Microbial and Metabolic Gut Profiling across Seven Malignancies Identifies Fecal Faecalibacillus intestinalis and Formic Acid as Commonly Altered in Cancer Patients.

The key association between gut dysbiosis and cancer is already known. Here, we used whole-genome shotgun sequencing (WGS) and gas chromatography/mass spectrometry (GC/MS) to conduct metagenomic and metabolomic analyses to identify common and distinct taxonomic configurations among 40, 45, 71, 34, 50, 60, and 40 patients with colorectal cancer, stomach cancer, breast cancer, lung cancer, melanoma, lymphoid neoplasms and acute myeloid leukemia (AML), respectively, and compared the data with those from sex- and age-matched healthy controls (HC). α-diversity differed only between the lymphoid neoplasm and AML groups and their respective HC, while ß-diversity differed between all groups and their HC. Of 203 unique species, 179 and 24 were under- and over-represented, respectively, in the case groups compared with HC. Of these, Faecalibacillus intestinalis was under-represented in each of the seven groups studied, Anaerostipes hadrus was under-represented in all but the stomach cancer group, and 22 species were under-represented in the remaining five case groups. There was a marked reduction in the gut microbiome cancer index in all case groups except the AML group. Of the short-chain fatty acids and amino acids tested, the relative concentration of formic acid was significantly higher in each of the case groups than in HC, and the abundance of seven species of Faecalibacterium correlated negatively with most amino acids and formic acid, and positively with the levels of acetic, propanoic, and butanoic acid. We found more differences than similarities between the studied malignancy groups, with large variations in diversity, taxonomic/metabolomic profiles, and functional assignments. While the results obtained may demonstrate trends rather than objective differences that correlate with different types of malignancy, the newly developed gut microbiota cancer index did distinguish most of the cancer cases from HC. We believe that these data are a promising step forward in the search for new diagnostic and predictive tests to assess intestinal dysbiosis among cancer patients.

Microbiome Sex-Related Diversity in Non-Muscle-Invasive Urothelial Bladder Cancer.

Sex-specific discrepancies in bladder cancer (BCa) are reported, and new studies imply that microbiome may partially explain the diversity. We aim to provide characterization of the bladder microbiome in both sexes diagnosed with non-muscle-invasive BCa with specific insight into cancer grade. In our study, 16S rRNA next-generation sequencing was performed on midstream urine, bladder tumor sample, and healthy-appearing bladder mucosa. Bacterial DNA was isolated using QIAamp Viral RNA Mini Kit. Metagenomic analysis was performed using hypervariable fragments of the 16S rRNA gene on Ion Torrent Personal Genome Machine platform. Of 41 sample triplets, 2153 taxa were discovered: 1739 in tumor samples, 1801 in healthy-appearing bladder mucosa and 1370 in midstream urine. Women were found to have smaller taxa richness in Chao1 index than men (p = 0.03). In comparison to low-grade tumors, patients with high-grade lesions had lower bacterial diversity and richness in urine. Significant differences between sexes in relative abundance of communities at family level were only observed in high-grade tumors.

Effects of Soluble Dextrin Fiber from Potato Starch on Body Weight and Associated Gut Dysbiosis Are Evident in Western Diet-Fed Mice but Not in Overweight/Obese Children.

BACKGROUND: The study investigated the impact of starch degradation products (SDexF) as prebiotics on obesity management in mice and overweight/obese children. METHODS: A total of 48 mice on a normal diet (ND) and 48 on a Western diet (WD) were divided into subgroups with or without 5% SDexF supplementation for 28 weeks. In a human study, 100 overweight/obese children were randomly assigned to prebiotic and control groups, consuming fruit and vegetable mousse with or without 10 g of SDexF for 24 weeks. Stool samples were analyzed for microbiota using 16S rRNA gene sequencing, and short-chain fatty acids (SCFA) and amino acids (AA) were assessed. RESULTS: Results showed SDexF slowed weight gain in female mice on both diets but only temporarily in males. It altered bacterial diversity and specific taxa abundances in mouse feces. In humans, SDexF did not influence weight loss or gut microbiota composition, showing minimal changes in individual taxa. The anti-obesity effect observed in mice with WD-induced obesity was not replicated in children undergoing a weight-loss program. CONCLUSIONS: SDexF exhibited sex-specific effects in mice but did not impact weight loss or microbiota composition in overweight/obese children.

