DNA Repair Pathway in Ovarian Cancer Patients Treated with HIPEC.

DNA repair pathways are essential for maintaining genome stability, and understanding the regulation of these mechanisms may help in the design of new strategies for treatments, the prevention of platinum-based chemoresistance, and the prolongation of overall patient survival not only with respect to ovarian cancer. The role of hyperthermic intraperitoneal chemotherapy (HIPEC) together with cytoreductive surgery (CRS) and adjuvant systemic chemotherapy is receiving more interest in ovarian cancer (OC) treatment because of the typical peritoneal spread of the disease. The aim of our study was to compare the expression level of 84 genes involved in the DNA repair pathway in tumors and the paired peritoneal metastasis tissue of patients treated with CRS/platinum-based HIPEC with respect to overall patient survival, presence of peritoneal carcinomatosis, treatment response, and alterations in the BRCA1 and BRCA2 genes. Tumors and metastatic tissue from 28 ovarian cancer patients collected during cytoreductive surgery before HIPEC with cisplatin were used for RNA isolation and subsequent cDNA synthesis. Quantitative real-time PCR followed. The most interesting findings of our study are undoubtedly the gene interactions among the genes CCNH, XPA, SLK, RAD51C, XPA, NEIL1, and ATR for primary tumor tissue and ATM, ATR, BRCA2, CDK7, MSH2, MUTYH, POLB, and XRCC4 for metastases. Another interesting finding is the correlation between gene expression and overall survival (OS), where a low expression correlates with a worse OS.

Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of TLC, chemical staining, carbohydrate-recognized ligand-binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4) and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches.

Identification of Recessively Inherited Genetic Variants Potentially Linked to Pancreatic Cancer Risk.

Although 21 pancreatic cancer susceptibility loci have been identified in individuals of European ancestry through genome-wide association studies (GWASs), much of the heritability of pancreatic cancer risk remains unidentified. A recessive genetic model could be a powerful tool for identifying additional risk variants. To discover recessively inherited pancreatic cancer risk loci, we performed a re-analysis of the largest pancreatic cancer GWAS, the Pancreatic Cancer Cohort Consortium (PanScan) and the Pancreatic Cancer Case-Control Consortium (PanC4), including 8,769 cases and 7,055 controls of European ancestry. Six single nucleotide polymorphisms (SNPs) showed associations with pancreatic cancer risk according to a recessive model of inheritance. We replicated these variants in 3,212 cases and 3,470 controls collected from the PANcreatic Disease ReseArch (PANDoRA) consortium. The results of the meta-analyses confirmed that rs4626538 (7q32.2), rs7008921 (8p23.2) and rs147904962 (17q21.31) showed specific recessive effects (p<10-5) compared with the additive effects (p>10-3), although none of the six SNPs reached the conventional threshold for genome-wide significance (p < 5×10-8). Additional bioinformatic analysis explored the functional annotations of the SNPs and indicated a possible relationship between rs36018702 and expression of the BCL2L11 and BUB1 genes, which are known to be involved in pancreatic biology. Our findings, while not conclusive, indicate the importance of considering non-additive genetic models when performing GWAS analysis. The SNPs associated with pancreatic cancer in this study could be used for further meta-analysis for recessive association of SNPs and pancreatic cancer risk and might be a useful addiction to improve the performance of polygenic risk scores.

Chromatographic method for the determination of inflammatory biomarkers and uric acid in human saliva.

Determination of concentration of biomarkers of the activation of immune system, uric acid, and creatinine in the saliva can be useful tool for the diagnosis and monitoring of early manifestations of diseases such as malignant, inflammatory, and periodontal disorders. We have developed and validated a high-performance liquid chromatographic method coupled with fluorescence and diode array detection for the separation and quantification of neopterin, tryptophan, creatinine, uric acid, and kynurenine in the human saliva. A separation of these analytes was achieved within 9 min by using second-generation monolithic stationary phase and elution with phosphate buffer. The present method involves very simple sample preparation requiring small amount of sample matrix. The internal standard 3-nitro-l-tyrosine was used for a more precise quantification. The sensitivity of the present method was demonstrated with lower limits of quantification of 0.6 × 10-3 µmol/L for neopterin, 0.725 µmol/L for tryptophan, 0.12 µmol/L for creatinine, 0.18 µmol/L for uric acid, and 0.135 µmol/L for kynurenine. The method was validated with 67 real-life saliva samples collected from patients suffering from breast, ovarian, colorectal, and renal cancer, and 19 saliva samples from patients with periodontal diseases and allowed monitoring of inflammatory response.

