Pipelines and Systems for Threshold-Avoiding Quantification of LC-MS/MS Data.

The accurate processing of complex liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) data from biological samples is a major challenge for metabolomics, proteomics, and related approaches. Here, we present the pipelines and systems for threshold-avoiding quantification (PASTAQ) LC-MS/MS preprocessing toolset, which allows highly accurate quantification of data-dependent acquisition LC-MS/MS datasets. PASTAQ performs compound quantification using single-stage (MS1) data and implements novel algorithms for high-performance and accurate quantification, retention time alignment, feature detection, and linking annotations from multiple identification engines. PASTAQ offers straightforward parameterization and automatic generation of quality control plots for data and preprocessing assessment. This design results in smaller variance when analyzing replicates of proteomes mixed with known ratios and allows the detection of peptides over a larger dynamic concentration range compared to widely used proteomics preprocessing tools. The performance of the pipeline is also demonstrated in a biological human serum dataset for the identification of gender-related proteins.

MSIWarp: A General Approach to Mass Alignment in Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is a technique that provides comprehensive molecular information with high spatial resolution from tissue. Today, there is a strong push toward sharing data sets through public repositories in many research fields where MSI is commonly applied; yet, there is no standardized protocol for analyzing these data sets in a reproducible manner. Shifts in the mass-to-charge ratio (m/z) of molecular peaks present a major obstacle that can make it impossible to distinguish one compound from another. Here, we present a label-free m/z alignment approach that is compatible with multiple instrument types and makes no assumptions on the sample's molecular composition. Our approach, MSIWarp (https://github.com/horvatovichlab/MSIWarp), finds an m/z recalibration function by maximizing a similarity score that considers both the intensity and m/z position of peaks matched between two spectra. MSIWarp requires only centroid spectra to find the recalibration function and is thereby readily applicable to almost any MSI data set. To deal with particularly misaligned or peak-sparse spectra, we provide an option to detect and exclude spurious peak matches with a tailored random sample consensus (RANSAC) procedure. We evaluate our approach with four publicly available data sets from both time-of-flight (TOF) and Orbitrap instruments and demonstrate up to 88% improvement in m/z alignment.

Novel functional proteins coded by the human genome discovered in metastases of melanoma patients.

In the advanced stages, malignant melanoma (MM) has a very poor prognosis. Due to tremendous efforts in cancer research over the last 10 years, and the introduction of novel therapies such as targeted therapies and immunomodulators, the rather dark horizon of the median survival has dramatically changed from under 1 year to several years. With the advent of proteomics, deep-mining studies can reach low-abundant expression levels. The complexity of the proteome, however, still surpasses the dynamic range capabilities of current analytical techniques. Consequently, many predicted protein products with potential biological functions have not yet been verified in experimental proteomic data. This category of 'missing proteins' (MP) is comprised of all proteins that have been predicted but are currently unverified. As part of the initiative launched in 2016 in the USA, the European Cancer Moonshot Center has performed numerous deep proteomics analyses on samples from MM patients. In this study, nine MPs were clearly identified by mass spectrometry in MM metastases. Some MPs significantly correlated with proteins that possess identical PFAM structural domains; and other MPs were significantly associated with cancer-related proteins. This is the first study to our knowledge, where unknown and novel proteins have been annotated in metastatic melanoma tumour tissue.

Clusterwise Peak Detection and Filtering Based on Spatial Distribution To Efficiently Mine Mass Spectrometry Imaging Data.

