Chondrosarcoma evaluation using hematein-based x-ray staining and high-resolution 3D micro-CT: a feasibility study.

BACKGROUND: Chondrosarcomas are rare malignant bone tumors diagnosed by analyzing radiological images and histology of tissue biopsies and evaluating features such as matrix calcification, cortical destruction, trabecular penetration, and tumor cell entrapment. METHODS: We retrospectively analyzed 16 cartilaginous tumor tissue samples from three patients (51-, 54-, and 70-year-old) diagnosed with a dedifferentiated chondrosarcoma at the femur, a moderately differentiated chondrosarcoma in the pelvis, and a predominantly moderately differentiated chondrosarcoma at the scapula, respectively. We combined a hematein-based x-ray staining with high-resolution three-dimensional (3D) microscopic x-ray computed tomography (micro-CT) for nondestructive 3D tumor assessment and tumor margin evaluation. RESULTS: We detected trabecular entrapment on 3D micro-CT images and followed bone destruction throughout the volume. In addition to staining cell nuclei, hematein-based staining also improved the visualization of the tumor matrix, allowing for the distinction between the tumor and the bone marrow cavity. The hematein-based staining did not interfere with further conventional histology. There was a 5.97 ± 7.17% difference between the relative tumor area measured using micro-CT and histopathology (p = 0.806) (Pearson correlation coefficient r = 0.92, p = 0.009). Signal intensity in the tumor matrix (4.85 ± 2.94) was significantly higher in the stained samples compared to the unstained counterparts (1.92 ± 0.11, p = 0.002). CONCLUSIONS: Using nondestructive 3D micro-CT, the simultaneous visualization of radiological and histopathological features is feasible. RELEVANCE STATEMENT: 3D micro-CT data supports modern radiological and histopathological investigations of human bone tumor specimens. It has the potential for being an integrative part of clinical preoperative diagnostics. KEY POINTS: • Matrix calcifications are a relevant diagnostic feature of bone tumors. • Micro-CT detects all clinically diagnostic relevant features of x-ray-stained chondrosarcoma. • Micro-CT has the potential to be an integrative part of clinical diagnostics.

Proteomic Characterization of Undifferentiated Small Round Cell Sarcomas with EWSR1- and CIC::DUX4-Translocations Reveals Diverging Tumor Biology and Distinct Diagnostic Markers.

Undifferentiated small round cell sarcomas of bone and soft tissue (USRS) are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n=42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n=24) or EWSR1::ERG - fusions (n=4), sarcomas with an EWSR1 - rearrangement (n=2), CIC::DUX4 fusion (n=8), as well as tumors classified as USRS with no genetic data available (n=4). Proteins extracted from formalin-fixed, paraffin-embedded (FFPE) pretherapeutic biopsies were analyzed qualitatively and quantitatively using shot gun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in two cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas and Bach2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expression of these 3 markers in the two genetic groups were further validated in an independent cohort of n= 34 USRS. Finally, our proteomic results point towards diverging signaling pathways in the different USRS subgroups.

Comparing CT-Like Images Based on Ultra-Short Echo Time and Gradient Echo T1-Weighted MRI Sequences for the Assessment of Vertebral Disorders Using Histology and True CT as the Reference Standard.

BACKGROUND: Several magnetic resonance (MR) techniques have been suggested for radiation-free imaging of osseous structures. PURPOSE: To compare the diagnostic value of ultra-short echo time and gradient echo T1-weighted MRI for the assessment of vertebral pathologies using histology and computed tomography (CT) as the reference standard. STUDY TYPE: Prospective. SUBJECTS: Fifty-nine lumbar vertebral bodies harvested from 20 human cadavers (donor age 73 ± 13 years; 9 male). FIELD STRENGTH/SEQUENCE: Ultra-short echo time sequence optimized for both bone (UTEb) and cartilage (UTEc) imaging and 3D T1-weighted gradient-echo sequence (T1GRE) at 3 T; susceptibility-weighted imaging (SWI) gradient echo sequence at 1.5 T. CT was performed on a dual-layer dual-energy CT scanner using a routine clinical protocol. ASSESSMENT: Histopathology and conventional CT were acquired as standard of reference. Semi-quantitative and quantitative morphological features of degenerative changes of the spines were evaluated by four radiologists independently on CT and MR images independently and blinded to all other information. Features assessed were osteophytes, endplate sclerosis, visualization of cartilaginous endplate, facet joint degeneration, presence of Schmorl's nodes, and vertebral dimensions. Vertebral disorders were assessed by a pathologist on histology. STATISTICAL TESTS: Agreement between T1GRE, SWI, UTEc, and UTEb sequences and CT imaging and histology as standard of reference were assessed using Fleiss' κ and intra-class correlation coefficients, respectively. RESULTS: For the morphological assessment of osteophytes and endplate sclerosis, the overall agreement between SWI, T1GRE, UTEb, and UTEc with the reference standard (histology combined with CT) was moderate to almost perfect for all readers (osteophytes: SWI, κ range: 0.68-0.76; T1GRE: 0.92-1.00; UTEb: 0.92-1.00; UTEc: 0.77-0.85; sclerosis: SWI, κ range: 0.60-0.70; T1GRE: 0.77-0.82; UTEb: 0.81-0.92; UTEc: 0.61-0.71). For the visualization of the cartilaginous endplate, UTEc showed the overall best agreement with the reference standard (histology) for all readers (κ range: 0.85-0.93). DATA CONCLUSIONS: Morphological assessment of vertebral pathologies was feasible and accurate using the MR-based bone imaging sequences compared to CT and histopathology. T1GRE showed the overall best performance for osseous changes and UTEc for the visualization of the cartilaginous endplate. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 2.

