Prediction of outcome of non-small cell lung cancer patients treated with chemotherapy and bortezomib by time-course MALDI-TOF-MS serum peptide profiling

Background: Only a minority of patients with advanced non-small cell lung cancer (NSCLC) benefit from chemotherapy. Serum peptide profiling of NSCLC patients was performed to investigate patterns associated with treatment outcome. Using magnetic bead-assisted serum peptide capture coupled to matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS), serum peptide mass profiles of 27 NSCLC patients treated with cisplatin-gemcitabine chemotherapy and bortezomib were obtained. Support vector machine-based algorithms to predict clinical outcome were established based on differential pre-treatment peptide profiles and dynamic changes in peptide abundance during treatment. Results: A 6-peptide ion signature distinguished with 82% accuracy, sensitivity and specificity patients with a relatively short vs. long progression-free survival (PFS) upon treatment. Prediction of long PFS was associated with longer overall survival. Inclusion of 7 peptide ions showing differential changes in abundance during treatment led to a 13-peptide ion signature with 86% accuracy at 100% sensitivity and 73% specificity. A 5-peptide ion signature could separate patients with a partial response vs. non-responders with 89% accuracy at 100% sensitivity and 83% specificity. Differential peptide profiles were also found when comparing the NSCLC serum profiles to those from cancer-free control subjects. Conclusion: This study shows that serum peptidome profiling using MALDI-TOF-MS coupled to pattern diagnostics may aid in prediction of treatment outcome of advanced NSCLC patients treated with chemotherap

Candidate biomarkers for treatment benefit from sunitinib in patients with advanced renal cell carcinoma using mass spectrometry-based (phospho)proteomics

The tyrosine kinase inhibitor sunitinib is an effective first-line treatment for patients with advanced renal cell carcinoma (RCC). Hypothesizing that a functional read-out by mass spectrometry-based (phospho, p-)proteomics will identify predictive biomarkers for treatment outcome of sunitinib, tumor tissues of 26 RCC patients were analyzed. Eight patients had primary resistant (RES) and 18 sensitive (SENS) RCC. A 78 phosphosite signature (p &lt; 0.05, fold-change &gt; 2) was identified; 22 p-sites were upregulated in RES (unique in RES: BCAR3, NOP58, EIF4A2, GDI1) and 56 in SENS (35 unique). EIF4A1/EIF4A2 were differentially expressed in RES at the (p-)proteome and, in an independent cohort, transcriptome level. Inferred kinase activity of MAPK3 (p = 0.026) and EGFR (p = 0.045) as determined by INKA was higher in SENS. Posttranslational modifications signature enrichment analysis showed that different p-site-centric signatures were enriched (p &lt; 0.05), of which FGF1 and prolactin pathways in RES and, in SENS, vanadate and thrombin treatment pathways, were most significant. In conclusion, the RCC (phospho)proteome revealed differential p-sites and kinase activities associated with sunitinib resistance and sensitivity. Independent validation is warranted to develop an assay for upfront identification of patients who are intrinsically resistant to sunitinib.</p

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.</p

Novel diagnostic cerebrospinal fluid biomarkers for pathologic subtypes of frontotemporal dementia identified by proteomics

Introduction: Reliable cerebrospinal fluid (CSF) biomarkers enabling identification of frontotemporal dementia (FTD) and its pathologic subtypes are lacking. Methods: Unbiased high-resolution mass spectrometry-based proteomics was applied on CSF of FTD patients with TAR DNA-binding protein 43 (TDP-43, FTD-TDP, n = 12) or tau pathology (FTD-tau, n = 8), and individuals with subjective memory complaints (SMC, n = 10). Validation was performed by applying enzyme-linked immunosorbent assay (ELISA) or enzymatic assays, when available, in a larger cohort (FTLD-TDP, n = 21, FTLD-tau, n = 10, SMC, n = 23) and in Alzheimer's disease (n = 20), dementia with Lewy bodies (DLB, n = 20), and vascular dementia (VaD, n = 18). Results: Of 1914 identified CSF proteins, 56 proteins were differentially regulated (fold change >1.2, P <.05) between the different patient groups: either between the two pathologic subtypes (10 proteins), or between at least one of these FTD subtypes and SMC (47 proteins). We confirmed the differential expression of YKL-40 by ELISA in a partly independent cohort. Furthermore, enzyme activity of catalase was decreased in FTD subtypes compared with SMC. Further validation in a larger cohort showed that the level of YKL-40 was twofold increased in both FTD pathologic subtypes compared with SMC and that the levels in FTLD-tau were higher compared to Alzheimer's dementia (AD), DLB, and VaD patients. Clinical validation furthermore showed that the catalase enzyme activity was decreased in the FTD subtypes compared to SMC, AD and DLB. Discussion: We identified promising CSF biomarkers for both FTD differential diagnosis and pathologic subtyping. YKL-40 and catalase enzyme activity should be validated further in similar pathology defined patient cohorts for their use for FTD diagnosis or treatment development