Mass spectrometry imaging (MSI) has the potential to reveal the localization of thousands of biomolecules such as metabolites and lipids in tissue sections. The increase in both mass and spatial resolution of today's instruments brings on considerable challenges in terms of data processing; accurately extracting meaningful signals from the large data sets generated by MSI without losing information that could be clinically relevant is one of the most fundamental tasks of analysis software. Ion images of the biomolecules are generated by visualizing their intensities in 2-D space using mass spectra collected across the tissue section. The intensities are often calculated by summing each compound's signal between predefined sets of borders (bins) in the m/z dimension. This approach, however, can result in mixed signals from different compounds in the same bin or splitting the signal from one compound between two adjacent bins, leading to low quality ion images. To remedy this problem, we propose a novel data processing approach. Our approach consists of a sensitive peak detection method able to discover both faint and localized signals by utilizing clusterwise kernel density estimates (KDEs) of peak distributions. We show that our method can recall more ground-truth molecules, molecule fragments, and isotopes than existing methods based on binning. Furthermore, it automatically detects previously reported molecular ions of lipids, including those close in m/z, in an experimental data set.

Clinical protein science in translational medicine targeting malignant melanoma.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

Test-retest variability of left ventricular 4D flow cardiovascular magnetic resonance measurements in healthy subjects.

BACKGROUND: Quantification and visualisation of left ventricular (LV) blood flow is afforded by three-dimensional, time resolved phase contrast cardiovascular magnetic resonance (CMR 4D flow). However, few data exist upon the repeatability and variability of these parameters in a healthy population. We aimed to assess the repeatability and variability over time of LV 4D CMR flow measurements. METHODS: Forty five controls underwent CMR 4D flow data acquisition. Of these, 10 underwent a second scan within the same visit (scan-rescan), 25 returned for a second visit (interval scan; median interval 52 days, IQR 28-57 days). The LV-end diastolic volume (EDV) was divided into four flow components: 1) Direct flow: inflow that passes directly to ejection; 2) Retained inflow: inflow that enters and resides within the LV; 3) Delayed ejection flow: starts within the LV and is ejected and 4) Residual volume: blood that resides within the LV for > 2 cardiac cycles. Each flow components' volume was related to the EDV (volume-ratio). The kinetic energy at end-diastole (ED) was measured and divided by the components' volume. RESULTS: The dominant flow component in all 45 controls was the direct flow (volume ratio 38 ± 4%) followed by the residual volume (30 ± 4%), then delayed ejection flow (16 ± 3%) and retained inflow (16 ± 4%). The kinetic energy at ED for each component was direct flow (7.8 ± 3.0 microJ/ml), retained inflow (4.1 ± 2.0 microJ/ml), delayed ejection flow (6.3 ± 2.3 microJ/ml) and the residual volume (1.2 ± 0.5 microJ/ml). The coefficients of variation for the scan-rescan ranged from 2.5%-9.2% for the flow components' volume ratio and between 13.5%-17.7% for the kinetic energy. The interval scan results showed higher coefficients of variation with values from 6.2-16.1% for the flow components' volume ratio and 16.9-29.0% for the kinetic energy of the flow components. CONCLUSION: LV flow components' volume and their associated kinetic energy values are repeatable and stable within a population over time. However, the variability of these measurements in individuals over time is greater than can be attributed to sources of error in the data acquisition and analysis, suggesting that additional physiological factors may influence LV flow measurements.

Creating hemodynamic atlases of cardiac 4D flow MRI.

PURPOSE: Hemodynamic atlases can add to the pathophysiological understanding of cardiac diseases. This study proposes a method to create hemodynamic atlases using 4D Flow magnetic resonance imaging (MRI). The method is demonstrated for kinetic energy (KE) and helicity density (Hd ). MATERIALS AND METHODS: Thirteen healthy subjects underwent 4D Flow MRI at 3T. Phase-contrast magnetic resonance cardioangiographies (PC-MRCAs) and an average heart were created and segmented. The PC-MRCAs, KE, and Hd were nonrigidly registered to the average heart to create atlases. The method was compared with 1) rigid, 2) affine registration of the PC-MRCAs, and 3) affine registration of segmentations. The peak and mean KE and Hd before and after registration were calculated to evaluate interpolation error due to nonrigid registration. RESULTS: The segmentations deformed using nonrigid registration overlapped (median: 92.3%) more than rigid (23.1%, P < 0.001), and affine registration of PC-MRCAs (38.5%, P < 0.001) and affine registration of segmentations (61.5%, P < 0.001). The peak KE was 4.9 mJ using the proposed method and affine registration of segmentations (P = 0.91), 3.5 mJ using rigid registration (P < 0.001), and 4.2 mJ using affine registration of the PC-MRCAs (P < 0.001). The mean KE was 1.1 mJ using the proposed method, 0.8 mJ using rigid registration (P < 0.001), 0.9 mJ using affine registration of the PC-MRCAs (P < 0.001), and 1.0 mJ using affine registration of segmentations (P = 0.028). The interpolation error was 5.2 ± 2.6% at mid-systole, 2.8 ± 3.8% at early diastole for peak KE; 9.6 ± 9.3% at mid-systole, 4.0 ± 4.6% at early diastole, and 4.9 ± 4.6% at late diastole for peak Hd . The mean KE and Hd were not affected by interpolation. CONCLUSION: Hemodynamic atlases can be obtained with minimal user interaction using nonrigid registration of 4D Flow MRI. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2017;46:1389-1399.

