Search

Monoclonal antibodies (mAbs) acting as immune checkpoint inhibitors (ICIs) are among the most frequently used immunotherapies in oncology. However, precision medicine approaches to adapt the treatment to the patient are still poorly exploited. Given the risk of severe adverse reactions, predicting patient eligibility for ICI therapy represents a great asset for precision medicine. Today, the extended panel of mass spectrometric approaches, accompanied by newly developed sample preparation methods is a strategy of choice for responder and non-responder stratification on a molecular basis, and early detection of resistance. In this perspective article, we review the biodisposition of mAbs, the interest in molecular stratification of patients treated with these mAbs, and the possible analytical strategies to achieve this goal, with a major emphasis on mass spectrometric approaches.

Clinical pharmacology is an important discipline for drug development aiming to define pharmacokinetics (PK), pharmacodynamics (PD) and optimum exposure to drugs, i.e. the concentration-response relationship and its modulators. For this purpose, information on drug concentrations at the anatomical, cellular and molecular sites of action is particularly valuable. In pharmacological assays, the limited accessibility of target cells in readily available samples (i.e. blood) often hampers mass spectrometry-based monitoring of the absolute quantity of a compound and the determination of its molecular action at the cellular level. Recently, new sample collection methods have been developed for the specific capture of rare circulating cells, especially for the diagnosis of circulating tumour cells. In parallel, new advances and developments in mass spectrometric instrumentation now allow analyses to be scaled down to the cellular level. Together, these developments may permit the monitoring of minute drug quantities and show their effect at the cellular level. In turn, such PK/PD associations on a cellular level would not only enrich our pharmacological knowledge of a given compound but also expand the basis for PK/PD simulations. In this review, we describe novel concepts supporting clinical pharmacology at the anatomical, cellular and molecular sites of action, and highlight the new challenges in mass spectrometry-based monitoring. Moreover, we present methods to tackle these challenges and define future needs.

Ion mobility spectrometry (IMS) represents a considerable asset for analytics of complex samples as it allows for rapid mass spectrometric separation of compounds. IMS is even more useful for the separation of isobaric compounds when classical separation methods such as liquid chromatography or electrophoresis cannot be used, e.g., during matrix-assisted laser desorption/ionization (MALDI) analyses of biological surfaces. In the present study, we proved the usefulness of IMS for pharmacological applications of MALDI analyses on tissue sections. To illustrate our proof-of-concept, we used the anthelmintic drug mebendazole (MBZ) as a model. Using this exemplary drug, we demonstrated the possibility of using ion mobility to discriminate a drug in tissues from the biological background that masked its signal at low concentrations. In this proof-of-concept, the IMS mode together with the use of a profiling approach for sample preparation enabled quantification of the model drug MBZ from tissue sections in the concentration range 5 to 5,000 ng/g and with a limit of detection of 1 ng/g of tissue, within 2 h. This study highlights the importance of IMS as a separation method for on-surface quantification of drugs in tissue sections.

Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog “in cascade”, between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is now the main analytical method for the identification and quantification of peptides and proteins in biological samples. In modern research, identification of biomarkers and their quantitative comparison between samples are becoming increasingly important for discovery, validation, and monitoring. Such data can be obtained following specific signals after fragmentation of peptides using multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) methods, with high specificity, accuracy, and reproducibility. In addition, these methods allow measurement of the amount of post-translationally modified forms and isoforms of proteins. This review article describes the basic principles of MRM assays, guidelines for sample preparation, recent advanced MRM-based strategies, applications and illustrative perspectives of MRM/PRM methods in clinical research and molecular biology.