Polygenic and multifactorial scores for pancreatic ductal adenocarcinoma risk prediction.

BACKGROUND: Most cases of pancreatic ductal adenocarcinoma (PDAC) are asymptomatic in early stages, and the disease is typically diagnosed in advanced phases, resulting in very high mortality. Tools to identify individuals at high risk of developing PDAC would be useful to improve chances of early detection. OBJECTIVE: We generated a polygenic risk score (PRS) for PDAC risk prediction, combining the effect of known risk SNPs, and carried out an exploratory analysis of a multifactorial score. METHODS: We tested the associations of the individual known risk SNPs on up to 2851 PDAC cases and 4810 controls of European origin from the PANcreatic Disease ReseArch (PANDoRA) consortium. Thirty risk SNPs were included in a PRS, which was computed on the subset of subjects that had 100% call rate, consisting of 839 cases and 2040 controls in PANDoRA and 6420 cases and 4889 controls from the previously published Pancreatic Cancer Cohort Consortium I-III and Pancreatic Cancer Case-Control Consortium genome-wide association studies. Additional exploratory multifactorial scores were constructed by complementing the genetic score with smoking and diabetes. RESULTS: The scores were associated with increased PDAC risk and reached high statistical significance (OR=2.70, 95% CI 1.99 to 3.68, p=2.54×10-10 highest vs lowest quintile of the weighted PRS, and OR=14.37, 95% CI 5.57 to 37.09, p=3.64×10-8, highest vs lowest quintile of the weighted multifactorial score). CONCLUSION: We found a highly significant association between a PRS and PDAC risk, which explains more than individual SNPs and is a step forward in the direction of the construction of a tool for risk stratification in the population.

Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques.

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 µm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.

Genetic analysis of subsequent second primary malignant neoplasms in long-term pancreatic cancer survivors suggests new potential hereditary genetic alterations.

BACKGROUND: The principal aim of this report was to study second primary malignant neoplasms (SMNs) in long-term survivors of pancreatic ductal adenocarcinoma (PDAC) with regard to the germline genetic background. PATIENTS AND METHODS: A total of 118 PDAC patients after a curative-intent surgery who were treated between 2006 and 2011 were analyzed. Of the 22 patients surviving for >5 years, six went on to develop SMNs. A genetic analysis of 219 hereditary cancer-predisposition and candidate genes was performed by targeted next-generation sequencing in germline DNA from 20 of these patients. RESULTS: Of all the radically resected PDAC patients, six patients went on to subsequently develop SMNs, which accounted for 27% of the long-term survivors. The median time to diagnosis of SMNs, which included two cases of rectal cancer, and one case each of prostate cancer, malignant melanoma, breast cancer, and urinary bladder cancer, was 52.5 months. At the time of analysis, none of these patients had died as a result of PDAC progression. We identified four carriers of germline pathogenic mutations in 20 analyzed long-term survivors. One carrier of the CHEK2 mutation was found among four analyzed patients who developed SMNs. Of the remaining 16 long-term PDAC survivors, 3 patients (19%) carried germline mutation(s) in the MLH1+ ATM, CHEK2, and RAD51D gene, respectively. CONCLUSION: This retrospective analysis indicates that SMNs in PDAC survivors are an important clinical problem and may be more common than has been acknowledged to be the case. In patients with good performance status, surgical therapy should be considered, as the SMNs often have a favorable prognosis.

Dysregulation of KRAS signaling in pancreatic cancer is not associated with KRAS mutations and outcome.

