Magnetic resonance linear accelerator technology and adaptive radiation therapy: An overview for clinicians.

Radiation therapy (RT) continues to play an important role in the treatment of cancer. Adaptive RT (ART) is a novel method through which RT treatments are evolving. With the ART approach, computed tomography or magnetic resonance (MR) images are obtained as part of the treatment delivery process. This enables the adaptation of the irradiated volume to account for changes in organ and/or tumor position, movement, size, or shape that may occur over the course of treatment. The advantages and challenges of ART maybe somewhat abstract to oncologists and clinicians outside of the specialty of radiation oncology. ART is positioned to affect many different types of cancer. There is a wide spectrum of hypothesized benefits, from small toxicity improvements to meaningful gains in overall survival. The use and application of this novel technology should be understood by the oncologic community at large, such that it can be appropriately contextualized within the landscape of cancer therapies. Likewise, the need to test these advances is pressing. MR-guided ART (MRgART) is an emerging, extended modality of ART that expands upon and further advances the capabilities of ART. MRgART presents unique opportunities to iteratively improve adaptive image guidance. However, although the MRgART adaptive process advances ART to previously unattained levels, it can be more expensive, time-consuming, and complex. In this review, the authors present an overview for clinicians describing the process of ART and specifically MRgART.

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay.

BACKGROUND: After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M). RESULTS: The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance. CONCLUSIONS: Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic preparedness.

Throughout the SARS-CoV-2 pandemic, limited diagnostic capacities prevented sentinel testing, demonstrating the need for novel testing infrastructures. Here, we describe the setup of a cost-effective platform that can be employed in a high-throughput manner, which allows surveillance testing as an acute pandemic control and preparedness tool, exemplified by SARS-CoV-2 diagnostics in an academic environment. The strategy involves self-sampling based on gargling saline, pseudonymized sample handling, automated RNA extraction, and viral RNA detection using a semiquantitative multiplexed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with an analytical sensitivity comparable with RT-qPCR. We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, analysis by colorimetry or sequencing, and communication of results. We evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT-LAMP assay. In parallel, we estimated the economic costs of setting up and running the test station. We performed > 35,000 tests, with an average turnover time of < 6 h from sample arrival to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost- and labor-efficient RT-LAMP diagnostics, which is independent of potentially limiting clinical diagnostics supply chains.

Tissue-like environments shape functional interactions of HIV-1 with immature dendritic cells.

Immature dendritic cells (iDCs) migrate in microenvironments with distinct cell and extracellular matrix densities in vivo and contribute to HIV-1 dissemination and mounting of antiviral immune responses. Here, we find that, compared to standard 2D suspension cultures, 3D collagen as tissue-like environment alters iDC properties and their response to HIV-1 infection. iDCs adopt an elongated morphology with increased deformability in 3D collagen at unaltered activation, differentiation, cytokine secretion, or responsiveness to LPS. While 3D collagen reduces HIV-1 particle uptake by iDCs, fusion efficiency is increased to elevate productive infection rates due to elevated cell surface exposure of the HIV-1-binding receptor DC-SIGN. In contrast, 3D collagen reduces HIV transfer to CD4 T cells from iDCs. iDC adaptations to 3D collagen include increased pro-inflammatory cytokine production and reduced antiviral gene expression in response to HIV-1 infection. Adhesion to a 2D collagen matrix is sufficient to increase iDC deformability, DC-SIGN exposure, and permissivity to HIV-1 infection. Thus, mechano-physical cues of 2D and 3D tissue-like collagen environments regulate iDC function and shape divergent roles during HIV-1 infection.

Test accuracy of rapid diagnostic tests and reverse-transcription polymerase chain reaction against virus isolation in cell culture for assessing SARS-CoV-2 infectivity: Systematic review and meta-analysis.

We aimed to assess the performance of Ag-RDT and RT-qPCR with regard to detecting infectious SARS-CoV-2 in cell cultures, as their diagnostic test accuracy (DTA) compared to virus isolation remains largely unknown. We searched three databases up to 15 December 2021 for DTA studies. The bivariate model was used to synthesise the estimates. Risk of bias was assessed using QUADAS-2/C. Twenty studies (2605 respiratory samples) using cell culture and at least one molecular test were identified. All studies were at high or unclear risk of bias in at least one domain. Three comparative DTA studies reported results on Ag-RDT and RT-qPCR against cell culture. Two studies evaluated RT-qPCR against cell culture only. Fifteen studies evaluated Ag-RDT against cell culture as reference standard in RT-qPCR-positive samples. For Ag-RDT, summary sensitivity was 93% (95% CI 78; 98%) and specificity 87% (95% CI 70; 95%). For RT-qPCR, summary sensitivity (continuity-corrected) was 98% (95% CI 95; 99%) and specificity 45% (95% CI 28; 63%). In studies relying on RT-qPCR-positive subsamples (n = 15), the summary sensitivity of Ag-RDT was 93% (95% CI 92; 93%) and specificity 63% (95% CI 63; 63%). Ag-RDT show moderately high sensitivity, detecting most but not all samples demonstrated to be infectious based on virus isolation. Although RT-qPCR exhibits high sensitivity across studies, its low specificity to indicate infectivity raises the question of its general superiority in all clinical settings. Study findings should be interpreted with caution due to the risk of bias, heterogeneity and the imperfect reference standard for infectivity.