4D flow MRI can detect subtle right ventricular dysfunction in primary left ventricular disease.

PURPOSE: To investigate whether 4D flow magnetic resonance imaging (MRI) can detect subtle right ventricular (RV) dysfunction in primary left ventricular (LV) disease. MATERIALS AND METHODS: 4D flow and morphological 3T MRI data were acquired in 22 patients with mild ischemic heart disease who were stratified into two groups based on LV end-diastolic volume index (EDVI): lower-LVEDVI and higher-LVEDVI, as well as in 11 healthy controls. The RV volume was segmented at end-diastole (ED) and end-systole (ES). Pathlines were emitted from the ED volume and traced forwards and backwards in time to ES. The blood volume was separated into flow components. The Direct Flow (DF) component was defined as RV inflow passing directly to outflow. The kinetic energy (KE) of the DF component was calculated. Echocardiographic conventional RV indices were also assessed. RESULTS: The higher-LVEDVI group had larger LVEDVI and lower LV ejection fraction (98 ± 32 ml/m(2) ; 48 ± 13%) compared to the healthy (67 ± 12, P = 0.002; 64 ± 7, P < 0.001) and lower-LVEDI groups (62 ± 10; 68 ± 7, both P < 0.001). The RV 4D flow-specific measures "DF/EDV volume-ratio" and "DF/EDV KE-ratio at ED" were lower in the higher-LVEDVI group (38 ± 5%; 52 ± 6%) compared to the healthy (44 ± 6; 65 ± 7, P = 0.018 and P < 0.001) and lower-LVEDVI groups (44 ± 6; 64 ± 7, P = 0.011 and P < 0.001). There was no difference in any of the conventional MRI and echocardiographic RV indices between the three groups. CONCLUSION: We found that in primary LV disease mild impairment of RV function can be detected by 4D flow-specific measures, but not by the conventional MRI and echocardiographic indices.

Merging clinical chemistry biomarker data with a COPD database - building a clinical infrastructure for proteomic studies.

BACKGROUND: Data from biological samples and medical evaluations plays an essential part in clinical decision making. This data is equally important in clinical studies and it is critical to have an infrastructure that ensures that its quality is preserved throughout its entire lifetime. We are running a 5-year longitudinal clinical study, KOL-Örestad, with the objective to identify new COPD (Chronic Obstructive Pulmonary Disease) biomarkers in blood. In the study, clinical data and blood samples are collected from both private and public health-care institutions and stored at our research center in databases and biobanks, respectively. The blood is analyzed by Mass Spectrometry and the results from this analysis then linked to the clinical data. METHOD: We built an infrastructure that allows us to efficiently collect and analyze the data. We chose to use REDCap as the EDC (Electronic Data Capture) tool for the study due to its short setup-time, ease of use, and flexibility. REDCap allows users to easily design data collection modules based on existing templates. In addition, it provides two functions that allow users to import batches of data; through a web API (Application Programming Interface) as well as by uploading CSV-files (Comma Separated Values). RESULTS: We created a software, DART (Data Rapid Translation), that translates our biomarker data into a format that fits REDCap's CSV-templates. In addition, DART is configurable to work with many other data formats as well. We use DART to import our clinical chemistry data to the REDCap database. CONCLUSION: We have shown that a powerful and internationally adopted EDC tool such as REDCap can be extended so that it can be used efficiently in proteomic studies. In our study, we accomplish this by using DART to translate our clinical chemistry data to a format that fits the templates of REDCap.

