Serum Metabolite Biomarkers for Pancreatic Tumors: Neuroendocrine and Pancreatic Ductal Adenocarcinomas-A Preliminary Study.

BACKGROUND: Pancreatic cancer is the most common pancreatic solid malignancy with an aggressive clinical course and low survival rate. There are a limited number of reliable prognostic biomarkers and a need to understand the pathogenesis of pancreatic tumors; neuroendocrine (PNET) and pancreatic ductal adenocarcinomas (PDAC) encouraged us to analyze the serum metabolome of pancreatic tumors and disturbances in the metabolism of PDAC and PNET. METHODS: Using the AbsoluteIDQ® p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria) with liquid chromatography-mass spectrometry (LC-MS), we identified changes in metabolite profiles and disrupted metabolic pathways serum of NET and PDAC patients. RESULTS: The concentration of six metabolites showed statistically significant differences between the control group and PDAC patients (p.adj < 0.05). Glutamine (Gln), acetylcarnitine (C2), and citrulline (Cit) presented a lower concentration in the serum of PDAC patients, while phosphatidylcholine aa C32:0 (PC aa C32:0), sphingomyelin C26:1 (SM C26:1), and glutamic acid (Glu) achieved higher concentrations compared to serum samples from healthy individuals. Five of the tested metabolites: C2 (FC = 8.67), and serotonin (FC = 2.68) reached higher concentration values in the PNET serum samples compared to PDAC, while phosphatidylcholine aa C34:1 (PC aa C34:1) (FC = -1.46 (0.68)) had a higher concentration in the PDAC samples. The area under the curves (AUC) of the receiver operating characteristic (ROC) curves presented diagnostic power to discriminate pancreatic tumor patients, which were highest for acylcarnitines: C2 with AUC = 0.93, serotonin with AUC = 0.85, and PC aa C34:1 with AUC = 0.86. CONCLUSIONS: The observations presented provide better insight into the metabolism of pancreatic tumors, and improve the diagnosis and classification of tumors. Serum-circulating metabolites can be easily monitored without invasive procedures and show the present clinical patients' condition, helping with pharmacological treatment or dietary strategies.

Obesity as a Risk Factor for Breast Cancer-The Role of miRNA.

Breast cancer (BC) is the most common cancer diagnosed among women in the world, with an ever-increasing incidence rate. Due to the dynamic increase in the occurrence of risk factors, including obesity and related metabolic disorders, the search for new regulatory mechanisms is necessary. This will help a complete understanding of the pathogenesis of breast cancer. The review presents the mechanisms of obesity as a factor that increases the risk of developing breast cancer and that even initiates the cancer process in the female population. The mechanisms presented in the paper relate to the inflammatory process resulting from current or progressive obesity leading to cell metabolism disorders and disturbed hormonal metabolism. All these processes are widely regulated by the action of microRNAs (miRNAs), which may constitute potential biomarkers influencing the pathogenesis of breast cancer and may be a promising target of anti-cancer therapies.

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.

Combination of HLA-DQ2/-DQ8 Haplotypes and a Single MSH5 Gene Variant in a Polish Population of Patients with Type 1 Diabetes as a First Line Screening for Celiac Disease?

Patients with type 1 diabetes (T1D) are at increased risk for developing celiac disease (CD). The aim of the study was to assess the usefulness of celiac-specific human leukocyte antigen (HLA) haplotype and the rs3130484 variant of MSH5 gene, a previously described non-HLA variant associated with CD in the Polish population as a first-line screening for CD in T1D pediatric patients. Serological CD screening performed in the T1D group (n = 248) and healthy controls (n = 551) allowed for CD recognition in 20 patients (8.1%) with T1D (T1D + CD group). HLA-DQ2, HLA-DQ8 and the rs3130484 variant were genotyped with TaqMan SNP Genotyping Assays. The T1D + CD group presented a higher, but not statistically significant, frequency of HLA-DQ2 in comparison with T1D subjects. Combining the rs3130484 with HLA-DQ2/HLA-DQ8 typing significantly increased the sensitivity of HLA testing from 32.7% to 68.7%, and the accuracy of estimating CD prediction from 51.7% to 86.4% but decreased the specificity from 100% to 78.2%. The receiver operating characteristic curve analysis confirmed the best discrimination for the combination of both genetic tests with an area under curve reaching 0.735 (95% CI: 0.700-0.7690) in comparison with 0.664 (95% CI: 0.632-0.696) for HLA typing alone. Results show the low utility of HLA-DQ2/HLA-DQ8 typing for CD screening in T1D pediatric patients. Combination of the rs3130484 variant of the MSH5 gene and HLA testing increases both the sensitivity and the predictive value of the test accuracy, but still, the obtained values are not satisfactory for recommending such testing as the first-line screening for CD in T1D patients.