BACKGROUND: Targeted genetic profiling of tissue samples is paramount to detect druggable genetic aberrations in patients with non-squamous non-small cell lung cancer (NSCLC). Accurate upfront estimation of tumor cell content (TCC) is a crucial pre-analytical step for reliable testing and to avoid false-negative results. As of now, TCC is usually estimated on hematoxylin-eosin (H&E) stained tissue sections by a pathologist, a methodology that may be prone to substantial intra- and interobserver variability. Here we the investigate suitability of digital pathology for TCC estimation in a clinical setting by evaluating the concordance between semi-automatic and conventional TCC quantification. METHODS: TCC was analyzed in 120 H&E and thyroid transcription factor 1 (TTF-1) stained high-resolution images by 19 participants with different levels of pathological expertise as well as by applying two semi-automatic digital pathology image analysis tools (HALO and QuPath). RESULTS: Agreement of TCC estimations [intra-class correlation coefficients (ICC)] between the two software tools (H&E: 0.87; TTF-1: 0.93) was higher compared to that between conventional observers (0.48; 0.47). Digital TCC estimations were in good agreement with the average of human TCC estimations (0.78; 0.96). Conventional TCC estimators tended to overestimate TCC, especially in H&E stainings, in tumors with solid patterns and in tumors with an actual TCC close to 50%. CONCLUSIONS: Our results determine factors that influence TCC estimation. Computer-assisted analysis can improve the accuracy of TCC estimates prior to molecular diagnostic workflows. In addition, we provide a free web application to support self-training and quality improvement initiatives at other institutions.

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.

Ulixertinib (BVD-523) is a well-tolerated, orally delivered, catalytic ERK1/2 inhibitor, which has shown promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors. Pediatric low grade gliomas (pLGGs) are the most common pediatric brain tumors, with the most frequent driving alterations (KIAA:BRAF fusion, BRAF V600E mutation) occurring in the MAPK pathway. To investigate the anti-tumoral activity of ulixertinib in pLGG, cell lines recapitulating both main MAPK alterations were used: DKFZ-BT66 (pilocytic astrocytoma; KIAA:BRAF fusion) and BT40 (pleomorphic xanthoastrocytoma; BRAF V600E mutation and CDKN2A/B deletion). The potential synergism of combinations with MEK inhibitors, senolytics, and chemotherapy was investigated in vitro using metabolic activity and MAPK activity assays. The most promising combinations were validated in vitro by analysis of viable, dead, and apoptotic cells through high-content microscopy. The most clinically relevant combinations were further validated in vivo: 1) in two zebrafish embryo models (respectively, BT40 and DKFZ-BT66 yolk sac injection) and 2) in NSG mice (BT40 orthotopic PDX) including in vivo pharmacokinetic and -dynamic analyses. Our data demonstrate ulixertinib’s ability to inhibit MAPK pathway activity in all used models. Ulixertinib treatment reduced cell viability in the BRAF V600E mutated cell line at a remarkably low concentration of 62.7 nM (compared to other cell lines’ IC50 from the Genomics of Drug Sensitivity in Cancer database). In vivo pharmacokinetic and -dynamic analyses showed good penetrance of the drug into mouse brain tissue, with concentrations above the in vitro IC50 and reduction of MAPK activity as assessed by Western blot. Furthermore, ulixertinib treatment slowed tumor growth and significantly increased survival in NSG mice with orthotopic BT40 xenografts. Ulixertinib showed indications for anti-proliferative synergy in vitro, according to the Loewe and Bliss independence models, in combination with MEK inhibitors (trametinib, binimetinib) or senolytics (navitoclax, A1331852). Combinations with chemotherapy (carboplatin, vinblastine) were at most additive. Indications for synergy with binimetinib and navitoclax were confirmed in the zebrafish embryo xenograft models for both MAPK-altered backgrounds. The combination of ulixertinib with navitoclax was further investigated in the BT40 PDX mouse model, where tumor growth and survival were comparable to ulixertinib monotherapy. In conclusion, our data indicate a strong potential for ulixertinib as a clinically relevant therapeutic option for the treatment of pLGG to be further investigated in upcoming clinical trials. Potential synergism with MEK inhibitors and senolytics was noted and warrants further investigation. Citation Format: Romain Sigaud, Lisa Rösch, Charlotte Gatzweiler, Julia Benzel, Laura von Soosten, Heike Peterziel, Sara Najafi, Simay Ayhan, Nina Hofmann, Kathrin I. Förster, Jürgen Burhenne, Rémi Longuespée, Cornelis M. van Tilburg, David T. Jones, Stefan M. Pfister, Deborah Knoerzer, Brent Kreider, Max Sauter, Kristian W. Pajtler, Marc Zuckermann, Ina Oehme, Olaf Witt, Till Milde. The first-in-class ERK inhibitor ulixertinib (BVD-523) shows activity in MAPK-driven pediatric low-grade glioma models as single agent and in combination with MEK inhibitors or senolytics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5221.