Characterization of Hormone Receptor and HER2 Status in Breast Cancer Using Mass Spectrometry Imaging.

Immunohistochemical evaluation of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 status stratify the different subtypes of breast cancer and define the treatment course. Triple-negative breast cancer (TNBC), which does not register receptor overexpression, is often associated with worse patient prognosis. Mass spectrometry imaging transcribes the molecular content of tissue specimens without requiring additional tags or preliminary analysis of the samples, being therefore an excellent methodology for an unbiased determination of tissue constituents, in particular tumor markers. In this study, the proteomic content of 1191 human breast cancer samples was characterized by mass spectrometry imaging and the epithelial regions were employed to train and test machine-learning models to characterize the individual receptor status and to classify TNBC. The classification models presented yielded high accuracies for estrogen and progesterone receptors and over 95% accuracy for classification of TNBC. Analysis of the molecular features revealed that vimentin overexpression is associated with TNBC, supported by immunohistochemistry validation, revealing a new potential target for diagnosis and treatment.

Differentiating Enchondromas and Atypical Cartilaginous Tumors in Long Bones with Computed Tomography and Magnetic Resonance Imaging.

The differentiation between the atypical cartilaginous tumor (ACT) and the enchondromas is crucial as ACTs require a curettage and clinical as well as imaging follow-ups, whereas in the majority of cases enchondromas require neither a treatment nor follow-ups. Differentiating enchondromas from ACTs radiologically remains challenging. Therefore, this study evaluated imaging criteria in a combination of computed tomography (CT) and magnetic resonance (MR) imaging for the differentiation between enchondromas and ACTs in long bones. A total of 82 patients who presented consecutively at our institution with either an ACT (23, age 52.7 ±18.8 years; 14 women) or an enchondroma (59, age 46.0 ± 11.1 years; 37 women) over a period of 10 years, who had undergone preoperative MR and CT imaging and subsequent biopsy or/and surgical removal, were included in this study. A histopathological diagnosis was available in all cases. Two experienced radiologists evaluated several imaging criteria on CT and MR images. Likelihood of an ACT was significantly increased if either edema within the bone (p = 0.049), within the adjacent soft tissue (p = 0.006) or continuous growth pattern (p = 0.077) were present or if the fat entrapment (p = 0.027) was absent on MR images. Analyzing imaging features on CT, the likelihood of the diagnosis of an ACT was significantly increased if endosteal scalloping >2/3 (p < 0.001), cortical penetration (p < 0.001) and expansion of bone (p = 0.002) were present and if matrix calcifications were observed in less than 1/3 of the tumor (p = 0.013). All other imaging criteria evaluated showed no significant influence on likelihood of ACT or enchondroma (p > 0.05). In conclusion, both CT and MR imaging show suggestive signs which can help to adequately differentiate enchondromas from ACTs in long bones and therefore can improve diagnostics and consequently patient management. Nevertheless, these features are rare and a combination of CT and MR imaging features did not improve the diagnostic performance substantially.

MALDI-MSI: A Powerful Approach to Understand Primary Pancreatic Ductal Adenocarcinoma and Metastases.

Cancer-related deaths are very commonly attributed to complications from metastases to neighboring as well as distant organs. Dissociate response in the treatment of pancreatic adenocarcinoma is one of the main causes of low treatment success and low survival rates. This behavior could not be explained by transcriptomics or genomics; however, differences in the composition at the protein level could be observed. We have characterized the proteomic composition of primary pancreatic adenocarcinoma and distant metastasis directly in human tissue samples, utilizing mass spectrometry imaging. The mass spectrometry data was used to train and validate machine learning models that could distinguish both tissue entities with an accuracy above 90%. Model validation on samples from another collection yielded a correct classification of both entities. Tentative identification of the discriminative molecular features showed that collagen fragments (COL1A1, COL1A2, and COL3A1) play a fundamental role in tumor development. From the analysis of the receiver operating characteristic, we could further advance some potential targets, such as histone and histone variations, that could provide a better understanding of tumor development, and consequently, more effective treatments.