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.

In search for interplay between stool microRNAs, microbiota and short chain fatty acids in Crohn's disease - a preliminary study.

BACKGROUND: Inflammatory bowel diseases are classic polygenic disorders, with genetic loads that reflect immunopathological processes in response to the intestinal microbiota. Herein we performed the multiomics analysis by combining the large scale surveys of gut bacterial community, stool microRNA (miRNA) and short chain fatty acid (SCFA) signatures to correlate their association with the activity of Crohn's disease (CD). METHODS: DNA, miRNA, and metabolites were extracted from stool samples of 15 CD patients, eight with active disease and seven in remission, and nine healthy individuals. Microbial, miRNA and SCFA profiles were assessed using datasets from 16S rRNA sequencing, Nanostring miRNA and GC-MS targeted analysis, respectively. RESULTS: Pairwise comparisons showed that 9 and 23 taxa differed between controls and CD patients with active and inactive disease, respectively. Six taxa were common to both comparisons, whereas four taxa differed in CD patients. α-Diversity was lower in both CD groups than in controls. The levels of 13 miRNAs differed (p-value < 0.05; FC > 1.5) in CD patients and controls before FDR correction and 4 after. Of six SCFAs, the levels of two differed significantly (p-value < 0.05, FC > 1.5) in CD patients and controls, and the levels of four differed in patients with active and inactive CD. PLS-DA revealed models with smallest error rate for controls in bacterial component and inactive disease in metabolites. CONCLUSION: A complex interrelationship may exist between gut dysbiosis, miRNA profiling and SCFA level in response to intestinal inflammation.

Differences between Well-Differentiated Neuroendocrine Tumors and Ductal Adenocarcinomas of the Pancreas Assessed by Multi-Omics Profiling.

Most pancreatic neuroendocrine tumors (PNETs) are indolent, while pancreatic ductal adenocarcinomas (PDACs) are particularly aggressive. To elucidate the basis for this difference and to establish the biomarkers, by using the deep sequencing, we analyzed somatic variants across coding regions of 409 cancer genes and measured mRNA/miRNA expression in nine PNETs, eight PDACs, and four intestinal neuroendocrine tumors (INETs). There were 153 unique somatic variants considered pathogenic or likely pathogenic, found in 50, 57, and 24 genes in PDACs, PNETs, and INETs, respectively. Ten and 11 genes contained a pathogenic mutation in at least one sample of all tumor types and in PDACs and PNETs, respectively, while 28, 34, and 11 genes were found to be mutated exclusively in PDACs, PNETs, and INETs, respectively. The mRNA and miRNA transcriptomes of PDACs and NETs were distinct: from 54 to 1659 differentially expressed mRNAs and from 117 to 250 differentially expressed miRNAs exhibited high discrimination ability and resulted in models with an area under the receiver operating characteristics curve (AUC-ROC) >0.9 for both miRNA and mRNA. Given the miRNAs high stability, we proposed exploring that class of RNA as new pancreatic tumor biomarkers.

Challenges in Cancer Biomarker Discovery Exemplified by the Identification of Diagnostic MicroRNAs in Prostate Tissues.

