Aptamer Proteomics for Biomarker Discovery in Heart Failure With Preserved Ejection Fraction: The PARAGON-HF Proteomic Substudy.

BACKGROUND: Prognostic markers and biological pathways linked to detrimental clinical outcomes in heart failure with preserved ejection fraction (HFpEF) remain incompletely defined. METHODS AND RESULTS: We measured serum levels of 4123 unique proteins in 1117 patients with HFpEF enrolled in the PARAGON-HF (Efficacy and Safety of LCZ696 Compared to Valsartan, on Morbidity and Mortality in Heart Failure Patients With Preserved Ejection Fraction) trial using a modified aptamer proteomic assay. Baseline circulating protein concentrations significantly associated with the primary end point and the timing and occurrence of total heart failure hospitalization and cardiovascular death were identified by recurrent events regression, accounting for multiple testing, adjusted for age, sex, treatment, and anticoagulant use, and compared with published analyses in 2515 patients with heart failure with reduced ejection fraction from the PARADIGM-HF (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) and ATMOSPHERE (Efficacy and Safety of Aliskiren and Aliskiren/Enalapril Combination on Morbidity-Mortality in Patients With Chronic Heart Failure) clinical trials. We identified 288 proteins that were robustly associated with the risk of heart failure hospitalization and cardiovascular death in patients with HFpEF. The baseline proteins most strongly related to outcomes included B2M (ß-2 microglobulin), TIMP1 (tissue inhibitor of matrix metalloproteinase 1), SERPINA4 (serpin family A member 4), and SVEP1 (sushi, von Willebrand factor type A, EGF, and pentraxin domain containing 1). Overall, the protein-outcome associations in patients with HFpEF did not markedly differ as compared with patients with heart failure with reduced ejection fraction. A proteomic risk score derived in patients with HFpEF was not superior to a previous proteomic score derived in heart failure with reduced ejection fraction nor to clinical risk factors, NT-proBNP (N-terminal pro-B-type natriuretic peptide), or high-sensitivity cardiac troponin. CONCLUSIONS: Numerous serum proteins linked to metabolic, coagulation, and extracellular matrix regulatory pathways were associated with worse HFpEF prognosis in the PARAGON-HF proteomic substudy. Our results demonstrate substantial similarities among serum proteomic risk markers for heart failure hospitalization and cardiovascular death when comparing clinical trial participants with heart failure across the ejection fraction spectrum. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifiers: NCT01920711, NCT01035255, NCT00853658.

High-Throughput CSF Proteomics and Machine Learning to Identify Proteomic Signatures for Parkinson Disease Development and Progression.

BACKGROUND AND OBJECTIVES: This study aimed to identify CSF proteomic signatures characteristic of Parkinson disease (PD) and evaluate their clinical utility. METHODS: This observational study used data from the Parkinson's Progression Markers Initiative (PPMI), which enrolled patients with PD, healthy controls (HCs), and non-PD participants carrying GBA1, LRRK2, and/or SNCA pathogenic variants (genetic prodromals) at international sites. Study participants were chosen from PPMI enrollees based on the availability of aptamer-based CSF proteomic data, quantifying 4,071 proteins, and classified as patients with PD without GBA1, LRRK2, and/or SNCA pathogenic variants (nongenetic PD), HCs, patients with PD carrying the aforementioned pathogenic variants (genetic PD), or genetic prodromals. Differentially expressed protein (DEP) analysis and the least absolute shrinkage and selection operator (LASSO) were applied to the data from nongenetic PD and HCs. Signatures characteristics of nongenetic PD were quantified as a PD proteomic score (PD-ProS), validated internally and then externally using data of 1,556 CSF proteins from the LRRK2 Cohort Consortium (LCC). We further tested the PD-ProS in genetic PD and genetic prodromals and examined associations with clinical progression. RESULTS: Data from 279 patients with nongenetic PD (mean ± SD, age 62.0 ± 9.6 years; male 67.7%) and 141 HCs (age 60.5 ± 11.9 years; male 64.5%) were used for PD-ProS derivation. From 23 DEPs, LASSO determined weights of 14 DEPs for the PD-ProS (area under the curve [AUC] 0.83, 95% CI 0.78-0.87), validated in an independent internal validation cohort of 71 patients with nongenetic PD and 35 HCs (AUC 0.81, 95% CI 0.73-0.90). In the LCC, only 5 of the 14 DEPs were also measured. Notably, these 5 DEPs still distinguished 34 patients with nongenetic PD from 31 HCs with the same weights (AUC 0.75, 95% CI 0.63-0.87). Furthermore, the PD-ProS distinguished 258 patients with genetic PD from 365 genetic prodromals. Finally, regardless of genetic status, the PD-ProS independently predicted both cognitive and motor decline in PD (dementia, adjusted hazard ratio in the highest quintile [aHR-Q5] 2.8 [95% CI 1.6-5.0]; Hoehn and Yahr stage IV, aHR-Q5 2.1 [95% CI 1.1-4.0]). DISCUSSION: By integrating high-throughput proteomics with machine learning, we identified PD-associated CSF proteomic signatures crucial for PD development and progression. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov (NCT01176565). A link to the trial registry page is clinicaltrials.gov/ct2/show/NCT01141023. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that the CSF proteome contains clinically important information regarding the development and progression of Parkinson disease that can be deciphered by a combination of high-throughput proteomics and machine learning.