Ulixertinib (BVD-523) is a catalytic ERK1/2 inhibitor that showed promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors. Pediatric low-grade gliomas (pLGG) are the most common pediatric brain tumors, with the most frequent driving alterations in the MAPK pathway. The anti-tumor activity of ulixertinib in pLGG and its potential synergism in combination with MEK inhibitors, senolytics, and chemotherapy were investigated in vitro using metabolic activity, MAPK reporter assay and high-content microscopy in pLGG-derived cell lines (DKFZ-BT66 - KIAA:BRAF fusion; BT40 - BRAF V600E mutation and CDKN2A/B deletion). The most clinically relevant combinations were further validated in vivo: 1) in zebrafish embryo models (BT40 and DKFZ-BT66 yolk sac injection) and 2) in NSG mice (BT40 orthotopic PDX) including in vivo pharmacokinetic and -dynamic analyses. Ulixertinib inhibited MAPK pathway activity in all models and reduced cell viability in the BRAF V600E mutated cell line at concentrations in the nanomolar range. In vivo pharmacokinetic and -dynamic analyses showed penetrance of the drug into mouse brain tissue and on-target activity, with concentrations above the in vitro IC50 and reduction of MAPK activity. Ulixertinib treatment slowed tumor growth and significantly increased survival in NSG mice with BT40 xenografts. Ulixertinib showed indications for anti-proliferative synergy in vitro in combination with MEK inhibitors (trametinib, binimetinib) or BH3 mimetics (navitoclax, A-1331852). Combinations with chemotherapy (carboplatin, vinblastine) were at most additive. Indications for synergy with binimetinib and navitoclax were confirmed in the zebrafish embryo models. In the NSG mouse model, the combination of ulixertinib with senolytics induced effects on tumor growth and survival comparable to ulixertinib monotherapy. Ulixertinib shows promising potential as a clinically relevant therapeutic option for the treatment of pLGG to be further investigated in upcoming clinical trials. Potential synergism with MEK inhibitors and BH3 mimetics was noted and warrants further investigation.

Human peripheral blood mononuclear cells (PBMCs) represent a sentinel blood sample which reacts to different pathophysiological stimuli in the form of immunological responses/immunophenotypic changes. The study of molecular content of PBMCs can provide better understanding of immune processes giving the possibility of monitoring the health conditions of the host organism. Proteomic analysis of PBMCs can achieve mentioned goal as important immune-related biomarkers are easily accessible for analysis. PBMCs have been gaining attention in different research areas including preclinical or clinical investigations. In this review, recent applications of proteomic analysis of PBMCs are described and discussed. Approaches are divided based on different proteomic workflows such as in-gel, in-solution and on-filter modes. The effect of various diseases such as autoimmune, cancer, neurodegenerative, viral, metabolic, and various immune stimulations such as radiation, vaccine, corticosteroids over PBMCs proteome, are described with emphasis on promising protein biomarker candidates.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has rarely been used in the field of therapeutic drug monitoring, partly because of the complexity of the ionization processes between the compounds to be quantified and the many MALDI matrices available. The development of a viable MALDI-MS method that meets regulatory guidelines for bioanalytical method validation requires prior knowledge of the suitability of (i) the MALDI matrix with the analyte class and properties for ionization, (ii) the crystallization properties of the MALDI matrix with automation features, and (iii) the MS instrumentation used to achieve sensitive and specific measurements in order to determine low pharmacological drug concentrations in biological matrices. In the present hybrid article/white paper, we review the developments required for the establishment of MALDI-MS assays for the quantification of drugs in tissues and plasma, illustrated with concrete results for the different steps. We summarize the necessary parameters that need to be controlled for the successful development of fully validated MALDI-MS methods according to regulatory authorities, as well as currently unsolved problems and promising ways to address them. Finally, we propose an expert opinion on future perspectives and needs in order to establish MALDI-MS as a universal method for therapeutic drug monitoring.

