volume (rCBVmax ) at each habitat with OS. Moreover, the stratification capabilities of the markers to divide patients into "vascular" groups were tested. The variability in the markers between individual centers was also assessed. STATISTICAL TESTS: Uniparametric Cox regression; Kaplan-Meier test; Mann-Whitney test. RESULTS: The rCBVmax derived from the HAT, LAT, and IPE habitats were significantly associated with patient OS (P < 0.05; hazard ratio [HR]: 1.05, 1.11, 1.28, respectively). Moreover, these markers can stratify patients into "moderate-" and "high-vascular" groups (P < 0.05). The Mann-Whitney test did not find significant differences among most of the centers in markers (HAT: P = 0.02-0.685; LAT: P = 0.010-0.769; IPE: P = 0.093-0.939; VPE: P = 0.016-1.000). DATA CONCLUSION: The rCBVmax calculated in HAT, LAT, and IPE habitats have been validated as clinically relevant prognostic biomarkers for glioblastoma patients in the pretreatment stage. This study demonstrates the robustness of the hemodynamic tissue signature (HTS) habitats to assess the GBM vascular heterogeneity and their association with patient prognosis independently of intercenter variability. LEVEL OF EVIDENCE: 3 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2020;51:1478-1486.

Evaluation of the expanding spectrum of sertraline against uncommon fungal pathogens.

The emergence of non-Aspergillus mold pathogens has increased notoriously in the last decades with serious health consequences. The options of treatment for these microorganisms often resistant to a wide variety of antifungals is limited. Sertraline is an antidepressant with in vitro and in vivo antifungal properties which has been recently studied as an adjuvant in the treatment of invasive infections. In this study, we evaluated the in vitro interaction of sertraline with voriconazole and amphotericin B against Lomentospora prolificans, Scedosporium spp., Fusarium spp., Paecilomyces spp., Alternaria spp. and Curvularia spp. The minimum inhibitory concentration and minimum fungicidal concentration for sertraline were in the range of 8-32 µg/mL. Sertraline showed antifungal capacity against all fungi tested and synergism in combination with amphotericin B against some strains of Lomentospora prolificans, Scedosporium apiospermum and Alternaria alternata, antagonism with voriconazole against Purpureocillium lilacinum and indifference in both combinations for most of the other strains tested. These results suggest a potential role of sertraline as an adjuvant in the treatment of some of these serious mycoses.

MTHFR and VDR Polymorphisms Improve the Prognostic Value of MYCN Status on Overall Survival in Neuroblastoma Patients.

Single nucleotide polymorphisms (SNPs) in Pharmacogenetics can play an important role in the outcomes of the chemotherapy treatment in Neuroblastoma, helping doctors maximize efficacy and minimize toxicity. Employing AgenaBioscience MassArray, 96 SNPs were genotyped in 95 patients looking for associations of SNP with response to induction therapy (RIT) and grade 3-4 toxicities, in High Risk patients. Associations of SNPs with overall (OS) and event-free (EFS) survival in the whole cohort were also explored. Cox and logistic regression models with Elastic net penalty were employed. Association with grade 3-4 gastrointestinal and infectious toxicities was found for 8 different SNPs. Better RIT was correlated with rs726501 AG, rs3740066 GG, rs2010963 GG and rs1143684 TT (OR = 2.87, 1.79, 1.23, 1.14, respectively). EFS was affected by rs2032582, rs4880, rs3814058, rs45511401, rs1544410 and rs6539870. OS was influenced by rs 1801133, rs7186128 and rs1544410. Remarkably, rs1801133 in MTHFR (p = 0.02) and rs1544410 in VDR (p = 0.006) also added an important predictive value for OS to the MYCN status, with a more accurate substratification of the patients. Although validation studies in independent cohorts will be required, the data obtained supports the utility of Pharmacogenetics for predicting Neuroblastoma treatment outcomes.

Analysis of genome sequence and symbiotic ability of rhizobial strains isolated from seeds of common bean (Phaseolus vulgaris).