Diarrheal-associated gut dysbiosis in cancer and inflammatory bowel disease patients is exacerbated by Clostridioides difficile infection.

Introduction: Low diversity gut dysbiosis can take different forms depending on the disease context. In this study, we used shotgun metagenomic sequencing and gas chromatography-mass spectrometry (GC-MS) to compared the metagenomic and metabolomic profiles of Clostridioides (Clostridium) difficile diarrheal cancer and inflammatory bowel disease (IBD) patients and defined the additive effect of C. difficile infection (CDI) on intestinal dysbiosis. Results: The study cohort consisted of 138 case-mix cancer patients, 43 IBD patients, and 45 healthy control individuals. Thirty-three patients were also infected with C. difficile. In the control group, three well-known enterotypes were identified, while the other groups presented with an additional Escherichia-driven enterotype. Bacterial diversity was significantly lower in all groups than in healthy controls, while the highest level of bacterial species richness was observed in cancer patients. Fifty-six bacterial species had abundance levels that differentiated diarrheal patient groups from the control group. Of these species, 52 and 4 (Bacteroides fragilis, Escherichia coli, Klebsiella pneumoniae, and Ruminococcus gnavus) were under-represented and over-represented, respectively, in all diarrheal patient groups. The relative abundances of propionate and butyrate were significantly lower in fecal samples from IBD and CDI patients than in control samples. Isobutyrate, propanate, and butyrate concentrations were lower in cancer, IBD, and CDI samples, respectively. Glycine and valine amino acids were over- represented in diarrheal patients. Conclusion: Our data indicate that different external and internal factors drive comparable profiles of low diversity dysbiosis. While diarrheal-related low diversity dysbiosis may be a consequence of systemic cancer therapy, a similar phenotype is observed in cases of moderate to severe IBD, and in both cases, dysbiosis is exacerbated by incidence of CDI.

Genetic associations with lymphomas in Polish patients: A pooled-DNA genome-wide association analysis.

Several single nucleotide polymorphisms (SNPs) associated with susceptibility to Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL) have been identified. The aim of this study was to identify susceptibility loci for HL and DLBCL in Polish patients. Altogether, DLBCL (n = 218 and HL patients (n = 224) and healthy individuals (n = 1181) were recruited. Lymphoma diagnosis was based on standard criteria. Genome-wide association study (GWAS) was performed using pooled-DNA samples on llumina Infinium Omni2.5 Exome-8 v1.3, and selected loci were replicated by TaqMan SNP genotyping of individuals. GWAS detected thirteen and seven SNPs associated with DLBCL and HL, respectively. In the replication study, six and seven SNPs reached significance after correction for multiple testing in the DLBCL and HL cohorts, respectively. One and four SNPs associated with DLBCL and HL, respectively, were localized within, and two SNPs-near the major histocompatibility complex (MHC) region. In conclusion, the majority of loci associated with HL and DLBCL aetiology in previous studies have potential roles in immune function. Our pooled-DNA GWAS enabled the identification of several susceptibility loci for DLBCL and HL in the Polish population; some of them were mapped within or adjacent to the MHC, and other associated SNPs were located outside the MHC.

Gene Expression-Based Functional Differences between the Bladder Body and Trigonal Urothelium in Adolescent Female Patients with Micturition Dysfunction.

The aim of this study is to determine the molecular differences between the urothelial transcriptomes of the bladder body and trigone. The transcriptomes of the bladder body and trigonal epithelia were analyzed by massive sequencing of total epithelial RNA. The profiles of urothelial and urinal microbiomes were assessed by amplicon sequencing of bacterial 16S rRNA genes in 17 adolescent females with pain and micturition dysfunction and control female subjects. The RNA sequencing identified 10,261 differentially expressed genes (DEGs) in the urothelia of the bladder body and trigone, with the top 1000 DEGs at these locations annotated to 36 and 77 of the Reactome-related pathways in the bladder body and trigone, respectively. These pathways represented 11 categories enriched in the bladder body urothelium, including extracellular matrix organization, the neuronal system, and 15 categories enriched in the trigonal epithelium, including RHO GTPase effectors, cornified envelope formation, and neutrophil degranulation. Five bacterial taxa in urine differed significantly in patients and healthy adolescent controls. The evaluation of their transcriptomes indicated that the bladder body and trigonal urothelia were functionally different tissues. The molecular differences between the body and trigonal urothelia responsible for clinical symptoms in adolescents with bladder pain syndrome/interstitial cystitis remain unclear.