The third-generation tyrosine kinase inhibitor (TKI), osimertinib, has revolutionized the treatment of patients with non-small cell lung carcinoma with epidermal growth factor receptor (EGFR)-activating mutation, and resistant to first- and second-generation TKIs. Osimertinib is now also proposed as a first-line therapy, thus extending the scope of applications in lung oncology. Personalized medicine approaches are still necessary to monitor if patients are exposed to adequate concentrations of osimertinib during their treatment. It would also help to understand the appearance of new resistances in patients after several months of dosing with osimertinib. Liquid chromatography&ndash, tandem mass spectrometry (LC&ndash, MS/MS) is currently the gold standard for the quantification of drugs in plasma enabling pharmacokinetic analyses and patient monitoring. In the present study, we propose an alternative to LC&ndash, MS/MS methods for the rapid and sensitive quantification of osimertinib in plasma using matrix-assisted laser desorption/ionization (MALDI) &ndash, MS. The presented assay requires only 3 min per sample for their preparation, analysis, and data extraction, and less than 3 h for quantification. A lower limit of quantification (LLOQ) of 5 ng/mL in plasma was retrieved. The method was fully validated, following the guidelines of the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for bioanalytical method validation. The present developments prove the importance to consider alternative MS assays for time-efficient quantification of small molecule inhibitors in plasma in the context of personalized medicine for targeted therapies.

Multiple therapeutic monoclonal antibodies (mAbs) are currently under development or in (pre)clinical study phases to reach regulatory approval. Among these, a new mAb against herpes simplex virus, HDIT101, was recently tested in healthy volunteers during a phase I clinical trial (first-in-human, dose escalation). In the frame of the pharmacokinetic evaluation of this new therapy, a mass spectrometric (MS)-based method was developed for the quantification of HDIT101 in human plasma using liquid chromatography coupled to tandem mass spectrometry. In this work, we describe the development of this bioanalytical assay using the quantification of a HDIT101 surrogate peptide, the assay validation procedure according to the FDA guidelines within the calibration range from 20 to 5000 μg/mL, and its application to plasma samples from the first-in-human clinical trial. This work presents a generic workflow for the development of MS-based quantification assays of new therapeutic antibodies that allows reaching high immunopurification recovery (98% for HDIT101 over the full calibration range with a precision of 6.9% CV). Surrogate peptide and stable isotopically labeled internal standard were stable, and batch-to-batch accuracies and precisions at the four quality standard levels ranged between -2 and 5% bias and 8 and 11% CV, respectively.

Receptor tyrosine kinases (RTKs) are key regulatory signaling proteins governing cancer cell growth and metastasis. During the last two decades, several molecules targeting RTKs were used in oncology as a first or second line therapy in different types of cancer. However, their effectiveness is limited by the appearance of resistance or adverse effects. In this review, we summarize the main features of RTKs and their inhibitors (RTKIs), their current use in oncology, and mechanisms of resistance. We also describe the technological advances of artificial intelligence, chemoproteomics, and microfluidics in elaborating powerful strategies that could be used in providing more efficient and selective small molecules inhibitors of RTKs. Finally, we discuss the interest of therapeutic combination of different RTKIs or with other molecules for personalized treatments, and the challenge for effective combination with less toxic and off-target effects.

High‐throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh‐frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single‐pot solid‐phase‐enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96‐well format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands‐on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low‐input samples, reproducibly quantifying 500–1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end‐to‐end sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost‐effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clinical and non‐clinical applications., The study presents an automated sample preparation pipeline for low‐input proteomics based on the SP3 method. The seamless integration of tissue lysis with autoSP3 in a 96‐well format features low variability, high sensitivity and longitudinal reproducibility for clinical studies.

Dysregulation of neuropeptides may play an important role in aging-induced impairments. Among them, pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. We hypothesized that the progressive decline of PACAP throughout life and the well-known general cytoprotective effects of PACAP lead to age-related pathophysiological changes in PACAP deficiency, supported by the increased vulnerability to various stressors of animals partially or totally lacking PACAP. Using young and aging CD1 PACAP knockout (KO) and wild type (WT) mice, we demonstrated pre-senile amyloidosis in young PACAP KO animals and showed that senile amyloidosis appeared accelerated, more generalized, more severe, and affected more individuals. Histopathology showed age-related systemic amyloidosis with mainly kidney, spleen, liver, skin, thyroid, intestinal, tracheal, and esophageal involvement. Mass spectrometry-based proteomic analysis, reconfirmed with immunohistochemistry, revealed that apolipoprotein-AIV was the main amyloid protein in the deposits together with several accompanying proteins. Although the local amyloidogenic protein expression was disturbed in KO animals, no difference was found in laboratory lipid parameters, suggesting a complex pathway leading to increased age-related degeneration with amyloid deposits in the absence of PACAP. In spite of no marked inflammatory histological changes or blood test parameters, we detected a disturbed cytokine profile that possibly creates a pro-inflammatory milieu favoring amyloid deposition. In summary, here we describe accelerated systemic senile amyloidosis in PACAP gene-deficient mice, which might indicate an early aging phenomenon in this mouse strain. Thus, PACAP KO mice could serve as a model of accelerated aging with human relevance. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