BACKGROUND: Rhizobia are alpha-proteobacteria commonly found in soil and root nodules of legumes. It was recently reported that nitrogen-fixing rhizobia also inhabit legume seeds. In this study, we examined whole-genome sequences of seven strains of rhizobia isolated from seeds of common bean (Phaseolus vulgaris). RESULTS: Rhizobial strains included in this study belonged to three different species, including Rhizobium phaseoli, R. leguminosarum, and R. grahamii. Genome sequence analyses revealed that six of the strains formed three pairs of highly related strains. Both strains comprising a pair shared all but one plasmid. In two out of three pairs, one of the member strains was effective in nodulation and nitrogen fixation, whereas the other was ineffective. The genome of the ineffective strain in each pair lacked several genes responsible for symbiosis, including nod, nif, and fix genes, whereas that of the effective strain harbored the corresponding genes in clusters, suggesting that recombination events provoked gene loss in ineffective strains. Comparisons of genomic sequences between seed strains and nodule strains of the same species showed high conservation of chromosomal sequences and lower conservation of plasmid sequences. Approximately 70% of all genes were shared among the strains of each species. However, paralogs were more abundant in seed strains than in nodule strains. Functional analysis showed that seed strains were particularly enriched in genes involved in the transport and metabolism of amino acids and carbohydrates, biosynthesis of cofactors and in transposons and prophages. Genomes of seed strains harbored several intact prophages, one of which was inserted at exactly the same genomic position in three strains of R. phaseoli and R. leguminosarum. The R. grahamii strain carried a prophage similar to a gene transfer agent (GTA); this represents the first GTA reported for this genus. CONCLUSIONS: Seeds represent a niche for bacteria; their access by rhizobia possibly triggered the infection of phages, recombination, loss or gain of plasmids, and loss of symbiosis genes. This process probably represents ongoing evolution that will eventually convert these strains into obligate endophytes.

Genomic studies of nitrogen-fixing rhizobial strains from Phaseolus vulgaris seeds and nodules.

BACKGROUND: Rhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. americanum (CCGM7) strains from seeds and compared them with those of the closely related strains CIAT652 and CFNEI73, respectively, isolated only from nodules. RESULTS: In a fine structural study of the S. americanum genomes, the chromosomes, megaplasmids and symbiotic plasmids were highly conserved and syntenic, with the exception of the smaller plasmid, which appeared unrelated. The symbiotic tract of CCGM7 appeared more disperse, possibly due to the action of transposases. The chromosomes of seed strains had less transposases and strain-specific genes. The seed strains CCGM1 and CCGM7 shared about half of their genomes with their closest strains (3353 and 3472 orthologs respectively), but a large fraction of the rest also had homology with other rhizobia. They contained 315 and 204 strain-specific genes, respectively, particularly abundant in the functions of transcription, motility, energy generation and cofactor biosynthesis. The proteomes of seed and nodule strains were obtained and showed a particular profile for each of the strains. About 82 % of the proteins in the comparisons appeared similar. Forty of the most abundant proteins in each strain were identified; these proteins in seed strains were involved in stress responses and coenzyme and cofactor biosynthesis and in the nodule strains mainly in central processes. Only 3 % of the abundant proteins had hypothetical functions. CONCLUSIONS: Functions that were enriched in the genomes and proteomes of seed strains possibly participate in the successful occupancy of the new niche. The genome of the strains had features possibly related to their presence in the seeds. This study helps to understand traits of rhizobia involved in seed adaptation.

Nitrogen-fixing rhizobial strains isolated from common bean seeds: phylogeny, physiology, and genome analysis.

Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobium and Sinorhizobium groups. The strains showed several large plasmids, except for a Sinorhizobium americanum isolate. Two strains, one Rhizobium phaseoli and one S. americanum strain, were thoroughly characterized. Optimal symbiotic performance was observed for both of these strains. The S. americanum strain showed biotin prototrophy when subcultured, as well as high pyruvate dehydrogenase (PDH) activity, both of which are key factors in maintaining optimal growth. The R. phaseoli strain was a biotin auxotroph, did not grow when subcultured, accumulated a large amount of poly-ß-hydroxybutyrate, and exhibited low PDH activity. The physiology and genomes of these strains showed features that may have resulted from their lifestyle inside the seeds: stress sensitivity, a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, a homocitrate synthase (usually present only in free-living diazotrophs), a hydrogenase uptake cluster, and the presence of prophages. We propose that colonization by rhizobia and their presence in Phaseolus seeds may be part of a persistence mechanism that helps to retain and disperse rhizobial strains.

Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia.

BACKGROUND: Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. RESULTS: Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. CONCLUSION: Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.

Role of platelets as mediators that link inflammation and thrombosis in atherosclerosis.

Platelets, crucial mediators of the acute complications of atherosclerosis that cause life-threatening ischemic events at late stages of the disease, are also key effectors of inflammation throughout plaque development through their interaction with endothelial and immune cells in the injured vessel wall. During the first steps of atherosclerosis, blood inflammatory leukocytes interact with the damaged endothelium in areas rich in platelet aggregates. In late stages of the disease, platelets secrete several inflammatory molecules, even without forming aggregates. These molecules exacerbate the inflammation and induce the transition from chronic to acute disease, featuring increased instability of the atherosclerotic lesion that results in plaque rupture and thrombosis. Moreover, platelets play an important role in vascular wall remodeling induced by chronic inflammation by controlling vascular cell differentiation and proliferation. In this review, we discuss the role of platelets as cell mediators that link inflammation and thrombosis in atherosclerotic disease and their potential in the development of new therapeutic tools to fight cardiovascular disease.

Evaluation of circulating tumor DNA by electropherogram analysis and methylome profiling in high-risk neuroblastomas.

Background: Liquid biopsy has emerged as a promising, non-invasive diagnostic approach in oncology because the analysis of circulating tumor DNA (ctDNA) reflects the precise status of the disease at diagnosis, progression, and response to treatment. DNA methylation profiling is also a potential solution for sensitive and specific detection of many cancers. The combination of both approaches, DNA methylation analysis from ctDNA, provides an extremely useful and minimally invasive tool with high relevance in patients with childhood cancer. Neuroblastoma is an extracranial solid tumor most common in children and responsible for up to 15% of cancer-related deaths. This high death rate has prompted the scientific community to search for new therapeutic targets. DNA methylation also offers a new source for identifying these molecules. However, the limited blood sample size which can be obtained from children with cancer and the fact that ctDNA content may occasionally be diluted by non-tumor cell-free DNA (cfDNA) complicate optimal quantities of material for high-throughput sequencing studies. Methods: In this article, we present an improved method for ctDNA methylome studies of blood-derived plasma from high-risk neuroblastoma patients. We assessed the electropherogram profiles of ctDNA-containing samples suitable for methylome studies, using 10 ng of plasma-derived ctDNA from 126 samples of 86 high-risk neuroblastoma patients, and evaluated several bioinformatic approaches to analyze DNA methylation sequencing data. Results: We demonstrated that enzymatic methyl-sequencing (EM-seq) outperformed bisulfite conversion-based method, based on the lower proportion of PCR duplicates and the higher percentage of unique mapping reads, mean coverage, and genome coverage. The analysis of the electropherogram profiles revealed the presence of nucleosomal multimers, and occasionally high molecular weight DNA. We established that 10% content of the mono-nucleosomal peak is sufficient ctDNA for successful detection of copy number variations and methylation profiles. Quantification of mono-nucleosomal peak also showed that samples at diagnosis contained a higher amount of ctDNA than relapse samples. Conclusions: Our results refine the use of electropherogram profiles to optimize sample selection for subsequent high-throughput analysis and support the use of liquid biopsy followed by enzymatic conversion of unmethylated cysteines to assess the methylomes of neuroblastoma patients.