Integration of stool microbiota, proteome and amino acid profiles to discriminate patients with adenomas and colorectal cancer

BACKGROUND: Screening for colorectal cancer (CRC) reduces its mortality but has limited sensitivity and specificity. Aims We aimed to explore potential biomarker panels for CRC and adenoma detection and to gain insight into the interaction between gut microbiota and human metabolism in the presence of these lesions. METHODS: This multicenter case-control cohort was performed between February 2016 and November 2019. Consecutive patients ≥18 years with a scheduled colonoscopy were asked to participate and divided into three age, gender, body-mass index and smoking status-matched subgroups: CRC (n = 12), adenomas (n = 21) and controls (n = 20). Participants collected fecal samples prior to bowel preparation on which proteome (LC-MS/MS), microbiota (16S rRNA profiling) and amino acid (HPLC) composition were assessed. Best predictive markers were combined to create diagnostic biomarker panels. Pearson correlation-based analysis on selected markers was performed to create networks of all platforms. RESULTS: Combining omics platforms provided new panels which outperformed hemoglobin in this cohort, currently used for screening (AUC 0.98, 0.95 and 0.87 for CRC vs controls, adenoma vs controls and CRC vs adenoma, respectively). Integration of data sets revealed markers associated with increased blood excretion, stress- and inflammatory responses and pointed toward downregulation of epithelial integrity. CONCLUSIONS: Integrating fecal microbiota, proteome and amino acids platforms provides for new biomarker panels that may improve noninvasive screening for adenomas and CRC, and may subsequently lead to lower incidence and mortality of colon cancer

Postoperative serum proteomic profiles may predict recurrence-free survival in high-risk primary breast cancer

Item does not contain fulltextPURPOSE: Better breast cancer prognostication may improve selection of patients for adjuvant therapy. We conducted a retrospective longitudinal study in which we investigated sera of high-risk primary breast cancer patients, to search for proteins predictive of recurrence-free survival. METHODS: Sera of 82 breast cancer patients obtained after surgery, but prior to the administration of adjuvant therapy, were fractionated using anion-exchange chromatography, to facilitate the detection of the low-abundant serum peptides. Selected fractions were subsequently analysed by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS), and the resulting protein profiles were searched for prognostic markers by appropriate bioinformatics tools. RESULTS: Four peak clusters (i.e. m/z 3073, m/z 3274, m/z 4405 and m/z 7973) were found to bear significant prognostic value (P </= 0.01). The m/z 3274 candidate marker was structurally identified as inter-alpha-trypsin inhibitor heavy chain 4 fragment(658-688) in serum. Except for the m/z 7973 peak cluster, these peaks remained independently associated with recurrence-free survival upon multivariate Cox regression analysis, including clinical parameters of known prognostic value in this study population. CONCLUSION: Investigation of the postoperative serum proteome by, e.g., anion-exchange fractionation followed by SELDI-TOF MS analysis is promising for the detection of novel prognostic factors. However, regarding the rather limited study population, validation of these results by analysis of independent study populations is warranted to assess the true clinical applicability of discovered prognostic markers. In addition, structural identification of the other markers will aid in elucidation of their role in breast cancer prognosis, as well as enable development of absolute quantitative assays

Proteins in stool as biomarkers for non-invasive detection of colorectal adenomas with high risk of progression

