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.

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.

Mutational landscape of primary and recurrent Ewing sarcoma.

INTRODUCTION: Ewing sarcoma (ES) is a highly aggressive malignancy of bone and soft tissues characterized by the presence of a genetic fusion involving the EWSR1 gene. More than one-third of patients develop distant metastases, which are associated with unfavorable prognosis. Knowledge about the disease's genetic landscape may help foster progress in using targeted therapies in the treatment of ES. AIM OF THE STUDY: The objective is to assess the mutational landscape of ES in pretreatment samples, tumor samples after neoadjuvant chemotherapy, and in metastatic/recurrent tumors in children and adults. MATERIAL AND METHODS: DNA from 39 formalin-fixed paraffin-embedded tumor samples of 22 patients (17 adults, 5 children) were analyzed by targeted next generation sequencing (NGS) using the Oncomine Comprehensive Assay v3gene panel. Additional functional analyses were performed between patient subgroups. RESULTS: All samples were characterized by low tumor mutation burden (< 10 mut/Mb). The most commonly mutated genes were PIK3R1 (59%) and POLE (50%). The most widely detected variants in biopsy samples were PIK3R1 T369I (50%), FGFR1 E159K, and TP53 at codon 72 (both in 27.3%). Additionally, the ATR,BRCA1, RAD50,ATM,CHEK1, and NBN genes showed a significantly higher number of mutations in ES. Mutations in PIK3R1 were significantly more frequent in adults, while mutations in the pathways responsible for cell cycle control, DNA repair, and transcriptional regulation were more frequent in children. CONCLUSIONS: Besides EWSR1 fusion, ES is characterized by numerous point mutations that are potential targets for precision medicine. There is high genomic heterogeneity that may explain differences in outcomes between patient subgroups.

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.

GWAS links variants in neuronal development and actin remodeling related loci with pseudoexfoliation syndrome without glaucoma.

Pseudoexfoliation syndrome (PEXS) is an age-related elastosis, strongly associated with the development of secondary glaucoma. It is clearly suggested that PEXS has a genetic component, but this has not been extensively studied. Here, a genome-wide association study (GWAS) using a DNA-pooling approach was conducted to explore the potential association of genetic variants with PEXS in a Polish population, including 103 PEXS patients without glaucoma and 106 perfectly (age- and gender-) matched controls. Individual sample TaqMan genotyping was used to validate GWAS-selected single-nucleotide polymorphism (SNP) associations. Multivariate binary logistic regression analysis was applied to develop a prediction model for PEXS. In total, 15 SNPs representing independent PEXS susceptibility loci were selected for further validation in individual samples. For 14 of these variants, significant differences in the allele and genotype frequencies between cases and controls were identified, of which 12 remained significant after Benjamini-Hochberg adjustment. The minor allele of five SNPs was associated with an increased risk of PEXS development, while for nine SNPs, it showed a protective effect. Beyond the known LOXL1 variant rs2165241, nine other SNPs were located within gene regions, including in OR11L1, CD80, TNIK, CADM2, SORBS2, RNF180, FGF14, FMN1, and RBFOX1 genes. None of these associations with PEXS has previously been reported. Selected SNPs were found to explain nearly 69% of the total risk of PEXS development. The overall risk prediction accuracy for PEXS, expressed by the area under the ROC curve (AUC) value, increased by 0.218, from 0.672 for LOXL1 rs2165241 alone to 0.89 when seven additional SNPs were included in the proposed 8-SNP prediction model. In conclusion, several new susceptibility loci for PEXS without glaucoma suggested that neuronal development and actin remodeling are potentially involved in either PEXS onset or inhibition or delay of its conversion to glaucoma.

Limited predictive value of achieving beneficial plasma (Z)-endoxifen threshold level by CYP2D6 genotyping in tamoxifen-treated Polish women with breast cancer.