Prediction of fetal blood group antigens from maternal plasma using Ion AmpliSeq HD technology.

BACKGROUND: Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT. AIM: To design NIPT of clinically important antigens using Ion AmpliSeq HD technology. METHODS: Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers. RESULTS: Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism. CONCLUSION: The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.

Characteristics of the gut microbiome in esports players compared with those in physical education students and professional athletes.

Introduction: Esports is a category of competitive video games that, in many aspects, may be similar to traditional sports; however, the gut microbiota composition of players has not been yet studied. Materials and methods: Here, we investigated the composition and function of the gut microbiota, as well as short chain fatty acids (SCFAs), and amino acids, in a group of 109 well-characterized Polish male esports players. The results were compared with two reference groups: 25 endurance athletes and 36 healthy students of physical education. DNA and metabolites isolated from fecal samples were analyzed using shotgun metagenomic sequencing and mass spectrometry, respectively. Physical activity and nutritional measures were evaluated by questionnaire. Results: Although anthropometric, physical activity and nutritional measures differentiated esports players from students, there were no differences in bacterial diversity, the Bacteroidetes/Firmicutes ratio, the composition of enterotype clusters, metagenome functional content, or SCFA concentrations. However, there were significant differences between esports players and students with respect to nine bacterial species and nine amino acids. By contrast, all of the above-mentioned measures differentiated professional athletes from esports players and students, with 45 bacteria differentiating professional athletes from the former and 31 from the latter. The only species differentiating all three experimental groups was Parabacteroides distasonis, showing the lowest and highest abundance in esports players and athletes, respectively. Conclusion: Our study confirms the marked impact of intense exercise training on gut microbial structure and function. Differences in lifestyle and dietary habits between esports players and physical education students appear to not have a major effect on the gut microbiota.

A Set of 17 microRNAs Common for Brain and Cerebrospinal Fluid Differentiates Primary Central Nervous System Lymphoma from Non-Malignant Brain Tumors.

The diagnosis of primary central nervous system (CNS) lymphoma, which is predominantly of the diffuse large B-cell lymphoma type (CNS DLBCL), is challenging. MicroRNAs (miRs) are gene expression-regulating non-coding RNAs that are potential biomarkers. We aimed to distinguish miR expression patterns differentiating CNS DLBCL and non-malignant CNS diseases with tumor presentation (n-ML). Next generation sequencing-based miR profiling of cerebrospinal fluids (CSFs) and brain tumors was performed. Sample source-specific (CSF vs. brain tumor) miR patterns were revealed. Even so, a set of 17 miRs differentiating CNS DLBCL from n-ML, no matter if assessed in CSF or in a tumor, was identified. Along with the results of pathway analyses, this suggests their pathogenic role in CNS DLBCL. A combination of just four of those miRs (miR-16-5p, miR-21-5p, miR-92a-3p, and miR-423-5p), assessed in CSFs, discriminated CNS DLBCL from n-ML samples with 100% specificity and 67.0% sensitivity. Analyses of paired CSF-tumor samples from patients with CNS DLBCL showed significantly lower CSF levels of miR-26a, and higher CSF levels of miR-15a-5p, miR-15b-5p, miR-19a-3p, miR-106b-3p, miR-221-3p, and miR-423-5p. Noteworthy, the same miRs belonged to the abovementioned set differentiating CNS DLBCL from non-malignant CNS diseases. Our results not only add to the basic knowledge, but also hold significant translational potential.

GWAS Links New Variant in Long Non-Coding RNA LINC02006 with Colorectal Cancer Susceptibility.

Despite great efforts, most of the genetic factors contributing to the risk of colorectal cancer (CRC) remain undetermined. Including small but homogenous populations in genome-wide association studies (GWAS) can help us discover new common risk variants specific to the studied population. In this study, including 465 CRC patients and 1548 controls, a pooled DNA samples-based GWAS was conducted in search of genetic variants associated with CRC in a Polish population. Combined with a new method of selecting single-nucleotide polymorphisms (SNPs) for verification in individual DNA samples, this approach allowed the detection of five new susceptibility loci not previously reported for CRC. The discovered loci were found to explain 10% of the overall risk of developing CRC. The strongest association was observed for rs10935945 in long non-coding RNA LINC02006 (3q25.2). Three other SNPs were also located within genes (rs17575184 in NEGR1, rs11060839 in PIWIL1, rs12935896 in BCAS3), while one was intergenic (rs9927668 at 16p13.2). An expression quantitative trait locus (eQTL) bioinformatic analysis suggested that these polymorphisms may affect transcription factor binding sites. In conclusion, four of the identified variants were located within genes likely involved in tumor invasiveness and metastasis. Therefore, they could possibly be markers of poor prognosis in CRC patients.