All isocitrate dehydrogenase (IDH) mutant solid neoplasms exhibit highly elevated levels of D-2-hydroxyglutarate (D-2HG). Detection of 2HG in tumor tissues currently is performed by gas or liquid chromatography-mass spectrometry (GC- or LC-MS) or biochemical detection. While these methods are highly accurate, a considerable amount of time for tissue preparation and a relatively high amount of tissue is required for testing. We here present a rapid approach to detect 2HG in brain tumor tissue based on matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF). We analyzed 26 brain tumor samples with known IDH1 or IDH2 mutation and compared readouts to those from 28 brain tumor samples of wildtype IDH status. IDH mutant samples exhibited a clear positive signal for 2HG which was not observed in any of the IDH wildtype tumors. Our analytical pipeline allowed for 2HG detection in less than 5 min. Data were validated by determining 2HG levels in all tissues with a biochemical assay. In conclusion, we developed a protocol for rapid detection of 2HG levels and illustrate the possibility to use MALDI-TOF for the detection of metabolites on frozen tissue sections in a diagnostic setting. Electronic supplementary material The online version of this article (10.1186/s40478-018-0523-3) contains supplementary material, which is available to authorized users.

In the last decade it became evident that many lung adenocarcinomas (ADC) harbor key genetic alterations such as KRAS, EGFR or BRAF mutations as well as rearrangements of ROS1 or ALK that drive these tumors. In the present study we investigated whether different driver mutations of ADC result in different proliferation rates, which might have clinical impact, including resistance to therapy, recurrence and prognosis. We analyzed the proliferation index (PI) on full slides of surgically resected ADC (n = 230) with known genetic aberrations by means of immunohistochemistry and subsequent digital image analysis and correlated the results with clinicopathological variables including overall (OS) and disease free survival (DFS). We did not observe significant differences in OS or DFS regarding the KRAS or EGFR mutational status (P = 0.56). However, KRAS mutated ADC showed an increased PI compared to EGFR mutated ADC, and ADC with ALK translocations (P 0.01). Subgroup analysis of EGFR mutated ADC showed a higher PI for tumors harboring a mutation in exon 18 and 20, compared to tumors with a mutation in exon 19 or 21. A PI of 11.5% was the best possible prognostic stratificator for OS (P = 0.01 in KRAS mutated and P 0.01 in EGFR mutated ADC). In conclusion, the PI differs significantly among ADC with distinct driver mutations. This might explain the varying indications for a prognostic relevance of the PI observed in prior studies. Our study provides a basis for the establishment of a reliable and clinically meaningful PI threshold.

Molecular heterogeneity exists at different spatial scales in biological samples and is an important parameter in the development of pathologies and resistances to therapies. When aiming to reach molecular heterogeneity of cells at extremely low spatial scales, single-cell analysis can be the ultimate choice. Proteomics performed in bulk population of cells (macroproteomics) is prone to mask molecular heterogeneity. Mass spectrometry-based single cell proteomics (SCP-MS) is the right solution to overcome this issue. Three main problems can be identified using SCP-MS: (i) analytical loss during sample preparation, (ii) inefficient microinjection/delivery of proteins/peptides from samples to MS and (iii) low analytical throughput. Technologies for automation of SCP have recently gained attention to improve methods accuracy, sensitivity, throughput and in-depth and low-biased proteome analysis. In this minireview, we therefore overview the state-of-the-art of automation of SCP-MS sample preparation approaches.