Identification and clinical translation of routinely tested biomarkers require a complex and multistep workflow. Here, we described a confirmatory process estimating the utility of previously identified candidate tissue miRNAs for diagnosis of prostate cancer (PCa). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) prostate tissue surgically resected from 44 patients with PCa and 24 patients with benign prostate hyperplasia (BPH). Of the 92 RNA samples obtained, 68 represented 42 malignant (PCa) areas and 26 represented nonmalignant (PCa 0%) areas of the prostate tissue sections. The levels of miR-32-5p, miR-183-5p, miR-141-5p, miR-187-3p, miR-375, miR-663b, miR-615-3p, miR-205-5p, miR-221-3p, and miR-222-3p were evaluated using Exiqon chemistry. Five (miR-32-5p, miR-141-5p, miR-187-3p, miR-375, and miR-615-3p), one (miR-32-5p), and two (miR-32-5p and miR-141-5p) miRNAs discriminated between BPH and areas of cancer-bearing prostate tissue harboring different numbers of cancer cells (PCa 15-70%, PCa 2-10%, and PCA 0%, respectively), with an area under the receiver operating characteristics curve (AUC-ROC) > 0.9. Only miRNA 32-5p discriminated BPH specimens from sections of cancer-bearing prostate tissue with a low percentage, a high percentage, or no dysplastic cells. miR-32-5p could be considered as potential diagnostic biomarker discriminating BPH from noncancerous areas within cancer-bearing prostate tissue. However, further clinical studies are warranted to confirm its diagnostic utility.

The composition and richness of the gut microbiota differentiate the top Polish endurance athletes from sedentary controls.

BACKGROUND: Little data are available on the subject of gut microbiota composition in endurance athletes as well as connections between diet and specific bacteria abundance. However, most studies suggest that athletes' microbiota undergoes major alterations, which may contribute to increased physical performance. Therefore, we decided to investigate differences in gut microbiota between healthy controls and endurance athletes. MATERIALS AND METHODS: Stools samples were collected from 14 marathon runners, 11 cross-country skiers and 46 sedentary healthy controls. The athletes' diet evaluation was performed with 24-h diet recall, using the Aliant programme. The 16S gene sequencing was performed using the Ion 16S Metagenomics Kit on Ion Torrent PGM sequencer. Taxonomic classification and diversity indices computation was performed with Mothur. RESULTS: 20 and 5 taxa differentiated healthy controls from marathon runners and cross-country skiers, respectively. Both groups presented a lowered abundance of major gut microbiota genus, Bacteroidetes and higher abundance of Prevotella. The athletes' microbiome was also more diverse in cross-country skiers than the one of sedentary controls (Simpson index p-value at 0.025). Thirty-one strong correlations (Spearman's coefficient > 0.6) were uncovered between bacteria abundance and diet, including inverse correlation of Prevotella with sucrose intake, Phascolarctobacterium with polyunsaturated fatty acids as well as positive correlation of Christensenellaceae with folic acid intake and Agathobacter with fiber amount in diet. CONCLUSIONS: The excessive training associates with both differences in composition and promotion of higher bacterial diversity. Taxons enriched in athletes are known to participate in fiber fermentation.

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.

Mutational Mosaics of Cell-Free DNA from Pancreatic Cyst Fluids.

BACKGROUND: Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts. METHODS: The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR. RESULTS: The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types. CONCLUSIONS: We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.

Mutation Profiling of Premalignant Colorectal Neoplasia.

Accumulation of allelic variants in genes that regulate cellular proliferation, differentiation, and apoptosis may result in expansion of the aberrant intestinal epithelium, generating adenomas. Herein, we compared the mutation profiles of conventional colorectal adenomas (CNADs) across stages of progression towards early carcinoma. DNA was isolated from 17 invasive adenocarcinomas (ACs) and 58 large CNADs, including 19 with low-grade dysplasia (LGD), 21 with LGD adjacent to areas of high-grade dysplasia and/or carcinoma (LGD-H), and 28 with high-grade dysplasia (HGD). Ion AmpliSeq Comprehensive Cancer Panel libraries were prepared and sequenced on the Ion Proton. We identified 956 unique allelic variants; of these, 499 were considered nonsynonymous variants. Eleven genes (APC, KRAS, SYNE1, NOTCH4, BLNK, FBXW7, GNAS, KMT2D, TAF1L, TCF7L2, and TP53) were mutated in at least 15% of all samples. Out of frequently mutated genes, TP53 and BCL2 had a consistent trend in mutation prevalence towards malignancy, while two other genes (HNF1A and FBXW7) exhibited the opposite trend. HGD adenomas had significantly higher mutation rates than LGD adenomas, while LGD-H adenomas exhibited mutation frequencies similar to those of LGD adenomas. A significant increase in copy number variant frequency was observed from LGD through HGD to malignant samples. The profiling of advanced CNADs demonstrated variations in mutation patterns among colorectal premalignancies. Only limited numbers of genes were repeatedly mutated while the majority were altered in single cases. Most genetic alterations in adenomas can be considered early contributors to colorectal carcinogenesis.