Spatial heterogeneity of four-dimensional relative pressure fields in the human left ventricle.

PURPOSE: To assess the spatial heterogeneity of the four-dimensional (4D) relative pressure fields in the healthy human left ventricle (LV) and provide reference data for normal LV relative pressure. METHODS: Twelve healthy subjects underwent a cardiac MRI examination where 4D flow and morphological data were acquired. The latter data were segmented and used to define the borders of the LV for computation of relative pressure fields using the pressure Poisson equation. The LV lumen was divided into 17 pie-shaped segments. RESULTS: In the normal left ventricle, the relative pressure in the apical segments was significantly higher relative to the basal segments (P < 0.0005) along both the anteroseptal and inferolateral sides after the peaks of early (E-wave) and late (A-wave) diastolic filling. The basal anteroseptal segment showed significantly lower median pressure than the opposite basal inferolateral segment during both E-wave (P < 0.0005) and A-wave (P = 0.0024). CONCLUSION: Relative pressure in the left ventricle is heterogeneous. During diastole, the main pressure differences in the LV occur along the basal-apical axis. However, pressure differences were also found in the short axis direction and may reflect important aspects of atrioventricular coupling. Additionally, this study provides reference data on LV pressure dynamics for a group of healthy subjects.

Turbulent kinetic energy in normal and myopathic left ventricles.

PURPOSE: To assess turbulent kinetic energy (TKE) within the left ventricle (LV) of healthy subjects using novel 4D flow magnetic resonance imaging (MRI) methods and to compare TKE values to those from a limited group of patients with a spectrum of dilated cardiomyopathy (DCM). MATERIALS AND METHODS: 4D flow and morphological MRI data were acquired in 11 healthy subjects and 9 patients with different degrees of diastolic dysfunction. TKELV was calculated within the LV at each diastolic timeframe. At peak early (E) and late (A) diastolic filling, the TKELV was compared to transmitral peak velocity, LV diameter, and mitral annular diameter. RESULTS: In the majority of subjects, TKELV peaks were observed at E and A. Peak TKELV at E was not different between the groups (P = 0.33), and correlated with mitral annular dimensions (r(2) = 0.32, P = 0.01). Peak TKELV at A was higher in DCM patients compared to healthy subjects (3.0 ± 1.8 vs. 1.5 ± 0.8 mJ, P = 0.02), and correlated with LV diameter and transmitral velocity (r(2) = 0.36, P = 0.01 and r(2) = 0.47, P < 0.01, respectively). CONCLUSION: In LVs of healthy subjects, TKE values are low. Values are highest during early diastole, and diminish with increasing LV size. In a heterogeneous group of DCM patients, late diastolic TKE values are higher than in healthy subjects.

Atlas-based analysis of 4D flow CMR: automated vessel segmentation and flow quantification.