Mass Spectrometry Imaging Spatial Tissue Analysis toward Personalized Medicine.

Novel profiling methodologies are redefining the diagnostic capabilities and therapeutic approaches towards more precise and personalized healthcare. Complementary information can be obtained from different omic approaches in combination with the traditional macro- and microscopic analysis of the tissue, providing a more complete assessment of the disease. Mass spectrometry imaging, as a tissue typing approach, provides information on the molecular level directly measured from the tissue. Lipids, metabolites, glycans, and proteins can be used for better understanding imbalances in the DNA to RNA to protein translation, which leads to aberrant cellular behavior. Several studies have explored the capabilities of this technology to be applied to tumor subtyping, patient prognosis, and tissue profiling for intraoperative tissue evaluation. In the future, intercenter studies may provide the needed confirmation on the reproducibility, robustness, and applicability of the developed classification models for tissue characterization to assist in disease management.

Multicenter Evaluation of Tissue Classification by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Many studies have demonstrated that tissue phenotyping (tissue typing) based on mass spectrometric imaging data is possible; however, comprehensive studies assessing variation and classifier transferability are largely lacking. This study evaluated the generalization of tissue classification based on Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometric imaging (MSI) across measurements performed at different sites. Sections of a tissue microarray (TMA) consisting of different formalin-fixed and paraffin-embedded (FFPE) human tissue samples from different tumor entities (leiomyoma, seminoma, mantle cell lymphoma, melanoma, breast cancer, and squamous cell carcinoma of the lung) were prepared and measured by MALDI-MSI at different sites using a standard protocol (SOP). Technical variation was deliberately introduced on two separate measurements via a different sample preparation protocol and a MALDI Time of Flight mass spectrometer that was not tuned to optimal performance. Using standard data preprocessing, a classification accuracy of 91.4% per pixel was achieved for intrasite classifications. When applying a leave-one-site-out cross-validation strategy, accuracy per pixel over sites was 78.6% for the SOP-compliant data sets and as low as 36.1% for the mistuned instrument data set. Data preprocessing designed to remove technical variation while retaining biological information substantially increased classification accuracy for all data sets with SOP-compliant data sets improved to 94.3%. In particular, classification accuracy of the mistuned instrument data set improved to 81.3% and from 67.0% to 87.8% per pixel for the non-SOP-compliant data set. We demonstrate that MALDI-MSI-based tissue classification is possible across sites when applying histological annotation and an optimized data preprocessing pipeline to improve generalization of classifications over technical variation and increasing overall robustness.

MALDI Mass Spectrometry Imaging for the Distinction of Adenocarcinomas of the Pancreas and Biliary Tree.

Pancreatic ductal adenocarcinoma and cholangiocarcinoma constitute two aggressive tumor types that originate from the epithelial lining of the excretory ducts of the pancreatobiliary tract. Given their close histomorphological resemblance, a correct diagnosis can be challenging and almost impossible without clinical information. In this study, we investigated whether mass spectrometric peptide features could be employed to distinguish pancreatic ductal adenocarcinoma from cholangiocarcinoma. Three tissue microarrays of formalin-fixed and paraffin-embedded material (FFPE) comprising 41 cases of pancreatic ductal adenocarcinoma and 41 cases of cholangiocarcinoma were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The derived peptide features and respective intensities were used to build different supervised classification algorithms: gradient boosting (GB), support vector machine (SVM), and k-nearest neighbors (KNN). On a pixel-by-pixel level, a classification accuracy of up to 95% could be achieved. The tentative identification of discriminative tryptic peptide signatures revealed proteins that are involved in the epigenetic regulation of the genome and tumor microenvironment. Despite their histomorphological similarities, mass spectrometry imaging represents an efficient and reliable approach for the distinction of PDAC from CC, offering a promising complementary or alternative approach to the existing tools used in diagnostics such as immunohistochemistry.

Diagnosis of joint invasion in patients with malignant bone tumors: value and reproducibility of direct and indirect signs on MR imaging.