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.

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.

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.

Challenges in Stratifying the Molecular Variability of Patient-Derived Colon Tumor Xenografts.

Colorectal cancer (CRC) is the second most common cancer in Europe and a leading cause of death worldwide. Patient-derived xenograft (PDX) models maintain complex intratumoral biology and heterogeneity and therefore remain the platform of choice for translational drug discovery. In this study, we implanted 37 primary CRC tumors and five CRC cell lines into NU/J mice to develop xenograft models. Primary tumors and established xenografts were histologically assessed and surveyed for genetic variants and gene expression using a panel of 409 cancer-related genes and RNA-seq, respectively. More than half of CRC tumors (20 out of 37, 54%) developed into a PDX. Histological assessment confirmed that PDX grading, stromal components, inflammation, and budding were consistent with those of the primary tumors. DNA sequencing identified an average of 0.14 variants per gene per sample. The percentage of mutated variants in PDXs increased with successive passages, indicating a decrease in clonal heterogeneity. Gene Ontology analyses of 4180 differentially expressed transcripts (adj. p value < 0.05) revealed overrepresentation of genes involved in cell division and catabolic processes among the transcripts upregulated in PDXs; downregulated transcripts were associated with GO terms related to extracellular matrix organization, immune responses, and angiogenesis. Neither a transcriptome-based consensus molecular subtype (CMS) classifier nor three other predictors reliably matched PDX molecular subtypes with those of the primary tumors. In sum, both genetic and transcriptomic profiles differed between donor tumors and PDXs, likely as a consequence of subclonal evolution at the early phase of xenograft development, making molecular stratification of PDXs challenging.

Mass Spectrometry-Based Comprehensive Analysis of Pancreatic Cyst Fluids.

Pancreatic cyst fluids (PCFs) enriched in tumour-derived proteins are considered a potential source of new biomarkers. This study aimed to determine compositional and quantitative differences between the degradome and proteome of PCFs aspirated from different types of pancreatic cyst lesions (PCLs). 91 patients who underwent endoscopic ultrasound-fine needle aspiration under routine clinical diagnosis of PCLs were enrolled. Four cysts were malignant (CAs), and 87 were nonmalignant and consisted of 18 intraductal papillary mucinous neoplasms (IPMNs), 14 mucinous cystic neoplasms (MCNs), nine serous cystic neoplasms (SCNs), 29 pseudocysts (PCs), and 17 unclassified. Profiles of the <5 kDa fraction, the degradome, and the trypsin-digested proteome were analysed using an LTQ-Orbitrap Elite mass spectrometer coupled with a nanoACQUITY LC system. Qualitative analyses identified 796 and 366 proteins in degradome and proteome, respectively, and 689 (77%) and 285 (78%) of them were present in the Plasma Proteome Database. Gene Ontology analysis showed a significant overrepresentation of peptidases and peptidases inhibitors in both datasets. In the degradome fraction, quantitative values were obtained for 6996 peptides originating from 657 proteins. Of these, 2287 peptides were unique to a single type, and 515 peptides, derived from 126 proteins, were shared across cyst types. 32 peptides originating from 12 proteins had differential (adjusted p-value ≤0.05, FC ≥1.5) abundance in at least one of the five cysts types. In proteome, relative expression was measured for 330 proteins. Of them, 33 proteins had significantly (adjusted p-value ≤0.05, FC ≥1.5) altered abundance in at least one of the studied groups and 19 proteins appeared to be unique to a given cyst type. PCFs are dominated by blood proteins and proteolytic enzymes. Although differences in PCF peptide composition and abundance could aid classification of PCLs, the unpredictable inherent PCF proteolytic activity may limit the practical applications of PCF protein profiling.