Peripheral Blood Cells from Patients with Hodgkin's and Diffuse Large B Cell Lymphomas May Be a Better Source of Candidate Diagnostic miRNAs Than Circulating miRNAs.

Hodgkin lymphoma (HL) and diffuse large B cell lymphoma (DLBCL) represent 15% and 20%, respectively, of all lymphoma types. The aim of this study was to identify and compare circulating serum miRNA (c-miRNA) and peripheral whole blood miRNA (wb-miRNA) profiles in patients with these lymphomas. Serum samples (20 HL, 21 DLBCL, and 30 healthy controls) and whole blood samples (21 HL, 17 DLBCL patients, and 30 healthy controls) were collected at the time of diagnosis. Serum and whole blood were also collected from 18 HL/17 DLBCL and eight HL/nine DLBCL patients, respectively, after treatment. Pairwise comparisons identified 125 c-miRNAs (adjusted P value < 0.05) showing significant dysregulation between 30 healthy controls and patients; of these, 47 and 55 differentiated controls from pretherapeutic HL and DLBCL patients, respectively. In addition, 60 and 16 c-miRNAs differentiated controls from posttherapeutic HL and DLBCL, respectively. Pairwise comparisons identified 292 wb-miRNAs (adjusted P value < 0.05) showing significant dysregulation between 30 controls and patients; of these, 103 and 169 differentiated controls from pretherapeutic HL and DLBCL, respectively, and 142 and 151 wb-miRNAs differentiated controls from posttherapeutic HL and DLBCL, respectively. Thus, lymphoma-associated miRNAs may be a better source of noninvasive candidate biomarkers than miRNAs in serum. It is unclear whether miRNA alterations in lymphoma cells are similar to those observed in white blood cells.

Limited Practical Utility of Liquid Biopsy in the Treated Patients with Advanced Breast Cancer.

Recently, liquid biopsy has emerged as a tool to monitor oncologic disease progression and the effects of treatment. In this study we aimed to determine the clinical utility of liquid biopsy relative to conventional oncological post-treatment surveillance. Plasma cell-free (cf) DNA was collected from six healthy women and 37 patients with breast cancer (18 and 19 with stage III and IV tumors, respectively). CfDNA was assessed using the Oncomine Pan-Cancer Cell-Free Assay. In cfDNA samples from patients with BC, 1112 variants were identified, with only a few recurrent or hotspot mutations within specific regions of cancer genes. Of 65 potentially pathogenic variants detected in tumors, only 19 were also discovered in at least one blood sample. The allele frequencies of detected variants (VAFs) were <1% in cfDNA from all controls and patients with stage III BC, and 24/85 (28.2%) variants had VAFs > 1% in only 8 of 25 (32%) patients with stage IV BC. Copy number variations (CNVs) spanning CDK4, MET, FGFR1, FGFR2, ERBB2, MYC, and CCND3 were found in 1 of 12 (8%) and 8 of 25 (32%) patients with stage III and IV tumors, respectively. In healthy controls and patients without BC progression after treatment, VAFs were <1%, while in patients with metastatic disease and/or more advanced genomic alterations, VAFs > 1% and/or CNV were detected in approximately 30%. Therefore, most patients with stage IV BC could not be distinguished from those with stage III disease following therapy, based on liquid biopsy results.

Signatures of circulating microRNA in four sarcoma subtypes.