BACKGROUND: Tamoxifen, the most frequently used drug for treating estrogen receptor-positive breast cancer, must be converted into active metabolites to exert its therapeutic efficacy, mainly through CYP2D6 enzymes. The objective of this study was to investigate the impact of CYP2D6 polymorphisms on (Z)-endoxifen-directed tamoxifen metabolism and to assess the usefulness of CYP2D6 genotyping for identifying patients who are likely to have insufficient (Z)-endoxifen concentrations to benefit from standard therapy. METHODS: Blood samples from 279 Polish women with breast cancer receiving tamoxifen 20 mg daily were analyzed for CYP2D6 genotype and drug metabolite concentration. Steady-state plasma levels of tamoxifen and its 14 metabolites were measured by using the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. RESULTS: In nearly 60 % of patients, including over 30 % of patients with fully functional CYP2D6, (Z)-endoxifen concentration was below the predefined threshold of therapeutic efficacy. The most frequently observed CYP2D6 genotype was EM/PM (34.8 %), among which 83.5 % of patients had a combination of wild-type and *4 alleles. Plasma concentration of five metabolites was significantly correlated with CYP2D6 genotype. For the first time, we identified an association between decreased (E/Z)-4-OH-N-desmethyl-tamoxifen-ß-D-glucuronide levels (r (2) = 0.23; p < 10(-16)) and increased CYP2D6 functional impairment. The strongest correlation was observed for (Z)-endoxifen, whose concentration was significantly lower in groups of patients carrying at least one CYP2D6 null allele, compared with EM/EM patients. The CYP2D6 genotype accounted for plasma level variability of (Z)-endoxifen by 27 % (p < 10(-16)) and for the variability of metabolic ratio indicating (Z)-endoxifen-directed metabolism of tamoxifen by 51 % (p < 10(-43)). CONCLUSIONS: The majority of breast cancer patients in Poland may not achieve a therapeutic level of (Z)-endoxifen upon receiving a standard dose of tamoxifen. This finding emphasizes the limited value of CYP2D6 genotyping in routine clinical practice for identifying patients who might not benefit from the therapy. In its place, direct monitoring of plasma steady-state (Z)-endoxifen concentration should be performed to personalize and optimize the treatment.

Breast cancer but not the menopausal status is associated with small changes of the gut microbiota.

Background: Possible relationships between gut dysbiosis and breast cancer (BC) development and progression have been previously reported. However, the results of these metagenomics studies are inconsistent. Our study involved 88 patients diagnosed with breast cancer and 86 cancer-free control women. Participants were divided into groups based on their menopausal status. Fecal samples were collected from 47 and 41 pre- and postmenopausal newly diagnosed breast cancer patients and 51 and 35 pre- and postmenopausal controls, respectively. In this study, we performed shotgun metagenomic analyses to compare the gut microbial community between pre- and postmenopausal BC patients and the corresponding controls. Results: Firstly, we identified 12, 64, 158, and 455 bacterial taxa on the taxonomy level of phyla, families, genera, and species, respectively. Insignificant differences of the Shannon index and ß-diversity were found at the genus and species levels between pre- and postmenopausal controls; the differences concerned only the Chao index at the species level. No differences in α-diversity indexes were found between pre- and postmenopausal BC patients, although ß-diversity differed these subgroups at the genus and species levels. Consistently, only the abundance of single taxa differed between pre- and postmenopausal controls and cases, while the abundances of 14 and 23 taxa differed or tended to differ between premenopausal cases and controls, and between postmenopausal cases and controls, respectively. There were similar differences in the distribution of enterotypes. Of 460 bacterial MetaCyc pathways discovered, no pathways differentiated pre- and postmenopausal controls or BC patients, while two and one pathways differentiated cases from controls in the pre- and postmenopausal subgroups, respectively. Conclusion: While our findings did not reveal an association of changes in the overall microbiota composition and selected taxa with the menopausal status in cases and controls, they confirmed differences of the gut microbiota between pre- and postmenopausal BC patients and the corresponding controls. However, these differences were less extensive than those described previously.

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.