A heterozygous mutation in GOT1 is associated with familial macro-aspartate aminotransferase.

BACKGROUND & AIMS: Macro-aspartate aminotransferase (macro-AST) manifests as a persistent elevation of AST levels, because of association of the protein with immunoglobulins in the circulation. Macro-AST is a rare, benign condition without a previously confirmed genetic basis. METHODS: Whole exome sequencing (WES)-based screening was performed on 32 participants with suspected familial macro-AST, while validation of variants was performed on an extended cohort of 92 probands and 1,644 healthy controls using Taqman genotyping. RESULTS: A missense variant (p.Gln208Glu, rs374966349) in glutamate oxaloacetate transaminase 1 (GOT1) was found, as a putative causal variant predisposing to familial macro-AST. The GOT1 p.Gln208Glu mutation was detected in 50 (54.3%) of 92 probands from 20 of 29 (69%) families, while its prevalence in healthy controls was only 0.18%. In silico analysis demonstrated that the amino acid at this position is not conserved among different species and that, functionally, a negatively charged glutamate on the GOT1 surface could strongly anchor serum immunoglobulins. CONCLUSIONS: Our data highlight that testing for the p.Gln208Glu genetic variant may be useful in diagnosis of macro-AST. LAY SUMMARY: Higher than normal levels of aspartate aminotransferase (AST) in the bloodstream may be a sign of a health problem. Individuals with macro-AST have elevated blood AST levels, without ongoing disease and often undergo unnecessary medical tests before the diagnosis of macro-AST is established. We found a genetic variant in the GOT1 gene associated with macro-AST. Genetic testing for this variant may aid diagnosis of macro-AST.

A novel approach to genome-wide association analysis identifies genetic associations with primary biliary cholangitis and primary sclerosing cholangitis in Polish patients.

BACKGROUND: Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are forms of hepatic autoimmunity, and risk for both diseases has a strong genetic component. This study aimed to define the genetic architecture of PBC and PSC within the Polish population. METHODS: Subjects were 443 women with PBC, 120 patients with PSC, and 934 healthy controls recruited from Gastroenterology Departments in various Polish hospitals. Allelotyping employed a pooled-DNA sample-based genome-wide association study (GWAS) approach, using Illumina Human Omni2.5-Exome BeadChips and the following novel selection criteria for risk loci: blocks of at least 10 single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium, where the distance between each adjacent SNP pair in the block was less than 30 kb, and each SNP was associated with disease at a significance level of P < 0.005. A selected index SNP from each block was validated using TaqMan SNP genotyping assays. RESULTS: Nineteen and twenty-one SNPs were verified as associated with PBC and PSC, respectively, by individual genotyping; 19 (10/9, PBC/PSC) SNPs reached a stringent (corrected) significance threshold and a further 21 (9/12, PBC/PSC) reached a nominal level of significance (P < 0.05 with odds ratio (OR) > 1.2 or < 0.83), providing suggestive evidence of association. The SNPs mapped to seven (1p31.3, 3q13, 6p21, 7q32.1, 11q23.3, 17q12, 19q13.33) and one (6p21) chromosome region previously associated with PBC and PSC, respectively. The SNP, rs35730843, mapping to the POLR2G gene promoter (P = 1.2 × 10-5, OR = 0.39) demonstrated the highest effect size, and was protective for PBC, whereas for PSC respective SNPs were: rs13191240 in the intron of ADGRB3 gene (P = 0.0095, OR = 0.2) and rs3822659 (P = 0.0051, OR = 0.236) along with rs9686714 (P = 0.00077, OR = 0.2), both located in the WWC1 gene. CONCLUSIONS: Our cost-effective GWAS approach followed by individual genotyping confirmed several previously identified associations and discovered new susceptibility loci associated with PBC and/or PSC in Polish patients. However, further functional studies are warranted to understand the roles of these newly identified variants in the development of the two disorders.