Genetically modified ZIKA virus as a microRNA-sensitive oncolytic virus against central nervous system tumors.

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3'UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors-highly deadly diseases in urgent need of effective advanced therapies.

Unifying the order and disorder dynamics in photoexcited VO2.

Photoinduced phase transition (PIPT) is always treated as a coherent process, but ultrafast disordering in PIPT is observed in recent experiments. Utilizing the real-time time-dependent density functional theory method, here we track the motion of individual vanadium (V) ions during PIPT in VO2 and uncover that their coherent or disordered dynamics can be manipulated by tuning the laser fluence. We find that the photoexcited holes generate a force on each V-V dimer to drive their collective coherent motion, in competing with the thermal-induced vibrations. If the laser fluence is so weak that the photoexcited hole density is too low to drive the phase transition alone, the PIPT is a disordered process due to the interference of thermal phonons. We also reveal that the photoexcited holes populated by the V-V dimerized bonding states will become saturated if the laser fluence is too strong, limiting the timescale of photoinduced phase transition.

Assessment of the Zoonotic Potential of Atypical Scrapie Prions in Humanized Mice Reveals Rare Phenotypic Convergence but Not Identity With Sporadic Creutzfeldt-Jakob Disease Prions.

BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.

A novel qPCR-based technique for identifying avian sex: An illustration within embryonic craniofacial bone.

Sex is a biological variable important to consider in all biomedical experiments. However, doing so in avian embryos can be challenging as sex can be morphologically indistinguishable. Unlike humans, female birds are the heterogametic sex with Z and W sex chromosomes. The female-specific W chromosome has previously been identified in chick using a species-specific polymerase chain reaction (PCR) technique. We developed a novel reverse transcription quantitative PCR (RT-qPCR) technique that amplifies the W chromosome gene histidine triad nucleotide-binding protein W (HINTW) in chick, quail, and duck. Accuracy of the HINTW RT-qPCR primer set was confirmed in all three species using species-specific PCR, including a novel quail-specific HINTW PCR primer set. Bone development-related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as adult duck beak size is known to be sexually dimorphic while quail beak size is not. Trends toward sex differences were found in duck gene expression but not in quail, as expected. With these novel RT-qPCR and PCR embryo sexing methods, sex of chick, quail, and duck embryos can now be assessed by either/both RNA and DNA, which facilitates analysis of sex as a biological variable in studies using these model organisms.

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR.

BACKGROUND: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. RESULTS: We developed a software named "Gene Selector for Validation" (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. CONCLUSION: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.

A comparison of the TempO-Seq and Affymetrix microarray platform using RTqPCR validation.

Next-generation risk assessment relies on mechanistic data from new approach methods, including transcriptome data. Various technologies, such as high-throughput targeted sequencing methods and microarray technologies based on hybridization with complementary probes, are used to determine differentially expressed genes (DEGs). The integration of data from different technologies requires a good understanding of the differences arising from the use of various technologies.To better understand the differences between the TempO-Seq platform and Affymetrix chip technology, whole-genome data for the volatile compound dimethylamine were compared. Selected DEGs were also confirmed using RTqPCR validation. Although the overlap of DEGs between TempO-Seq and Affymetrix was no higher than 37%, a comparison of the gene regulation in terms of log2fold changes revealed a very high concordance. RTqPCR confirmed the majority of DEGs from either platform in the examined dataset. Only a few conflicts were found (11%), while 22% were not confirmed, and 3% were not detected.Despite the observed differences between the two platforms, both can be validated using RTqPCR. Here we highlight some of the differences between the two platforms and discuss their applications in toxicology.

ExonSurfer: a web-tool to design primers at exon-exon junctions.

BACKGROUND: Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design. RESULTS: ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets. CONCLUSION: ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in subtelomeric regions and micro-chromosomes with high G/C contents.