A proteogenomic view of Parkinson's disease causality and heterogeneity.

The pathogenesis and clinical heterogeneity of Parkinson's disease (PD) have been evaluated from molecular, pathophysiological, and clinical perspectives. High-throughput proteomic analysis of cerebrospinal fluid (CSF) opened new opportunities for scrutinizing this heterogeneity. To date, this is the most comprehensive CSF-based proteomics profiling study in PD with 569 patients (350 idiopathic patients, 65 GBA + mutation carriers and 154 LRRK2 + mutation carriers), 534 controls, and 4135 proteins analyzed. Combining CSF aptamer-based proteomics with genetics we determined protein quantitative trait loci (pQTLs). Analyses of pQTLs together with summary statistics from the largest PD genome wide association study (GWAS) identified 68 potential causal proteins by Mendelian randomization. The top causal protein, GPNMB, was previously reported to be upregulated in the substantia nigra of PD patients. We also compared the CSF proteomes of patients and controls. Proteome differences between GBA + patients and unaffected GBA + controls suggest degeneration of dopaminergic neurons, altered dopamine metabolism and increased brain inflammation. In the LRRK2 + subcohort we found dysregulated lysosomal degradation, altered alpha-synuclein processing, and neurotransmission. Proteome differences between idiopathic patients and controls suggest increased neuroinflammation, mitochondrial dysfunction/oxidative stress, altered iron metabolism and potential neuroprotection mediated by vasoactive substances. Finally, we used proteomic data to stratify idiopathic patients into "endotypes". The identified endotypes show differences in cognitive and motor disease progression based on previously reported protein-based risk scores.Our findings not only contribute to the identification of new therapeutic targets but also to shape personalized medicine in CNS neurodegeneration.

Serum Concentrations of Selenium and Copper in Patients Diagnosed with Pancreatic Cancer.

PURPOSE: Understanding of the etiology and pathogenesis of pancreatic cancer (PaCa) is still insufficient. This study evaluated the associations between concentrations of selenium (Se) and copper (Cu) in the serum of PaCa patients. MATERIALS AND METHODS: The study included 100 PaCa patients and 100 control subjects from the same geographical region in Poland. To determine the average concentration of Se, Cu, and ratio Cu:Se in the Polish population, assay for Se and Cu was performed in 480 healthy individuals. Serum levels of Se and Cu were measured using inductively coupled plasma mass spectrometry. RESULTS: In the control group, the average Se level was 76 µg/L and Cu 1,098 µg/L. The average Se level among PaCa patients was 60 µg/L and the mean Cu level was 1,432 µg/L. The threshold point at which any decrease in Se concentration was associated with PaCa was 67.45 µg/L. The threshold point of Cu level above which there was an increase in the prevalence of PaCa was 1,214.58 µg/L. In addition, a positive relationship was observed between increasing survival time and Se plasma level. CONCLUSION: This retrospective study suggests that low levels of Se and high levels of Cu might influence development of PaCa and that higher levels of Se are associated with longer survival in patients with PaCa. The results suggest that determining the level of Se and Cu could be incorporated into a risk stratification scheme for the selection and surveillance control examination to complement existing screening and diagnostic procedures.