Exome sequencing to explore the possibility of predicting genetic susceptibility to the joint occurrence of polycystic ovary syndrome and Hashimoto's thyroiditis.

A large body of evidence indicates that women with polycystic ovary syndrome (PCOS) have a higher risk of developing Hashimoto's thyroiditis (HT) than healthy individuals. Given the strong genetic impact on both diseases, common predisposing genetic factors are possibly involved but are not fully understood. Here, we performed whole-exome sequencing (WES) for 250 women with sporadic PCOS, HT, combined PCOS and HT (PCOS+HT), and healthy controls to explore the genetic background of the joint occurrence of PCOS and HT. Based on relevant comparative analyses, multivariate logistic regression prediction modeling, and the most informative feature selection using the Monte Carlo feature selection and interdependency discovery algorithm, 77 variants were selected for further validation by TaqMan genotyping in a group of 533 patients. In the allele frequency test, variants in RAB6A, GBP3, and FNDC7 genes were found to significantly (padjusted < 0.05) differentiated the PCOS+HT and PCOS groups, variant in HIF3A differentiated the PCOS+HT and HT groups, whereas variants in CDK20 and CCDC71 differentiated the PCOS+HT and both single disorder groups. TaqMan genotyping data were used to create final prediction models, which differentiated between PCOS+HT and PCOS or HT with a prediction accuracy of AUC = 0.78. Using a 70% cutoff of the prediction score improved the model parameters, increasing the AUC value to 0.87. In summary, we demonstrated the polygenic burden of both PCOS and HT, and many common and intersecting signaling pathways and biological processes whose disorders mutually predispose patients to the development of both diseases.

The comparative analysis of selected interleukins and proinflammatory factors in CSF among de novo diagnosed patients with RRMS.

OBJECTIVES: Cytokines play a key role in neuroinflammation, which is present in every subset of multiple sclerosis (MS). The aim of the study was to assess levels of selected interleukins and proinflammatory factors in cerebrospinal fluid (CSF) among patients diagnosed with relapsing-remitting multiple sclerosis (RRMS). METHODS: One hundred eighteen patients diagnosed de novo with RRMS were enrolled in the study. We analysed the relationships between selected cytokines' levels depending on the age at diagnosis, time from the first symptoms to diagnosis and presence of MRI lesions. RESULTS: Among the study group the levels of IL-5 and IL-13 increased with the age at the diagnosis of MS. The concentration of IL-10 was lower in group of patients over the age of 35. The levels of IFN-γ, TNF-α, IL-5, IL-10 and IL-15 increased with the longer time from the first symptoms to diagnosis. Positive correlations were found between the levels of IL-2 and IL-12, IL-17, IL-4, IL-1RA as well as IL-1 and IL-4, IL-17. The concentration of IL-5 correlated positively with IL-4, IL-9 and IL-13. The level of IL-10 increased with IL-6 and IL-9 concentrations. A negative correlation was found for IL-10 and IL-4. In turn, between IL-13 and both IL-5 and IL-9, the relationship was positive. The level of IL-2 was significantly higher among patients without gadolinium-enhanced (Gd(+)) MRI lesions. CONCLUSIONS: The results of the study provide new insight into the role of selected molecules in the development of inflammation in MS. It might be crucial in planning the most adequate immunomodulatory therapy.

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.

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.

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.

A novel germline PALB2 deletion in Polish breast and ovarian cancer patients.