Background: Sarcomas are rare malignant tumors of mesenchymal origin. The discovery of circulating biomarkers with high diagnostic value could supplement diagnosis of this heterogenous group of tumors. The aim of this study was to identify the profiles of circulating miRNA (c-miRNAs) in four groups of common bone and soft tissue sarcomas. Methods: At the time of diagnosis, blood samples were collected from 86 patients: 36 with locally advanced/unresectable/metastatic gastrointestinal stromal tumor (GIST) who received first-line treatment with imatinib; 16 with locally advanced osteosarcoma (OS); 26 with locally advanced synovial sarcoma (SS); and eight with locally advanced Ewing sarcoma (ES). In addition, samples were collected from 30 healthy controls. C-miRNAs were isolated using a miRCURY RNA Isolation Kit, followed by preparation of cDNA libraries and sequencing on the Ion Proton platform. Results: Pair-wise comparisons identified 156 unique c-miRNAs (adjusted P-value < 0.05) showing significant dysregulation between controls and patients; of these, 24, 36, 42, and 99 differentiated controls from pretherapeutic OS, SS, ES, and GIST, respectively. Ten c-miRNAs were commonly altered in at least three sarcoma types. Receiver operating characteristic curves and area under the curve (ROC-AUC) analyses revealed that a four-miRNA diagnostic classifier was able to differentiate controls from ES, GIST, OS, and SS, with AUC-ROC values of 1, 0.97, 0.95, and 0.94, respectively. Conclusions: Aberrant miRNA expression signatures were identified in serum from patients with four different sarcoma subtypes. Differences in miRNA expression profiles between sarcoma patients and healthy volunteers suggest that miRNAs may play a role in sarcoma development.

Common functional alterations identified in blood transcriptome of autoimmune cholestatic liver and inflammatory bowel diseases.

Primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), are heterogeneous chronic autoimmune diseases that may share underlying pathogenic mechanisms. Herein, we compared simultaneously analyzed blood transcriptomes from patients with PBC, PSC, and IBD. Microarray-based measurements were conducted using RNA isolated from whole blood samples from 90, 45, 95 and 93 patients with PBC, PSC, CD, and UC, respectively, and 47 healthy controls. Expression levels of selected transcripts were analyzed by quantitative reverse-transcribed PCR using an independent cohort of 292, 71 and 727 patients with PBC, PSC, and IBD, respectively. Of 4026, 2650 and 4967 probe sets differentially expressed (adjusted p-value < 0.05) in samples from patients with PBC, PSC, and IBD, respectively, compared with healthy controls, 1946 were common to all three comparisons. Functional analyses indicated that most terms enriched for genes differentially expressed in PBC, PSC, and IBD patients compared with healthy controls were related to mitochondrial function, the vesicle endomembrane system, and GTPase-mediated processes. This study indicates that microarray-based profiling of blood gene expression supports research into the molecular mechanisms underlying disease, rather than being useful for selection of diagnostic biomarkers for use in clinical practice.

Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas.

OBJECTIVE: DNA repair pathways are potential targets of molecular therapy in cancer patients. The FANCD2, BRIP1, BRCA1/2, and FANCF genes are involved in homologous recombination DNA repair, which implicates their possible role in cell response to DNA-damaging agents. We evaluated a clinical significance of pre-treatment expression of these genes at mRNA level in 99 primary, advanced-stage ovarian carcinomas from patients, who later received taxane-platinum (TP) or platinum-cyclophosphamide (PC) treatment. METHODS: Gene expression was determined with the use of Real-Time PCR. The BRCA2 and BRIP1 gene sequence was investigated with the use of SSCP, dHPLC, and PCR-sequencing. RESULTS: Increased FANCD2 expression occurred to be a negative prognostic factor for all patients (PC+TP:HR 3.85, p = 0.0003 for the risk of recurrence; HR 1.96, p = 0.02 for the risk of death), and this association was even stronger in the TP-treated group (HR 6.7, p = 0.0002 and HR 2.33, p = 0.01, respectively). Elevated BRIP1 expression was the only unfavorable molecular factor in the PC-treated patients (HR 8.37, p = 0.02 for the risk of recurrence). Additionally, an increased FANCD2 and BRCA1/2 expression levels were associated with poor ovarian cancer outcome in either TP53-positive or -negative subgroups of the TP-treated patients, however these groups were small. Sequence analysis identified one protein truncating variant (1/99) in BRCA2 and no mutations (0/56) in BRIP1. CONCLUSIONS: Our study shows for the first time that FANCD2 overexpression is a strong negative prognostic factor in ovarian cancer, particularly in patients treated with TP regimen. Moreover, increased mRNA level of the BRIP1 is a negative prognostic factor in the PC-treated patients. Next, changes in the BRCA2 and BRIP1 genes are rare and together with other analyzed FA genes considered as homologous recombination deficiency may not affect the expression level of analyzed genes.

Prediction of fetal blood group and platelet antigens from maternal plasma using next-generation sequencing.