Kocuria kristinae neuroinfection in an immunocompetent patient: a case report and review of the literature.

Few cases of disease by Kocuria kristinae have been reported, some in immunocompetent patients but mainly in immunocompromised. The current case report describes a 28-year-old female with an initial diagnosis of pituitary macroadenoma. After the initial surgery, the patient was readmitted due to tension pneumocephalus and cerebrospinal fluid (CSF) fistula. Cultures showed K. kristinae in the CSF and Candida albicans in the urine. The patient died after multiple complications. This is the first case of neuroinfection by K. kristinae in the American continent as reviewed. It was not determined as the main cause of death due to the sudden herniation, however, with active infection derived from the identification in two different samples, for this reason, we consider that it could be useful to take it as a cause of disease and a probable cause when the studies for detection of the most common pathogens have been negative.

High tumor incidence and activation of the PI3K/AKT pathway in transgenic mice define AIB1 as an oncogene.

The gene encoding AIB1, an estrogen receptor coactivator, is amplified in a subset of human breast cancers. Here we show that overexpression of AIB1 in transgenic mice (AIB1-tg) leads to mammary hypertrophy, hyperplasia, abnormal postweaning involution, and the development of malignant mammary tumors. Tumors are also increased in other organs, including the pituitary and uterus. AIB1 overexpression increases mammary IGF-I mRNA and serum IGF-I protein levels. In addition, IGF-I receptor and downstream signaling molecules are activated in primary mammary epithelial cells and mammary tumor cells derived from AIB1-tg mice. Knockdown of AIB1 expression in cultured AIB1-tg mammary tumor cells leads to reduced IGF-I mRNA levels and increased apoptosis, suggesting that an autocrine IGF-I loop underlies the mechanism of AIB1-induced oncogenesis.

Germline variant in Ctcf links mental retardation to Wilms tumor predisposition.

CTCF germline mutations have been related to MRD21. We report the first bilateral Wilms tumor suffered by a MRD21 patient carrying an unreported CTCF missense variant in a zinc finger domain of CTCF protein. We found that germline heterozygous variant I446K became homozygous in the tumor due to a loss of heterozygosity rearrangement affecting the whole q arm on chromosome 16. Our findings propose CTCF I446K variant as a link between MRD21 and Wilms tumor predisposition.

argC Orthologs from Rhizobiales show diverse profiles of transcriptional efficiency and functionality in Sinorhizobium meliloti.

Several factors can influence ortholog replacement between closely related species. We evaluated the transcriptional expression and metabolic performance of ortholog substitution complementing a Sinorhizobium meliloti argC mutant with argC from Rhizobiales (Agrobacterium tumefaciens, Rhizobium etli, and Mesorhizobium loti). The argC gene is necessary for the synthesis of arginine, an amino acid that is central to protein and cellular metabolism. Strains were obtained carrying plasmids with argC orthologs expressed under the speB and argC (S. meliloti) and lac (Escherichia coli) promoters. Complementation analysis was assessed by growth, transcriptional activity, enzymatic activity, mRNA levels, specific detection of ArgC proteomic protein, and translational efficiency. The argC orthologs performed differently in each complementation, reflecting the diverse factors influencing gene expression and the ability of the ortholog product to function in a foreign metabolic background. Optimal complementation was directly related to sequence similarity with S. meliloti, and was inversely related to species signature, with M. loti argC showing the poorest performance, followed by R. etli and A. tumefaciens. Different copy numbers of genes and amounts of mRNA and protein were produced, even with genes transcribed from the same promoter, indicating that coding sequences play a role in the transcription and translation processes. These results provide relevant information for further genomic analyses and suggest that orthologous gene substitutions between closely related species are not completely functionally equivalent.

Intratumoral immunosuppression profiles in 11q-deleted neuroblastomas provide new potential therapeutic targets.