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with a poor prognosis, and no targeted therapy is currently available. The aim of the present study was to investigate the prognostic significance of the expression of V-Ki-ras2 Κirsten rat sarcoma viral oncogene homolog (KRAS), downstream signaling pathway genes and the association with clinical characteristics in PDAC patients undergoing radical surgery. Tumors and adjacent non-neoplastic pancreatic tissues were examined in 45 patients with histologically verified PDAC. KRAS and B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene mutation analysis was performed using the KRAS/BRAF/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α array. The transcript profile of 52 KRAS downstream signaling pathway genes was assessed using quantitative-polymerase chain reaction. KRAS mutation was detected in 80% of cases. The genes of four signaling pathways downstream of KRAS, including the phosphoinositide 3-kinase/3-phosphoinositide-dependent protein kinase 1/V-akt murine thymoma viral oncogene homolog 1, RAL guanine nucleotide exchange factor, Ras and Rab interactor 1/ABL proto-oncogene-1, non-receptor tyrosine kinase, and RAF proto-oncogene serine/threonine-protein kinase/mitogen-activated protein kinase pathways, exhibited differential expression in PDAC compared with that in the adjacent normal tissues. However, no significant differences in expression were evident between patients with KRAS-mutated and wild-type tumors. The expression of KRAS downstream signaling pathways genes did not correlate with angioinvasion, perineural invasion, grade or presence of lymph node metastasis. Additionally, the presence of KRAS mutations was not associated with overall survival. Among the KRAS downstream effective signaling pathways molecules investigated, only v-raf-1 murine leukemia viral oncogene homolog 1 expression was predictive of prognosis. Overall, KRAS mutation is present in the majority of cases of PDAC, but is not associated with changes in the expression of KRAS downstream signaling pathways and the clinical outcome. This may partly explain the failure of KRAS-targeted therapies in PDAC.

SLC22A3 polymorphisms do not modify pancreatic cancer risk, but may influence overall patient survival.

Expression of the solute carrier (SLC) transporter SLC22A3 gene is associated with overall survival of pancreatic cancer patients. This study tested whether genetic variability in SLC22A3 associates with pancreatic cancer risk and prognosis. Twenty four single nucleotide polymorphisms (SNPs) tagging the SLC22A3 gene sequence and regulatory elements were selected for analysis. Of these, 22 were successfully evaluated in the discovery phase while six significant or suggestive variants entered the validation phase, comprising a total study number of 1,518 cases and 3,908 controls. In the discovery phase, rs2504938, rs9364554, and rs2457571 SNPs were significantly associated with pancreatic cancer risk. Moreover, rs7758229 associated with the presence of distant metastases, while rs512077 and rs2504956 correlated with overall survival of patients. Although replicated, the association for rs9364554 did not pass multiple testing corrections in the validation phase. Contrary to the discovery stage, rs2504938 associated with survival in the validation cohort, which was more pronounced in stage IV patients. In conclusion, common variation in the SLC22A3 gene is unlikely to significantly contribute to pancreatic cancer risk. The rs2504938 SNP in SLC22A3 significantly associates with an unfavorable prognosis of pancreatic cancer patients. Further investigation of this SNP effect on the molecular and clinical phenotype is warranted.

Structural characterization of a plant photosystem I and NAD(P)H dehydrogenase supercomplex.

Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI-NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI-NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.

Enhancement of immune response towards non-lipidized Borrelia burgdorferi recombinant OspC antigen by binding onto the surface of metallochelating nanoliposomes with entrapped lipophilic derivatives of norAbuMDP.

Lyme disease caused by spirochete Borrelia burgdorferi sensu lato, is a tick-born illness. If the infection is not eliminated by the host immune system and/or antibiotics, it may further disseminate and cause severe chronic complications. The immune response to Borrelia is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies associated with Th1 cell activation. A new experimental vaccine was constructed using non-lipidized form of recombinant B. burgdorferi s.s. OspC protein was anchored by metallochelating bond onto the surface of nanoliposomes containing novel nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After i.d. immunization, the experimental vaccines surpassed Alum with respect to OspC-specific titers of IgG2a, IgG2b isotypes when MT06 was used and IgG3, IgM isotypes when MT05 was used. Both adjuvants exerted a high adjuvant effect comparable or better than MDP and proved themselves as nonpyrogenic.

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests.

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.