Cumulative Small Effect Genetic Markers and the Risk of Colorectal Cancer in Poland, Estonia, Lithuania, and Latvia.

The continued identification of new low-penetrance genetic variants for colorectal cancer (CRC) raises the question of their potential cumulative effect among compound carriers. We focused on 6 SNPs (rs380284, rs4464148, rs4779584, rs4939827, rs6983267, and rs10795668), already described as risk markers, and tested their possible independent and combined contribution to CRC predisposition. Material and Methods. DNA was collected and genotyped from 2330 unselected consecutive CRC cases and controls from Estonia (166 cases and controls), Latvia (81 cases and controls), Lithuania (123 cases and controls), and Poland (795 cases and controls). Results. Beyond individual effects, the analysis revealed statistically significant linear cumulative effects for these 6 markers for all samples except of the Latvian one (corrected P value = 0.018 for the Estonian, corrected P value = 0.0034 for the Lithuanian, and corrected P value = 0.0076 for the Polish sample). Conclusions. The significant linear cumulative effects demonstrated here support the idea of using sets of low-risk markers for delimiting new groups with high-risk of CRC in clinical practice that are not carriers of the usual CRC high-risk markers.

Selenium as a marker of cancer risk and of selection for control examinations in surveillance.

Publication is summarization of existing data being results of literature review and our experience on usefulness of selenium as a diagnostic marker selection for control examinations in surveillance and as a marker of patients with high risk of cancers.

Management of ovarian and endometrial cancers in women belonging to HNPCC carrier families: review of the literature and results of cancer risk assessment in Polish HNPCC families.

BACKGROUND: Over half the cancer deaths in HNPCC families are due to extra-colonic malignancies that include endometrial and ovarian cancers. The benefits of surveillance for gynecological cancers are not yet proven and there is no consensus on the optimal surveillance recommendations for women with MMR mutations. METHODS: We performed a systematic review of the literature and evaluated gynecological cancer risk in a series of 631 Polish HNPCC families classified into either Lynch Syndrome (LS, MMR mutations detected) or HNPCC (fulfillment of the Amsterdam or modified Amsterdam criteria). RESULTS: Published data clearly indicates no benefit for ovarian cancer screening in contrast to risk reducing surgery. We confirmed a significantly increased risk of OC in Polish LS families (OR = 4,6, p < 0.001) and an especially high risk of OC was found for women under 50 years of age: OR = 32,6, p < 0.0001 (95% CI 12,96-81,87). The cumulative OC risk to 50 year of life was calculated to be 10%. Six out of 19 (32%) early-onset patients from LS families died from OC within 2 years of diagnosis. We confirmed a significantly increased risk of EC (OR = 26, 95% CI 11,36-58,8; p < 0,001). The cumulative risk for EC in Polish LS families was calculated to be 67%. CONCLUSIONS: Due to the increased risk of OC and absence of any benefit from gynecological screening reported in the literature it is recommended that prophylactic oophorectomy for female carriers of MMR mutations after 35 year of age should be considered as a risk reducing option. Annual transvaginal ultrasound supported by CA125 or HE4 marker testing should be performed after prophylactic surgery in these women. Due to the high risk of EC it is reasonable to offer, after the age of 35 years, annual clinical gynecologic examinations with transvaginal ultrasound supported by routine aspiration sampling of the endometrium for women from either LS or HNPCC families. An alternative option, which could be taken into consideration for women preferring surgical prevention, is risk reducing total hysterectomy (with bilateral salpingo-oophorectomy) for carriers after childbearing is complete.

Common variants of xeroderma pigmentosum genes and prostate cancer risk.