BACKGROUND: Fetuses whose mothers have produced antibodies to red blood cell (RBC) or platelet antigens are at risk of being affected by hemolytic disease or alloimmune thrombocytopenia, respectively, only if they inherit the incompatible antigen. Noninvasive diagnosis of the fetal antigen is employed for management of immunized pregnancies, but the specific detection of SNPs, encoding the majority of antigens, in maternal plasma is still a challenge. We applied targeted next-generation sequencing (NGS) to predict the fetal antigen based on the detection of fetomaternal chimerism. METHODS AND MATERIALS: The DNA of 13 pregnant women (with anti-K [3] anti-k [1], anti-Fya [1], anti-D + C + Jka [1], anti-D + E + K [1], anti-HPA-1a [1], anti-HPA-3b [1], anti-HPA-5b [1], and nonimmunized [3]) was sequenced using primers for regions encoding RhD, RhC, Rhc, RhE/e, K/k, Fya/b, Jka/b, MN, Ss, and HPA-1, 2, 3, 5, 15, 4 X-polymorphisms on the Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RESULTS: NGS results were in agreement with the phenotype/genotype of women and their neonates (except for the unsuccessful detection of MN and RhC). NGS determined fetal allele chimerism for K, k, Fya, Fyb, Jka, Jkb, S, RhE (from 0.42% to 6.08%); RhD, Rhc (100%); HPA-1a, -2b, -3a, 3b, -5b, -15a, 15b (from 0.23% to 4.11%). NGS revealed fetal chimerism for incompatible antigens (from 0.7% to 4.8%) in 7 immunized cases, excluded in 3 (with anti-K, anti-Fya , anti-HPA-3b). CONCLUSION: The designed NGS predicts the fetal RBC and platelet antigen status universally in cases with various clinically significant antibodies as well as providing confirmation of the presence of fetal DNA. However, some improvement of the unsuccessful primers is required.

Whole-exome sequencing identifies novel pathogenic variants across the ATP7B gene and some modifiers of Wilson's disease phenotype.

BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessive disorder associated with disease-causing alterations across the ATP7B gene, with highly variable symptoms and age of onset. We aimed to assess whether the clinical variability of WD relates to modifier genes. METHODS: A total of 248 WD patients were included, of whom 148 were diagnosed after age of 17. Human exome libraries were constructed using AmpliSeq technology and sequenced using the IonProton platform. RESULTS: ATP7B p.His1069Gln mutation was present in 215 patients, with 112 homozygotes and 103 heterozygotes. Three other mutations: p.Gln1351Ter, p.Trp779Ter and c.3402delC were identified in >10 patients. Among patients, 117 had a homozygous mutation, 101 were compound heterozygotes, 27 had one heterozygous mutation, and 3 other patients had no identifiable pathogenic variant of ATP7B. Sixteen mutations were novel, found as part of a compound mutation or as a sole, homozygous mutation. For disease phenotype prediction, age at diagnosis was a deciding factor, while frameshift allelic variants of ATP7B and being male increased the odds of developing a neurological phenotype. Rare allelic variants in ESD and INO80 increased and decreased chances for the neurological phenotype, respectively, while rare variants in APOE and MBD6 decreased the chances of WD early manifestation. Compound mutations contributed to earlier age of onset. CONCLUSIONS: In a Polish population, genetic screening for WD may help genotype for four variants (p.His1069Gln, p.Gln1351Ter, p.Trp779Ter and c.3402delC), with direct sequencing of all ATP7B amplicons as a second diagnostic step. We also identified some allelic variants that may modify a WD phenotype.

Somatic aberrations of BRCA1 gene are associated with ALDH1, EGFR, and tumor progression in prostate cancer.