Mass spectrometry imaging datasets are mostly analyzed in terms of average intensity in regions of interest. However, biological tissues have different morphologies with several sizes, shapes, and structures. The important biological information, contained in this highly heterogeneous cellular organization, could be hidden by analyzing the average intensities. Finding an analytical process of morphology would help to find such information, describe tissue model, and support identification of biomarkers. This study describes an informatics approach for the extraction and identification of mass spectrometry image features and its application to sample analysis and modeling. For the proof of concept, two different tissue types (healthy kidney and CT-26 xenograft tumor tissues) were imaged and analyzed. A mouse kidney model and tumor model were generated using morphometric - number of objects and total surface - information. The morphometric information was used to identify m/z that have a heterogeneous distribution. It seems to be a worthwhile pursuit as clonal heterogeneity in a tumor is of clinical relevance. This study provides a new approach to find biomarker or support tissue classification with more information. Graphical Abstract ᅟ.

Aetiologically linked to HPV infection, malignancies of the anal canal have substantially increased in incidence over the last 20 years. Although most anal squamous cell carcinomas (SCCs) respond well to chemoradiotherapy, about 30% of patients experience a poor outcome, for undetermined reasons. Despite cumulative efforts for discovering independent predictors of overall survival, both nodal status and tumour size are still the only reliable factors predicting patient outcome. Recent efforts have revealed that the biology of HPV-related lesions in the cervix is strongly linked to the originally infected cell population. To address the hypothesis that topography also influences both gene expression profile and behaviour of anal (pre)neoplastic lesions, we correlated both proteomic signatures and clinicopathological features of tumours arising from two distinct portions of the anal canal: the lower part (squamous zone) and the more proximal anal transitional zone. Although microdissected cancer cells appeared indistinguishable by morphology (squamous phenotype), unsupervised clustering analysis of the whole proteome significantly highlighted the heterogeneity that exists within anal canal tumours. More importantly, two region-specific subtypes of SCC were revealed. The expression profile (sensitivity/specificity) of several selected biomarkers (keratin filaments) further confirmed the subclassification of anal (pre)cancers based on their cellular origin. Less commonly detected compared to their counterparts located in the squamous mucosa, SCCs originating in the transitional zone more frequently displayed a poor or basaloid differentiation, and were significantly correlated with reduced disease-free and overall survivals. Taken together, we present direct evidence that anal canal SCC comprises two distinct entities with different cells of origin, proteomic signatures, and survival rates. This study forms the basis for a dualistic classification of anal carcinoma, with implications for management, outcome expectations, and possibly therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cystic echinococcosis (CE) is a pandemic infectious disease caused by the tapeworm Echinococcus granulosus that forms cysts in different organs such as lungs and liver. Imaging examination and serological tests have some drawbacks such as low sensitivity. In this study, we used an up-to-date workflow of laser microdissection-based microproteomics and matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry in order to depict the proteomic pattern of CE in the liver. This investigation revealed specific markers of a parasitic cyst in liver. This proteomic pattern could facilitate diagnosis of CE in the future.

Aims Human papilloma virus (HPV) infection is a causative agent for approximately 5% of all new cancer cases in humans. The virus is detected in cervical, anal, vaginal, penile, vulvar and head and neck cancers and has prognostic implications. Thus, test systems are required to detect high-risk but also low-risk HPV subtypes with high specificity and sensitivity in a time-effective and cost-effective manner. In the present study we developed a new mass spectrometry (MS)-based test system for the detection of HPV infections in formalin-fixed paraffin-embedded (FFPE) tissue samples. Methods A high-throughput matrix-assisted laser desorption ionisation time of flight MS-based assay was applied to genotype 19 HPV types in FFPE tissue specimens (n=46). The results from the MS assay were compared with the results obtained from two hybridisation-based test systems: the HPV 3.5 LCD-array kit and the EuroArrayHPV system. Results In 36 out of 46 (78%) tissue samples, a HPV infection could be detected by the MS-based HPV assay. In 16 samples (44%) only one and in 20 samples (56%) two to six HPV subtypes were identified. The overall agreement of all three assays was almost perfect (Cohen9s k value: 0.83). Conclusions The MS-based assay is highly sensitive, reliable as well as cost-effective and represents a suitable technology for the detection of HPV infections in FFPE tissue material.