The genetic basis of prostate cancer (PC) is complex and appears to involve multiple susceptibility genes. A number of studies have evaluated a possible correlation between several NER gene polymorphisms and PC risk, but most of them evaluated only single SNPs among XP genes and the results remain inconsistent. Out of 94 SNPs located in seven XP genes (XPA-XPG) a total of 15 SNPs were assayed in 720 unselected patients with PC and compared to 1121 healthy adults. An increased risk of disease was associated with the XPD SNP, rs1799793 (Asp312Asn) AG genotype (OR=2.60; p<0.001) and with the AA genotype (OR=531; p<0.0001) compared to the control population. Haplotype analysis of XPD revealed one protective haplotype and four associated with an increased disease risk, which showed that the A allele (XPD rs1799793) appeared to drive the main effect on promoting prostate cancer risk. Polymorphism in XPD gene appears to be associated with the risk of prostate cancer.

A common nonsense mutation of the BLM gene and prostate cancer risk and survival.

BACKGROUND: Germline mutations of BRCA2 and NBS1 genes cause inherited recessive chromosomal instability syndromes and predispose to prostate cancer of poor prognosis. Mutations of the BLM gene cause another chromosomal instability clinical syndrome, called Bloom syndrome. Recently, a recurrent truncating mutation of BLM (Q548X) has been associated with a 6-fold increased risk of breast cancer in Russia, Belarus and Ukraine, but its role in prostate cancer etiology and survival has not been investigated yet. METHODS: To establish whether the Q548X allele of the BLM gene is present in Poland, and whether this allele predisposes to poor prognosis prostate cancer, we genotyped 3337 men with prostate cancer and 2604 controls. RESULTS: Q548X was detected in 13 of 3337 (0.4%) men with prostate cancer compared to 15 of 2604 (0.6%) controls (OR=0.7; 95% CI 0.3-1.4). A positive family history of any cancer in a first- or second-degree relative was seen only in 4 of the 13 (30%) mutation positive families, compared to 49% (1485/3001) of the non-carrier families (p=0.3). The mean follow-up was 49months. Survival was similar among carriers of Q548X and non-carriers (HR=1.1; p=0.9). The 5-year survival for men with a BLM mutation was 83%, compared to 72% for mutation-negative cases. CONCLUSIONS: BLM Q548X is a common founder mutation in Poland. We found no evidence that this mutation predisposes one to prostate cancer or affect prostate cancer survival. However, based on the observed 0.6% population frequency of the Q548X allele, we estimate that one in 100,000 children should be affected by Bloom syndrome in Poland.

Can selenium levels act as a marker of colorectal cancer risk?

BACKGROUND: Selenium has attracted attention because of its antioxidant properties. Antioxidants protects cells from damage. Certain breakdown products of selenium are believed to prevent tumor growth by enhancing the immune cell activity and suppressing the development of tumor blood vessels. In this observational study, selenium level was measured in a series of patients from Poland and Estonia to determine a correlation between levels of this microelement and colorectal cancer risk. METHODS: A total of 169 colorectal cancer patients and 169 healthy controls were enrolled in the study after obtaining their informed consent. Selenium level in the blood serum was measured using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). The statistical analysis was performed by Fisher's exact test. RESULTS: The threshold point of selenium level was 55 µg/l and 65 µg/l for Poland and Estonia respectively, for an increase in cancer risk. The lower levels of selenium were associated with greater risk of colorectal cancer. CONCLUSIONS: The result reveals a significant strong association between low selenium level and the colorectal cancer risk in both Estonian and Polish populations.

DNA and RNA analyses in detection of genetic predisposition to cancer.

During the past decade many new molecular methods for DNA and RNA analysis have emerged. The most popular thus far have been SSCP, HET, CMC, DGGE, RFLP or ASA, which have now been replaced by methods that are more cost effective and less time consuming. Real-time amplification techniques and particularly those with the capacity of multiplexing have become commonly used in laboratory practice. Novel screening methods enable the very rapid examination of large patients series. Use of liquid handling robotics applied to the isolation of DNA or RNA, the normalisation of sample concentration, and standardization of target amplification by PCR have also contributed to a reduced risk of sample contamination and have resulted in laboratory analysis being easier and faster.The aim of this study is the introduction of a few modern techniques, most commonly used in detection of genetic predisposition to cancer.

Association of MMP8 gene variation with an increased risk of malignant melanoma.