BACKGROUND: Although multiple chicken genomes have been assembled and annotated, the numbers of protein-coding genes in chicken genomes and their variation among breeds are still uncertain due to the low quality of these genome assemblies and limited resources used in their gene annotations. To fill these gaps, we recently assembled genomes of four indigenous chicken breeds with distinct traits at chromosome-level. In this study, we annotated genes in each of these assembled genomes using a combination of RNA-seq- and homology-based approaches. RESULTS: We identified varying numbers (17,497-17,718) of protein-coding genes in the four indigenous chicken genomes, while recovering 51 of the 274 "missing" genes in birds in general, and 36 of the 174 "missing" genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in the four breeds, we found 571 ~ 627 protein-coding genes in each genome, which were missing in the annotations of the reference chicken genomes (GRCg6a and GRCg7b/w). After removing redundancy, we ended up with a total of 1,420 newly annotated genes (NAGs). The NAGs tend to be found in subtelomeric regions of macro-chromosomes (chr1 to chr5, plus chrZ) and middle chromosomes (chr6 to chr13, plus chrW), as well as in micro-chromosomes (chr14 to chr39) and unplaced contigs, where G/C contents are high. Moreover, the NAGs have elevated quadruplexes G frequencies, while both G/C contents and quadruplexes G frequencies in their surrounding regions are also high. The NAGs showed tissue-specific expression, and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. Most of the NAGs were also encoded in the reference chicken genomes, thus, these genomes might harbor more genes than previously thought. CONCLUSION: The NAGs are widely distributed in wild, indigenous and commercial chickens, and they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

Tumor cell-intrinsic MELK enhanced CCL2-dependent immunosuppression to exacerbate hepatocarcinogenesis and confer resistance of HCC to radiotherapy.

BACKGROUND: The outcome of hepatocellular carcinoma (HCC) is limited by its complex molecular characteristics and changeable tumor microenvironment (TME). Here we focused on elucidating the functional consequences of Maternal embryonic leucine zipper kinase (MELK) in the tumorigenesis, progression and metastasis of HCC, and exploring the effect of MELK on immune cell regulation in the TME, meanwhile clarifying the corresponding signaling networks. METHODS: Bioinformatic analysis was used to validate the prognostic value of MELK for HCC. Murine xenograft assays and HCC lung metastasis mouse model confirmed the role of MELK in tumorigenesis and metastasis in HCC. Luciferase assays, RNA sequencing, immunopurification-mass spectrometry (IP-MS) and coimmunoprecipitation (CoIP) were applied to explore the upstream regulators, downstream essential molecules and corresponding mechanisms of MELK in HCC. RESULTS: We confirmed MELK to be a reliable prognostic factor of HCC and identified MELK as an effective candidate in facilitating the tumorigenesis, progression, and metastasis of HCC; the effects of MELK depended on the targeted regulation of the upstream factor miR-505-3p and interaction with STAT3, which induced STAT3 phosphorylation and increased the expression of its target gene CCL2 in HCC. In addition, we confirmed that tumor cell-intrinsic MELK inhibition is beneficial in stimulating M1 macrophage polarization, hindering M2 macrophage polarization and inducing CD8 + T-cell recruitment, which are dependent on the alteration of CCL2 expression. Importantly, MELK inhibition amplified RT-related immune effects, thereby synergizing with RT to exert substantial antitumor effects. OTS167, an inhibitor of MELK, was also proven to effectively impair the growth and progression of HCC and exert a superior antitumor effect in combination with radiotherapy (RT). CONCLUSIONS: Altogether, our findings highlight the functional role of MELK as a promising target in molecular therapy and in the combination of RT therapy to improve antitumor effect for HCC.

Prenatal tetrahydrocannabinol and cannabidiol exposure produce sex-specific pathophysiological phenotypes in the adolescent prefrontal cortex and hippocampus.

Clinical and preclinical evidence has demonstrated an increased risk for neuropsychiatric disorders following prenatal cannabinoid exposure. However, given the phytochemical complexity of cannabis, there is a need to understand how specific components of cannabis may contribute to these neurodevelopmental risks later in life. To investigate this, a rat model of prenatal cannabinoid exposure was utilized to examine the impacts of specific cannabis constituents (Δ9-tetrahydrocannabinol [THC]; cannabidiol [CBD]) alone and in combination on future neuropsychiatric liability in male and female offspring. Prenatal THC and CBD exposure were associated with low birth weight. At adolescence, offspring displayed sex-specific behavioural changes in anxiety, temporal order and social cognition, and sensorimotor gating. These phenotypes were associated with sex and treatment-specific neuronal and gene transcriptional alterations in the prefrontal cortex, and ventral hippocampus, regions where the endocannabinoid system is implicated in affective and cognitive development. Electrophysiology and RT-qPCR analysis in these regions implicated dysregulation of the endocannabinoid system and balance of excitatory and inhibitory signalling in the developmental consequences of prenatal cannabinoids. These findings reveal critical insights into how specific cannabinoids can differentially impact the developing fetal brains of males and females to enhance subsequent neuropsychiatric risk.

Infective SARS-CoV-2 in Skull Sawdust at Autopsy, Finland.