BRCA1 is a pivotal tumor suppressor. Its dysfunction is known to play a role in different tumors. Among others, BRCA1 germline mutations account for higher risk and more aggressive course of prostate cancer (PCa). In addition, somatic BRCA1 gene loss was demonstrated to be a signature of PCa dissemination to lymph nodes and peripheral blood, and indicate worse clinical outcome. In order to substantiate the data for BRCA1 gene loss in PCa and reveal its phenotypical background, BRCA1 gene status was assessed in a large cohort of PCa patients and compared to different molecular factors. BRCA1 gene dosage was assessed in 2398 tumor samples from 1,199 PCa patients using fluorescent in situ hybridization. It was compared to clinico-pathological parameters, patients' outcome as well as selected proteins (Ki-67, apoptosis marker, cytokeratins, vimentin, E- and N-cadherin, ALDH1 and EGFR) examined immunohistochemically. BRCA1 losses were found in 10%, whereas gains appeared in 7% of 603 informative PCa patients. BRCA1 losses correlated to higher T stage (p = 0.027), Gleason score (p = 0.039), shorter time to biochemical recurrence in patients with Gleason score > 7 independently of other factors (multivariate analysis, p = 0.005) as well as expression of proteins regulating stemness and epithelial-mesenchymal transition, that is, ALDH1 (p = 0.021) and EGFR (p = 0.011), respectively. BRCA1 gains correlated to shorter time to metastasis (p = 0.012) and expression of ALDH1 (p = 0.014). These results support the assumption that BRCA1 gene losses contribute to a progressive and stem cell-like phenotype of PCa. Furthermore, they reveal that also BRCA1 gain conceivably representing loss-of-function might mark more invasive tumors.

Redefining the Practical Utility of Blood Transcriptome Biomarkers in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: The study investigates the practical utility of whole-blood gene expression profiling to diagnose inflammatory bowel diseases [IBDs]. METHODS: The discovery cohorts included 102 and 51 paediatric IBD patients and controls, and 95 and 46 adult IBD patients and controls, respectively. The replication cohorts included 447 and 76 paediatric IBD patients and controls, and 271 and 108 adult IBD patients and controls, respectively. In the discovery phase, RNA samples extracted from whole peripheral blood were analysed using RNA-Seq, and the predictive values of selected biomarkers were validated using quantitative polymerase chain reaction [qPCR]. RESULTS: In all, 15 differentially expressed transcripts [adjusted p ≤0.05] were selected from the discovery sequencing datasets. The receiver operating characteristic curves and area under the curve [ROC-AUC] in replication analyses showed high discriminative power [AUC range, 0.91-0.98] for 11 mRNAs in paediatric patients with active IBD. By contrast, the AUC-ROC values ranged from 0.63 to 0.75 in comparison among inactive paediatric IBDs and active/inactive adult IBDs, indicating a lack of discriminative power. The best multi-mRNA diagnostic classifier showed moderate discriminative power [AUC = 0.81] for paediatric inactive IBD, but was not able to discriminate active or inactive adult IBD patients from controls. The AUC-ROC values did not confirm an ability of the mRNAs abundances to discriminate between active ulcerative colitis and active Crohn's disease in paediatric or adult populations. CONCLUSIONS: This study identifies and validates blood transcriptional biomarkers that could be used in clinical settings as diagnostic predictors of IBD clinical activity in paediatric, but not adult, IBD patients.

IDH1/2 Mutations Predict Shorter Survival in Chondrosarcoma.

Background. Recent studies have shown that isocitrate dehydrogenase 1/2 (IDH1/2)- activating mutations occur in a variety of cancers, including acute myeloid leukaemia, gliomas, and chondrosarcomas (CHS)s. The effect of IDH1/2 mutation on overall survival (OS) has not been reported in CHS. The aim of our study was to assess the prevalence of known cancer-related gene mutations in CHS, as well as their prognostic role in patient survival. Methods. DNA from FFPE samples of 80 patients (F:M- 1:1.3; mean age: 58 years; range 27-86) with histologically confirmed CHS (G1:29; G2:34; G3:17) was subjected to library preparation with the Ion AmpliSeq Cancer Hotspot Panel v2 and sequenced on the PGM Ion Torrent. Results. Among the clinical features only histological grade influenced OS. Deep sequencing identified 1784 single nucleotide variants. Of them, 426 were considered to be pathogenic or probably pathogenic. Activating IDH1/2 mutations were found in 27 patients (34%) including 17 R132 IDH1 (21%), 10 R172 IDH2 (13%) and 3 R140 IDH2 variants (4%). Three patients had concurrent IDH1 and IDH2 mutations. The R140 IDH2 mutant has not been reported to date in CHS patients. OS for CHS patients with IDH1/2 mutations was significantly lower than in patients without mutations (93% vs 64%; p<0.001). No other genetic feature of the Cancer Hotspot Panel had an impact on OS. Conclusions. In CHS, IDH1/2-mutation status and the histological aggressiveness of the CHS are important predictors for OS. The R140 IDH2 may also be a novel target for the treatment of CHS patients.