Matrix metalloproteinases (MMPs) are implicated in the development of cancers including malignant melanoma (MM) and breast cancer. We tested the possible association of MMP1 and MMP8 gene variation with these two types of cancer. We genotyped 300 unselected patients with MM, 300 consecutive breast cancer cases, 300 controls for melanoma, and 300 controls for breast cancer (age-matched and sex-matched healthy adults with negative cancer family histories). Our study showed that the MMP8 gene rs11225395 polymorphism was associated with the risk of developing MM (odds ratio: 1.69; 95% confidence interval: 1.02-2.80; P=0.040) for the A/A genotype and 1.49 (95% confidence interval: 1.03-2.17; P=0.035) for the A/G genotype compared with the G/G genotype. The A allele was over-represented among MM cases compared with controls (odds ratio=1.54; P=0.017). In-vitro assays showed that the A allele had a higher promoter activity than the G allele in melanoma cells. No association was detected between this variant and breast cancer susceptibility. We found no strong association between MMP1 variation and the risk of MM or breast cancer. The finding of this study indicates an influence of MMP8 gene variation on melanoma susceptibility.

Variant alleles of the CYP1B1 gene are associated with colorectal cancer susceptibility.

BACKGROUND: CYP1B1 is a P450 enzyme which is involved in the activation of pro-carcinogens to carcinogens as well as sex hormone metabolism. Because differences in the activity of the enzyme have been correlated with variant alleles of single nucleotide polymorphisms (SNPs), it represents an attractive candidate gene for studies into colorectal cancer susceptibility. METHODS: We genotyped 597 cancer patients and 597controls for three CYP1B1 SNPs, which have previously been shown to be associated with altered enzymatic activity. Using the three SNPs, eight different haplotypes were constructed. The haplotype frequencies were estimated in cases and controls and then compared. The odds ratio for each tumour type, associated with each haplotype was estimated, with reference to the most common haplotype observed in the controls. RESULTS: The three SNPs rs10012, rs1056827 and rs1056836 alone did not provide any significant evidence of association with colorectal cancer risk. Haplotypes of rs1056827 and rs10012 or rs1056827 and rs1056836 revealed an association with colorectal cancer which was significantly stronger in the homozygous carriers. One haplotype was under represented in the colorectal cancer patient group compared to the control population suggesting a protective effect. CONCLUSION: Genetic variants within the CYP1B1 that are associated with altered function appear to influence susceptibility to a colorectal cancer in Poland. Three haplotypes were associated with altered cancer risk; one conferred protection and two were associated with an increased risk of disease. These observations should be confirmed in other populations.

Long-term genome-wide blood RNA expression profiles yield novel molecular response candidates for IFN-beta-1b treatment in relapsing remitting MS.

AIMS: In multiple sclerosis patients, treatment with recombinant IFN-beta (rIFN-beta) is partially efficient in reducing clinical exacerbations. However, its molecular mechanism of action is still under scrutiny. MATERIALS & METHODS: We used DNA microarrays (Affymetrix, CA, USA) and peripheral mononuclear blood cells from 25 relapsing remitting multiple sclerosis patients to analyze the longitudinal transcriptional profile within 2 years of rIFN-beta administration. Sets of differentially expressed genes were attained by applying a combination of independent criteria, thereby providing efficient data curation and gene filtering that accounted for technical and biological noise. Gene ontology term-association analysis and scientific literature text mining were used to explore evidence of gene interaction. RESULTS: Post-therapy initiation, we identified 42 (day 2), 175 (month 1), 103 (month 12) and 108 (month 24) differentially expressed genes. Increased expression of established IFN-beta marker genes, as well as differential expression of circulating IFN-beta-responsive candidate genes, were observed. MS4A1 (CD20), a known target of B-cell depletion therapy, was significantly downregulated after one month. CMPK2, FCER1A, and FFAR2 appeared as hitherto unrecognized multiple sclerosis treatment-related differentially expressed genes that were consistently modulated over time. Overall, 84 interactions between 54 genes were attained, of which two major gene networks were identified at an earlier stage of therapy: the first (n = 15 genes) consisted of mostly known IFN-beta-activated genes, whereas the second (n = 12) mainly contained downregulated genes that to date have not been associated with IFN-beta effects in multiple sclerosis array research. CONCLUSION: We achieved both a broadening of the knowledge of IFN-beta mechanism-of-action-related constituents and the identification of time-dependent interactions between IFN-beta regulated genes.