SummaryHigh-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples originating from a variety of sources, including fresh frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format, performing unbiased protein purification and digestion, and delivering peptides that can be directly analyzed by LCMS. AutoSP3 eliminates hands-on time and minimizes the risk of error, reduces variability in protein quantification and improves longitudinal performance and reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1000 proteins from 100-1000 cells (

Rationale The introduction of desorption electrospray ionization (DESI) - and ambient desorption/ionization (ADI) ion sources in general - in the 2000s has opened new possibilities for mass spectrometric (MS) analyses of biological sample surfaces. DESI allows for a rapid screening of solid samples because no sample preparation is needed and the analysis is performed at atmospheric pressure. In the present study, we used DESI as an ion source for the rapid detection of a small molecule in blood droplets deposited on glass slides. Methods Blood was spiked with different concentrations of a model drug, mebendazole. One microliter blood droplets of each preparation were deposited on the surface of a glass slide and analyzed by DESI, either in imaging or profiling mode. Results The results suggested that DESI imaging mode was not appropriate for the detection of mebendazole in blood droplets as an initial solvation time was necessary before the obtention of signal. A profiling approach consisting of analyzing a single position of the blood droplet was used for further analysis and allowed mebendazole to be detected in the fg range and to monitor the volume of sample analyzed. Conclusions The study suggests that profiling mode at a single position is adequate for DESI analyses in whole blood droplets. This proof-of-concept study illustrates the potential of DESI profiling as a possible alternative to liquid chromatography/MS analyses of whole blood, when analyses are needed within a restricted time. Rapid detection methods in blood at atmospheric pressure may find interesting applications in the fields of toxicology and pharmacology.

Purpose To facilitate the transition of MALDI-MS Imaging (MALDI-MSI) from basic science to clinical application, it is necessary to analyze formalin-fixed paraffin-embedded (FFPE) tissues. The aim is to improve in situ tryptic digestion for MALDI-MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites. Experimental design FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) are prepared for MALDI-MSI. Samples are coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples are sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50 µm spatial resolution. Data are analyzed using flexImaging, SCiLS, and R. Results Trypsin application and digestion are identified as sources of variation and loss of spatial resolution in the MALDI-MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA. Conclusions and clinical relevance Spatial resolution and site-to-site reproducibility can be maintained by adhering to a standardized MALDI-MSI workflow.

Objective Recognition of neuroendocrine differentiation is important for tumor classification and treatment stratification. To detect and confirm neuroendocrine differentiation, a combination of morphology and immunohistochemistry is often required. In this regard, synaptophysin, chromogranin A, and CD56 are established immunohistochemical markers. Insulinoma-associated protein 1 (INSM1) has been suggested as a novel stand-alone marker with the potential to replace the current standard panel. In this study, we compared the sensitivity and specificity of INSM1 and established markers. Materials and methods A cohort of 493 lung tumors including 112 typical, 39 atypical carcinoids, 77 large cell neuroendocrine carcinomas, 144 small cell lung cancers, 30 thoracic paragangliomas, 47 adenocarcinomas, and 44 squamous cell carcinomas were selected and tissue microarrays were constructed. Synaptophysin, chromogranin A, CD56, and INSM1 were stained on all cases and evaluated manually as well as with an analysis software. Positivity was defined as ≥1% stained tumor cells in at least 1 of 2 cores per patient. Results INSM1 was positive in 305 of 402 tumors with expected neuroendocrine differentiation (typical and atypical carcinoids, large cell neuroendocrine carcinomas, small cell lung cancers, and paraganglioma; sensitivity: 76%). INSM1 was negative in all but 1 of 91 analyzed non-neuroendocrine tumors (adenocarcinomas, squamous cell carcinomas; specificity: 99%). All conventional markers, as well as their combination, had a higher sensitivity (97%) and a lower specificity (78%) for neuroendocrine differentiation compared with INSM1. Conclusions Although INSM1 might be a meaningful adjunct in the differential diagnosis of neuroendocrine neoplasias, a general uncritical vote for replacing the traditional markers by INSM1 may not be justified.