BACKGROUND: Flow volume quantification in the great thoracic vessels is used in the assessment of several cardiovascular diseases. Clinically, it is often based on semi-automatic segmentation of a vessel throughout the cardiac cycle in 2D cine phase-contrast Cardiovascular Magnetic Resonance (CMR) images. Three-dimensional (3D), time-resolved phase-contrast CMR with three-directional velocity encoding (4D flow CMR) permits assessment of net flow volumes and flow patterns retrospectively at any location in a time-resolved 3D volume. However, analysis of these datasets can be demanding. The aim of this study is to develop and evaluate a fully automatic method for segmentation and analysis of 4D flow CMR data of the great thoracic vessels. METHODS: The proposed method utilizes atlas-based segmentation to segment the great thoracic vessels in systole, and registration between different time frames of the cardiac cycle in order to segment these vessels over time. Additionally, net flow volumes are calculated automatically at locations of interest. The method was applied on 4D flow CMR datasets obtained from 11 healthy volunteers and 10 patients with heart failure. Evaluation of the method was performed visually, and by comparison of net flow volumes in the ascending aorta obtained automatically (using the proposed method), and semi-automatically. Further evaluation was done by comparison of net flow volumes obtained automatically at different locations in the aorta, pulmonary artery, and caval veins. RESULTS: Visual evaluation of the generated segmentations resulted in good outcomes for all the major vessels in all but one dataset. The comparison between automatically and semi-automatically obtained net flow volumes in the ascending aorta resulted in very high correlation (r (2)=0.926). Moreover, comparison of the net flow volumes obtained automatically in other vessel locations also produced high correlations where expected: pulmonary trunk vs. proximal ascending aorta (r (2)=0.955), pulmonary trunk vs. pulmonary branches (r (2)=0.808), and pulmonary trunk vs. caval veins (r (2)=0.906). CONCLUSIONS: The proposed method allows for automatic analysis of 4D flow CMR data, including vessel segmentation, assessment of flow volumes at locations of interest, and 4D flow visualization. This constitutes an important step towards facilitating the clinical utility of 4D flow CMR.

Impact of dobutamine stress on diastolic energetic efficiency of healthy left ventricle: an in vivo kinetic energy analysis.

The total kinetic energy (KE) of blood can be decomposed into mean KE (MKE) and turbulent KE (TKE), which are associated with the phase-averaged fluid velocity field and the instantaneous velocity fluctuations, respectively. The aim of this study was to explore the effects of pharmacologically induced stress on MKE and TKE in the left ventricle (LV) in a cohort of healthy volunteers. 4D Flow MRI data were acquired in eleven subjects at rest and after dobutamine infusion, at a heart rate that was ∼60% higher than the one in rest conditions. MKE and TKE were computed as volume integrals over the whole LV and as data mapped to functional LV flow components, i.e., direct flow, retained inflow, delayed ejection flow and residual volume. Diastolic MKE and TKE increased under stress, in particular at peak early filling and peak atrial contraction. Augmented LV inotropy and cardiac frequency also caused an increase in direct flow and retained inflow MKE and TKE. However, the TKE/KE ratio remained comparable between rest and stress conditions, suggesting that LV intracavitary fluid dynamics can adapt to stress conditions without altering the TKE to KE balance of the normal left ventricle at rest.

Quantification of presystolic blood flow organization and energetics in the human left ventricle.

Intracardiac blood flow patterns are potentially important to cardiac pumping efficiency. However, these complex flow patterns remain incompletely characterized both in health and disease. We hypothesized that normal left ventricular (LV) blood flow patterns would preferentially optimize a portion of the end-diastolic volume (LVEDV) for effective and rapid systolic ejection by virtue of location near and motion towards the LV outflow tract (LVOT). Three-dimensional cine velocity and morphological data were acquired in 12 healthy persons and 1 patient with dilated cardiomyopathy using MRI. A previously validated method was used for analysis in which the LVEDV was separated into four functional flow components based on the blood's locations at the beginning and end of the cardiac cycle. Each component's volume, kinetic energy (KE), site, direction, and linear momentum relative to the LVOT were calculated. Of the four components, the LV inflow that passes directly to outflow in a single cardiac cycle (Direct Flow) had the largest volume. At the time of isovolumic contraction, Direct Flow had the greatest amount of KE and the most favorable combination of distance, angle, and linear momentum relative to the LVOT. Atrial contraction boosted the late diastolic KE of the ejected components. We conclude that normal diastolic LV flow creates favorable conditions for ensuing ejection, defined by proximity and energetics, for the Direct Flow, and that atrial contraction augments the end-diastolic KE of the ejection volume. The correlation of Direct Flow characteristics with ejection efficiency might be a relevant investigative target in cardiac failure.

4-D blood flow in the human right ventricle.