Screening to detect colorectal cancer (CRC) in an early or premalignant state is an effective method to reduce CRC mortality rates. Current stool-based screening tests, e.g. fecal immunochemical test (FIT), have a suboptimal sensitivity for colorectal adenomas and difficulty distinguishing adenomas at high risk of progressing to cancer from those at lower risk. We aimed to identify stool protein biomarker panels that can be used for the early detection of high-risk adenomas and CRC. Proteomics data (LC–MS/MS) were collected on stool samples from adenoma (n = 71) and CRC patients (n = 81) as well as controls (n = 129). Colorectal adenoma tissue samples were characterized by low-coverage whole-genome sequencing to determine their risk of progression based on specific DNA copy number changes. Proteomics data were used for logistic regression modeling to establish protein biomarker panels. In total, 15 of the adenomas (15.8%) were defined as high risk of progressing to cancer. A protein panel, consisting of haptoglobin (Hp), LAMP1, SYNE2, and ANXA6, was identified for the detection of high-risk adenomas (sensitivity of 53% at specificity of 95%). Two panels, one consisting of Hp and LRG1 and one of Hp, LRG1, RBP4, and FN1, were identified for high-risk adenomas and CRCs detection (sensitivity of 66% and 62%, respectively, at specificity of 95%). Validation of Hp as a biomarker for high-risk adenomas and CRCs was performed using an antibody-based assay in FIT samples from a subset of individuals from the discovery series (n = 158) and an independent validation series (n = 795). Hp protein was significantly more abundant in high-risk adenoma FIT samples compared to controls in the discovery (p = 0.036) and the validation series (p = 9e-5). We conclude that Hp, LAMP1, SYNE2, LRG1, RBP4, FN1, and ANXA6 may be of value as stool biomarkers for early detection of high-risk adenomas and CRCs

Vemurafenib plus cobimetinib in unresectable stage IIIc or stage IV melanoma:Response monitoring and resistance prediction with positron emission tomography and tumor characteristics (REPOSIT): study protocol of a phase II, open-label, multicenter study

BACKGROUND: In patients with BRAFV600 mutated unresectable stage IIIc or metastatic melanoma, molecular targeted therapy with combined BRAF/MEK-inhibitor vemurafenib plus cobimetinib has shown a significantly improved progression-free survival and overall survival compared to treatment with vemurafenib alone. Nevertheless, the majority of BRAFV600 mutation-positive melanoma patients will eventually develop resistance to treatment. Molecular imaging with F-18-Fluorodeoxyglucose (F-18-FDG) PET has been used to monitor response to vemurafenib in some BRAFV600 mutated metastatic melanoma patients, showing a rapid decline of F-18-FDG uptake within 2 weeks following treatment. Furthermore, preliminary results suggest that metabolic alterations might predict the development of resistance to treatment. F-18-Fluoro-3'-deoxy-3' L-fluorothymidine (F-18-FLT), a PET-tracer visualizing proliferation, might be more suitable to predict response or resistance to therapy than F-18-FDG. METHODS: This phase II, open-label, multicenter study evaluates whether metabolic response to treatment with vemurafenib plus cobimetinib in the first 7 weeks as assessed by F-18-FDG/F-18-FLT PET can predict progression-free survival and whether early changes in F-18-FDG/F-18-FLT can be used for early detection of treatment response compared to standard response assessment with RECISTv1.1 ceCT at 7 weeks. Ninety patients with BRAFV600E/K mutated unresectable stage IIIc/IV melanoma will be included. Prior to and during treatment all patients will undergo F-18-FDG PET/CT and in 25 patients additional F-18-FLT PET/CT is performed. Histopathological tumor characterization is assessed in a subset of 40 patients to unravel mechanisms of resistance. Furthermore, in all patients, blood samples are taken for pharmacokinetic analysis of vemurafenib/cobimetinib. Outcomes are correlated with PET/CT-imaging and therapy response. DISCUSSION: The results of this study will help in linking PET measured metabolic alterations induced by targeted therapy of BRAFV600 mutated melanoma to molecular changes within the tumor. We will be able to correlate both F-18-FDG and F-18-FLT PET to outcome and decide on the best modality to predict long-term remissions to combined BRAF/MEK-inhibitors. Results coming from this study may help in identifying responders from non-responders early after the initiation of therapy and reveal early development of resistance to vemurafenib/cobimetinib. Furthermore, we believe that the results can be fundamental for further optimizing individual patient treatment

Consensus Guidelines for CSF and Blood Biobanking for CNS Biomarker Studies

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in cerebrospinal fluid (CSF) are being used in clinical practice. Anti-aquaporin-4 antibodies in serum are currently useful for the diagnosis of neuromyelitis optica (NMO), but we could expect novel CSF biomarkers that help define prognosis and response to treatment for this disease. One of the most critical factors in biomarker research is the inadequate powering of studies performed by single centers. Collaboration between investigators is needed to establish large biobanks of well-defined samples. A key issue in collaboration is to establish standardized protocols for biobanking to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by pre-analytical factors. Here, consensus guidelines for CSF collection and biobanking are presented, based on the guidelines that have been published by the BioMS-eu network for CSF biomarker research. We focussed on CSF collection procedures, pre-analytical factors and high quality clinical and paraclinical information. Importantly, the biobanking protocols are applicable for CSF biobanks for research targeting any neurological disease