High-risk neuroblastoma (NB) patients with 11q deletion frequently undergo late but consecutive relapse cycles with fatal outcome. To date, no actionable targets to improve current multimodal treatment have been identified. We analyzed immune microenvironment and genetic profiles of high-risk NB correlating with 11q immune status. We show in two independent cohorts that 11q-deleted NB exhibits various immune inhibitory mechanisms, including increased CD4+ resting T cells and M2 macrophages, higher expression of programmed death-ligand 1, interleukin-10, transforming growth factor-beta-1, and indoleamine 2,3-dioxygenase 1 (P < 0.05), and also higher chromosomal breakages (P ≤ 0.02) and hemizygosity of immunosuppressive miRNAs than MYCN-amplified and other 11q-nondeleted high-risk NB. We also analyzed benefits of maintenance treatment in 83 high-risk stage M NB patients focusing on 11q status, either with standard anti-GD2 immunotherapy (n = 50) or previous retinoic acid-based therapy alone (n = 33). Immunotherapy associated with higher EFS (50 vs. 30, P = 0.028) and OS (72 vs. 52, P = 0.047) at 3 years in the overall population. Despite benefits from standard anti-GD2 immunotherapy in high-risk NB patients, those with 11q deletion still face poor outcome. This NB subgroup displays intratumoral immune suppression profiles, revealing a potential therapeutic strategy with combination immunotherapy to circumvent this immune checkpoint blockade.

Next-Generation Sequencing Identifies Potential Actionable Targets in Paediatric Sarcomas.

Background: Bone and soft-tissue sarcomas represent 13% of all paediatric malignancies. International contributions to introduce next-generation sequencing (NGS) approaches into clinical application are currently developing. We present the results from the Precision Medicine program for children with sarcomas at a reference centre. Results: Samples of 70 paediatric sarcomas were processed for histopathological analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and next-generation sequencing (NGS) with a consensus gene panel. Pathogenic alterations were reported and, if existing, targeted recommendations were translated to the clinic. Seventy paediatric patients with sarcomas from 10 centres were studied. Median age was 11.5 years (range 1-18). Twenty-two (31%) had at least one pathogenic alteration by NGS. Thirty pathogenic mutations in 18 different genes were detected amongst the 22 patients. The most frequent alterations were found in TP53, followed by FGFR4 and CTNNB1. Combining all biological studies, 18 actionable variants were detected and six patients received targeted treatment observing a disease control rate of 78%. Extrapolating the results to the whole cohort, 23% of the patients would obtain clinical benefit from this approach. Conclusions: Paediatric sarcomas have a different genomic landscape when compared to adult cohorts. Incorporating NGS targets into paediatric sarcomas' therapy is feasible and allows personalized treatments with clinical benefit in the relapse setting.

Germline Predisposition to Pediatric Cancer, from Next Generation Sequencing to Medical Care.

Knowledge about genetic predisposition to pediatric cancer is constantly expanding. The categorization and clinical management of the best-known syndromes has been refined over the years. Meanwhile, new genes for pediatric cancer susceptibility are discovered every year. Our current work shares the results of genetically studying the germline of 170 pediatric patients diagnosed with cancer. Patients were prospectively recruited and studied using a custom panel, OncoNano V2. The well-categorized predisposing syndromes incidence was 9.4%. Likely pathogenic variants for predisposition to the patient's tumor were identified in an additional 5.9% of cases. Additionally, a high number of pathogenic variants associated with recessive diseases was detected, which required family genetic counseling as well. The clinical utility of the Jongmans MC tool was evaluated, showing a high sensitivity for detecting the best-known predisposing syndromes. Our study confirms that the Jongmans MC tool is appropriate for a rapid assessment of patients; however, the updated version of Ripperger T criteria would be more accurate. Meaningfully, based on our findings, up to 9.4% of patients would present genetic alterations predisposing to cancer. Notably, up to 20% of all patients carry germline pathogenic or likely pathogenic variants in genes related to cancer and, thereby, they also require expert genetic counseling. The most important consideration is that the detection rate of genetic causality outside Jongmans MC et al. criteria was very low.