Right ventricular (RV) function is a powerful prognostic indicator in many forms of heart disease, but its assessment remains challenging and inexact. RV dysfunction may alter the normal patterns of RV blood flow, but those patterns have been incompletely characterized. We hypothesized that, based on anatomic differences, the proportions and energetics of RV flow components would differ from those identified in the left ventricle (LV) and that the portion of the RV inflow passing directly to outflow (Direct Flow) would be prepared for effective systolic ejection as a result of preserved kinetic energy (KE) compared with other RV flow components. Three-dimensional, time-resolved phase-contrast velocity, and balanced steady-state free-precession morphological data were acquired in 10 healthy subjects using MRI. A previously validated method was used to separate the RV and LV end-diastolic volumes into four flow components and measure their volume and KE over the cardiac cycle. The RV Direct Flow: 1) followed a smoothly curving route that did not extend into the apical region of the ventricle; 2) had a larger volume and possessed a larger presystolic KE (0.4 ± 0.3 mJ) than the other flow components (P < 0.001 and P < 0.01, respectively); and 3) represented a larger part of the end-diastolic blood volume compared with the LV Direct Flow (P < 0.01). These findings suggest that diastolic flow patterns distinct to the normal RV create favorable conditions for ensuing systolic ejection of the Direct Flow component. These flow-specific aspects of RV diastolic-systolic coupling provide novel perspectives on RV physiology and may add to the understanding of RV pathophysiology.

The Human Melanoma Proteome Atlas-Complementing the melanoma transcriptome.

The MM500 meta-study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass-spectrometry-based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well-annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein-coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease.

The human melanoma proteome atlas-Defining the molecular pathology.

The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in-depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients.

Semi-automatic quantification of 4D left ventricular blood flow.

BACKGROUND: The beating heart is the generator of blood flow through the cardiovascular system. Within the heart's own chambers, normal complex blood flow patterns can be disturbed by diseases. Methods for the quantification of intra-cardiac blood flow, with its 4D (3D+time) nature, are lacking. We sought to develop and validate a novel semi-automatic analysis approach that integrates flow and morphological data. METHOD: In six healthy subjects and three patients with dilated cardiomyopathy, three-directional, three-dimensional cine phase-contrast cardiovascular magnetic resonance (CMR) velocity data and balanced steady-state free-precession long- and short-axis images were acquired. The LV endocardium was segmented from the short-axis images at the times of isovolumetric contraction (IVC) and isovolumetric relaxation (IVR). At the time of IVC, pathlines were emitted from the IVC LV blood volume and traced forwards and backwards in time until IVR, thus including the entire cardiac cycle. The IVR volume was used to determine if and where the pathlines left the LV. This information was used to automatically separate the pathlines into four different components of flow: Direct Flow, Retained Inflow, Delayed Ejection Flow and Residual Volume. Blood volumes were calculated for every component by multiplying the number of pathlines with the blood volume represented by each pathline. The accuracy and inter- and intra-observer reproducibility of the approach were evaluated by analyzing volumes of LV inflow and outflow, the four flow components, and the end-diastolic volume. RESULTS: The volume and distribution of the LV flow components were determined in all subjects. The calculated LV outflow volumes [ml] (67 +/- 13) appeared to fall in between those obtained by through-plane phase-contrast CMR (77 +/- 16) and Doppler ultrasound (58 +/- 10), respectively. Calculated volumes of LV inflow (68 +/- 11) and outflow (67 +/- 13) were well matched (NS). Low inter- and intra-observer variability for the assessment of the volumes of the flow components was obtained. CONCLUSIONS: This semi-automatic analysis approach for the quantification of 4D blood flow resulted in accurate LV inflow and outflow volumes and a high reproducibility for the assessment of LV flow components.

Improved survival prognostication of node-positive malignant melanoma patients utilizing shotgun proteomics guided by histopathological characterization and genomic data.

Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency. Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles. This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients. Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.

The Hidden Story of Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma-Association with Clinical Outcome and Tumor Phenotypes.

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.