We assessed the distribution of SARS-CoV-2 at autopsy in 22 deceased persons with confirmed COVID-19. SARS-CoV-2 was found by PCR (2/22, 9.1%) and by culture (1/22, 4.5%) in skull sawdust, suggesting that live virus is present in tissues postmortem, including bone. Occupational exposure risk is low with appropriate personal protective equipment.

Selection of Reference Genes for Expression Normalization by RT-qPCR in Dracocephalum moldavica L.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes' stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.

Validation of Selected MicroRNA Transcriptome Data in the Bovine Corpus Luteum during Early Pregnancy by RT-qPCR.

In cattle, the corpus luteum (CL) is pivotal in maintaining early pregnancy by secreting progesterone. To establish pregnancy, the conceptus produces interferon-τ, preventing luteolysis and initiating the transformation of the CL spurium into a CL verum. Although this transformation is tightly regulated, limited data are available on the expression of microRNAs (miRNAs) during and after this process. To address this gap, we re-analyzed previously published RNA-Seq data of CL from pregnant cows and regressed CL from non-pregnant cows. This analysis identified 44 differentially expressed miRNAs. From this pool, three miRNAs-bta-miR-222-3p, bta-miR-29c, and bta-miR-2411-3p-were randomly selected for relative quantification. Using bovine ovaries (n = 14) obtained from an abattoir, total RNA (including miRNAs) was extracted and converted to cDNA for RT-qPCR. The results revealed that bta-miR-222-3p was downregulated (p = 0.016) in pregnant females compared to non-pregnant cows with regressed CL. However, no differences in miRNA expression were observed between CL of pregnant and non-pregnant cows for bta-miR-29c (p > 0.32) or bta-miR-2411-3p (p > 0.60). In silico prediction approaches indicated that these miRNAs are involved in pathways regulating pregnancy maintenance, such as the VEGF- and FoxO-signaling pathways. Additionally, their biogenesis is regulated by GABPA and E2F4 transcription factors. The validation of selected miRNA expression in the CL during pregnancy by RT-qPCR provides novel insights that could potentially lead to the identification of biomarkers related to CL physiology and pregnancy outcome.

Evaluation of HNF1B, KLK3, ELAC2, TMPRSS2-ERG, and CTNNB1 polymorphisms associated with prostate cancer in samples of patients from HUPE-UERJ.

PURPOSE: Prostate cancer (PCa) is the leading cause of death among men in 48 countries. Genetic alterations play a significant role in PCa carcinogenesis. For the hypothesis of this research, five unique polymorphisms (SNP) were investigated in different genes that showed to be associated in different ways with PCa: rs4430796, rs2735839, rs4792311, rs12329760, and rs28931588, respectively for the genes HNF1B, KLK3, ELAC2, TMPRSS2-ERG, and CTNNB1. PATIENTS AND METHODS: Blood samples from 426 subjects were evaluated: 290 controls (161 females and 129 males) and 136 PCa patients. SNP were determined by real-time polymerase chain reaction. TaqMan SNP genotyping assay. In the control samples, the SNPs were defined in association with the self-reported ethnicity, and in 218 control samples with markers with ancestry indicators. The genes were in Hardy-Weinberg equilibrium. One hundred and seventy control samples were matched by ethnicity for comparison with the PCa samples. RESULTS: The G allele at rs28931588 was monomorphic in both patients and controls studied. Significant differences were observed in allelic and genotypic frequencies between the control and Pca samples in rs2735839 (KLK3; p = 0.002 and χ2 = 8.73 and p = 0.01, respectively), by the global frequency and in the dominant model rs2735839_GG (odds ratio [OR] = 0.51, p = 0.02). AA and GA genotypes at rs4792311 (ELAC2) were more frequent in patients with Gleason 7(4 + 3), 8, and 9 (n = 37%-59.7%) compared to patients with Gleason 6 and 7(3 + 4) (n = 26%-40.0%) conferring a protective effect on the GG genotype (OR = 0.45, p = 0.02). The same genotype showed an OR = 2.71 (p = 0.01) for patients with low severity. The HNF1B-KLK3-ELAC2-TMPRSS2-ERG haplotypes: GAAT, AAAT, GAGT, and AAGT were more frequent in patients with Pca with OR ranging from 4.65 to 2.48. CONCLUSIONS: Higher frequencies of risk alleles were confirmed in the SNPs, KLK3 rs2735839_A, ELAC2 rs4792311_A, and TMPRSS2 rs12329760_T in patients with Pca. Rs2735839_A was associated with risk of Pca and rs4792311_A with severity and Gleason score of 7(4 + 3) or greater. There is a need for careful observation of rs2735839 and rs4792311 in association with the prostatic biopsy due to the increased risk of Pca.

A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.