BACKGROUND: PALB2 protein was recently identified as a partner of BRCA1 and BRCA2 which determines their proper function in DNA repair. METHODS: Initially, the entire coding sequence of the PALB2 gene with exon/intron boundaries was evaluated by the PCR-SSCP and direct sequencing methods on 70 ovarian carcinomas. Sequence variants of interest were further studied on enlarged groups of ovarian carcinomas (total 339 non-consecutive ovarian carcinomas), blood samples from 334 consecutive sporadic and 648 consecutive familial breast cancer patients, and 1310 healthy controls from central Poland. RESULTS: Ten types of sequence variants were detected, and among them four novel polymorphisms: c.2996+58T>C in intron 9; c.505C>A (p.L169I), c.618T>G (p.L206L), both in exon 4; and c.2135C>T (A712V) in exon 5 of the PALB2 gene. Another two polymorphisms, c.212-58A>C and c.2014G>C (E672Q) were always detected together, both in cancer (7.5% of patients) and control samples (4.9% of controls, p = 0.2). A novel germline truncating mutation, c.509_510delGA (p.R170fs) was found in exon 4: in 2 of 339 (0.6%) unrelated ovarian cancer patients, in 4 of 648 (0.6%) unrelated familial breast cancer patients, and in 1 of 1310 controls (0.08%, p = 0.1, p = 0.044, respectively). One ovarian cancer patient with the PALB2 mutation had also a germline nonsense mutation of the BRCA2 gene. CONCLUSIONS: The c.509_510delGA is a novel PALB2 mutation that increases the risk of familial breast cancer. Occurrence of the same PALB2 alteration in seven unrelated women suggests that c.509_510delGA (p.R170fs) is a recurrent mutation for Polish population.

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.

Age at onset of bilateral breast cancer, the presence of hereditary BRCA1, BRCA2, CHEK2 gene mutations and positive family history of cancer.

BACKGROUND: Our research focused on the relations between the age at onset of bilateral breast cancer and the prevalence of selected hereditary BRCA1, BRCA2 and CHEK2 gene mutations with reference to their positive family history of cancer. METHODS: The DNA of peripheral blood lymphocytes of patients was examined for the presence of selected hereditary mutations in the BRCA1, BRCA2 and CHEK2 genes, using molecular biology techniques. The family history of neoplasms was also analyzed. RESULTS: The following mutations in the BRCA1 gene were identified: 185delAG, C61G, 5382insC, 3875 del11ins7, and R1751X. In the BRCA2 gene, the 9631delC and A9599T mutations were found. In the CHEK2 gene, the 1100delC and I157T mutations were identified. BRCA1/2 gene mutations were identified in 19.4% of patients and CHEK2 gene mutations in 7%. CONCLUSIONS: It was shown that the presence of the mutations in the BRCA1/2 genes among patients with bilateral breast cancer is associated with an earlier occurrence of the first and the second breast cancer than in patients without hereditary mutations in these genes (a difference of 7.2-8.4 years). In patients with CHEK2 gene mutations, breast cancer occurred 2.1-3.8 years earlier than in patients without mutations in the CHEK2 gene.

Non-CYP2D6 Variants Selected by a GWAS Improve the Prediction of Impaired Tamoxifen Metabolism in Patients with Breast Cancer.

A certain minimum plasma concentration of (Z)-endoxifen is presumably required for breast cancer patients to benefit from tamoxifen therapy. In this study, we searched for DNA variants that could aid in the prediction of risk for insufficient (Z)-endoxifen exposure. A metabolic ratio (MR) corresponding to the (Z)-endoxifen efficacy threshold level was adopted as a cutoff value for a genome-wide association study comprised of 287 breast cancer patients. Multivariate regression was used to preselect variables exhibiting an independent impact on the MR and develop models to predict below-threshold MR values. In total, 15 single-nucleotide polymorphisms (SNPs) were significantly associated with below-threshold MR values. The strongest association was with rs8138080 (WBP2NL). Two alternative models for MR prediction were developed. The predictive accuracy of Model 1, including rs7245, rs6950784, rs1320308, and the CYP2D6 genotype, was considerably higher than that of the CYP2D6 genotype alone (AUC 0.879 vs 0.758). Model 2, which was developed using the same three SNPs as for Model 1 plus rs8138080, appeared as an interesting alternative to the full CYP2D6 genotype testing. In conclusion, the four novel SNPs, tested alone or in combination with the CYP2D6 genotype, improved the prediction of impaired tamoxifen-to-endoxifen metabolism, potentially allowing for treatment optimization.