Purpose Identification of proteolytic peptides from matrix-assisted laser desorption/ionization (MALDI) imaging remains a challenge. The low fragmentation yields obtained using in situ post source decay impairs identification. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an alternative to in situ MS/MS, but leads to multiple identification candidates for a given mass. The authors propose to use LC-MS/MS-based biomarker discovery results to reliably identify proteolytic peptides from MALDI imaging. Experimental design The authors defined m/z values of interest for high grade squamous intraepithelial lesion (HSIL) by MALDI imaging. In parallel the authors used data from a biomarker discovery study to correlate m/z from MALDI imaging with masses of peptides identified by LC-MS/MS in HSIL. The authors neglected candidates that were not significantly more abundant in HSIL according to the biomarker discovery investigation. Results The authors assigned identifications to three m/z of interest. The number of possible identifiers for MALDI imaging m/z peaks using LC-MS/MS-based biomarker discovery studies was reduced by about tenfold compared using a single LC-MS/MS experiment. One peptide identification candidate was validated by immunohistochemistry. Conclusion and clinical relevance This concept combines LC-MS/MS-based quantitative proteomics with MALDI imaging and allows reliable peptide identification. Public datasets from LC-MS/MS biomarker discovery experiments will be useful to identify MALDI imaging m/z peaks.

Multiple applications of proteomics in life and health science, pathology and pharmacology, require handling size-limited cell and tissue samples. During proteomic sample preparation, analyte loss in these samples arises when standard procedures are used. Thus, specific considerations have to be taken into account for processing, that are summarised under the term microproteomics (μPs). Microproteomic workflows include: sampling (e.g., flow cytometry, laser capture microdissection), sample preparation (possible disruption of cells or tissue pieces via lysis, protein extraction, digestion in bottom-up approaches, and sample clean-up) and analysis (chromatographic or electrophoretic separation, mass spectrometric measurements and statistical/bioinformatic evaluation). All these steps must be optimised to reach wide protein dynamic ranges and high numbers of identifications. Under optimal conditions, sampling is adapted to the studied sample types and nature, sample preparation isolates and enriches the whole protein content, clean-up removes salts and other interferences such as detergents or chaotropes, and analysis identifies as many analytes as the instrumental throughput and sensitivity allow. In the suggested review, we present and discuss the current state in μP applications for processing of small number of cells (cell μPs) and microscopic tissue regions (tissue μPs).

Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry–mass spectrometry (LC–MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest.

Histopathological diagnoses have been done in the last century based on hematoxylin and eosin staining. These methods were complemented by histochemistry, electron microscopy, immunohistochemistry (IHC), and molecular techniques. Mass spectrometry (MS) methods allow the thorough examination of various biocompounds in extracts and tissue sections. Today, mass spectrometry imaging (MSI), and especially matrix-assisted laser desorption ionization (MALDI) imaging links classical histology and molecular analyses. Direct mapping is a major advantage of the combination of molecular profiling and imaging. MSI can be considered as a cutting edge approach for molecular detection of proteins, peptides, carbohydrates, lipids, and small molecules in tissues. This review covers the detection of various biomolecules in histopathological sections by MSI. Proteomic methods will be introduced into clinical histopathology within the next few years.

Purpose Differential diagnosis of ulcerative colitis (UC) and Crohn's disease (CD) is of utmost importance for the decision making of respective therapeutic treatment strategies but in about 10-15% of cases, a clinical and histopathological assessment does not lead to a definite diagnosis. The aim of the study is to characterize proteomic differences between UC and CD. Experimental design Microproteomics is performed on formalin-fixed paraffin-embedded colonic tissue specimens from 9 UC and 9 CD patients. Protein validation is performed using immunohistochemistry (IHC) (nUC =51, nCD =62, nCTRL =10) followed by digital analysis. Results Microproteomic analyses reveal eight proteins with higher abundance in CD compared to UC including proteins related to neutrophil activity and damage-associated molecular patterns. Moreover, one protein, Aldo-keto reductase family 1 member C3 (AKR1C3), is present in eight out of nine CD and absent in all UC samples. Digital IHC analysis reveal a higher percentage and an increased expression intensity of AKR1C3-positive epithelial cells in CD compared to UC and in controls compared to inflammatory bowel disease (IBD). Conclusion and clinical relevance Overall, the results suggest that microproteomics is an adequate tool to highlight protein patterns in IBD. IHC and digital pathology might support future differential diagnosis of UC and CD.