Combined iPLEX and TaqMan assays to screen for 45 common mutations in Lynch syndrome and FAP patients.

Mutations of genes associated with the mismatch repair mechanism and mutations of the APC gene are the most frequent causes of hereditary colorectal cancer. An iPLEX test combined with TaqMan genotyping assays was therefore developed to identify common recurrent mutations of those genes in the Polish population. We analyzed 349 DNA samples from 95 positive controls previously identified by sequencing and 254 unexamined individuals. The iPLEX test included two plexes, which comprised seven mutations of the APC gene and 29 mutations of three of the mismatch repair genes. TaqMan assays were designed for nine mutations not covered by the iPLEX assays: one mutation in the APC gene and eight mutations in the mismatch repair genes. Results were then verified independently by sequencing. Our combination method allowed detection of all recurrent mutations occurring in group of patients, followed by full analysis by DNA sequencing. With the exception of one false positive in the iPLEX test in the positive control group that could be assigned to contamination from neighboring wells rather than a detection error, given sufficient DNA concentration and quality, the designed iPLEX/TaqMan test had an accuracy of 100% for the designed assays. These results suggest that the combined iPLEX/TaqMan test is an outstanding tool for identification of recurrent mutations among hereditary colorectal cancer patients.

Mutation analysis of hypoxia-inducible factors HIF1A and HIF2A in renal cell carcinoma.

BACKGROUND: Inactivation of the Von Hippel-Lindau (VHL) tumour suppressor gene leading to overexpression of hypoxia-inducible transcription factors (HIF)-1alpha and -2alpha is a critical event in the pathogenesis of most clear cell renal cell carcinomas (RCC). HIF-1alpha and HIF-2alpha share significant homology and regulate overlapping repertoires of hypoxia-inducible target genes but may have differing effects on RCC cell growth. Loss of HIF-1alpha expression has been described in RCC cell lines and primary tumours. Whether mutations in the alpha-subunits of HIF-1alpha and HIF-2alpha contribute to renal tumourigenesis was investigated here. MATERIALS AND METHODS: Mutation analysis of the complete coding sequence of HIF-1alpha and HIF-2alpha was carried out in primary RCC (n=40). RESULTS: The analysis revealed a somatic HIF1A missense substitution, p.Val116Glu, in a single RCC. Functional studies demonstrated that p.Val116Glu impaired HIF-1alpha transcriptional activity. Genotyping of HIF1A variants p.Pro582Ser and p.Ala588Thr demonstrated no significant differences between RCC patients and controls. CONCLUSION: The detection of a loss-of-function HIF1A mutation in a primary RCC is consistent with HIF-1 and HIF-2 having different roles in renal tumourigenesis, However, somatic mutations of HIF1A are not frequently implicated in the pathogenesis of RCC.

Clinical characteristics of laryngeal cancer in BRCA-1 mutation carriers.

BACKGROUND: The aim of this study was to analyze the occurrence of clinical features characteristic of breast cancer type 1 susceptibility protein (BRCA-1)-dependent tumors in a series of BRCA-1 mutation carriers with laryngeal cancer. PATIENTS AND METHODS: The clinical features of five laryngeal cancer patients with BRCA-1 mutations registered in our center were analyzed for: sex, age at diagnosis, age at operation, tumor size and localization, histopathological subtype and grading, lymph node and distant metastases, mode of treatment and long term results of the therapy. RESULTS: The five patients were all men, with an average age at diagnosis of 52.4 years. The majority of the patients had clinical features typical of BRCA-1-dependent tumors: four out of the five patients had advanced staging at the time of diagnosis and in three of them the disorder disseminated within one year of follow-up. CONCLUSION: Since laryngeal carcinomas in men with BRCA-1 mutations show clinical features characteristic of BRCA-1 dependent tumors, it is reasonable to consider treatment modifications appropriate for this sub-group of tumors.