OBJECTIVES: To evaluate the performance and reproducibility of MR imaging features in the diagnosis of joint invasion (JI) by malignant bone tumors. METHODS: MR images of patients with and without JI (n = 24 each), who underwent surgical resection at our institution, were read by three radiologists. Direct (intrasynovial tumor tissue (ITT), intraarticular destruction of cartilage/bone, invasion of capsular/ligamentous insertions) and indirect (tumor size, signal alterations of epiphyseal/transarticular bone (bone marrow replacement/edema-like), synovial contrast enhancement, joint effusion) signs of JI were assessed. Odds ratios, sensitivity, specificity, PPV, NPV, and reproducibilities (Cohen's and Fleiss' κ) were calculated for each feature. Moreover, the diagnostic performance of combinations of direct features was assessed. RESULTS: Forty-eight patients (28.7 ± 21.4 years, 26 men) were evaluated. All readers reliably assessed the presence of JI (sensitivity = 92-100 %; specificity = 88-100%, respectively). Best predictors for JI were direct visualization of ITT (OR = 186-229, p < 0.001) and destruction of intraarticular bone (69-324, p < 0.001). Direct visualization of ITT was also highly reliable in assessing JI (sensitivity, specificity, PPV, NPV = 92-100 %), with excellent reproducibility (κ = 0.83). Epiphyseal bone marrow replacement and synovial contrast enhancement were the most sensitive indirect signs, but lacked specificity (29-54%). By combining direct signs with high specificity, sensitivity was increased (96 %) and specificity (100 %) was maintained. CONCLUSION: JI by malignant bone tumors can reliably be assessed on preoperative MR images with high sensitivity, specificity, and reproducibility. Particularly direct visualization of ITT, destruction of intraarticular bone, and a combination of highly specific direct signs were valuable, while indirect signs were less predictive and specific. KEY POINTS: • Direct visualization of intrasynovial tumor was the single most sensitive and specific (92-100%) MR imaging sign of joint invasion. • Indirect signs of joint invasion, such as joint effusion or synovial enhancement, were less sensitive and specific compared to direct signs. • A combination of the most specific direct signs of joint invasion showed best results with perfect specificity and PPV (both 100%) and excellent sensitivity and NPV (both 96 %).

Engineering a better light sheet in an axicon-based system using a flattened Gaussian beam of low order.

Lasers are fundamental tools in research and development. The shape of an incident laser beam directly affects the results, when it propagates through complex structured meso-aspheric optical elements. In conic-based systems utilizing elements such as axicons, the impact of secondary lobes is mostly overlooked, although the intensity distributions at the central spot and the side-lobes directly affect the beam properties. We investigate the interaction of two axicons (160° and 170°) with incident beams approximated by Gaussian, high-order Flattened-Gaussian, and low-order Flattened-Gaussian functions. We demonstrate that replacing an incident Gaussian beam with a low-order Flattened-Gaussian beam reduces the secondary lobes and significantly improves the uniformity of the intensity profile. We practically applied this effect in engineering a conic-aspheric-based static light-sheet microscope producing markedly improved results.

The Impact of Histological Annotations for Accurate Tissue Classification Using Mass Spectrometry Imaging.

Knowing the precise location of analytes in the tissue has the potential to provide information about the organs' function and predict its behavior. It is especially powerful when used in diagnosis and prognosis prediction of pathologies, such as cancer. Spatial proteomics, in particular mass spectrometry imaging, together with machine learning approaches, has been proven to be a very helpful tool in answering some histopathology conundrums. To gain accurate information about the tissue, there is a need to build robust classification models. We have investigated the impact of histological annotation on the classification accuracy of different tumor tissues. Intrinsic tissue heterogeneity directly impacts the efficacy of the annotations, having a more pronounced effect on more heterogeneous tissues, as pancreatic ductal adenocarcinoma, where the impact is over 20% in accuracy. On the other hand, in more homogeneous samples, such as kidney tumors, histological annotations have a slenderer impact on the classification accuracy.

A Mass Spectrometry Imaging Based Approach for Prognosis Prediction in UICC Stage I/II Colon Cancer.

Currently, pathological evaluation of stage I/II colon cancer, following the Union Internationale Contre Le Cancer (UICC) guidelines, is insufficient to identify patients that would benefit from adjuvant treatment. In our study, we analyzed tissue samples from 276 patients with colon cancer utilizing mass spectrometry imaging. Two distinct approaches are herein presented for data processing and analysis. In one approach, four different machine learning algorithms were applied to predict the tendency to develop metastasis, which yielded accuracies over 90% for three of the models. In the other approach, 1007 m/z features were evaluated with regards to their prognostic capabilities, yielding two m/z features as promising prognostic markers. One feature was identified as a fragment from collagen (collagen 3A1), hinting that a higher collagen content within the tumor is associated with poorer outcomes. Identification of proteins that reflect changes in the tumor and its microenvironment could give a very much-needed prediction of a patient's prognosis, and subsequently assist in the choice of a more adequate treatment.

Implementation of Mass Spectrometry Imaging in Pathology: Advances and Challenges.

Mass spectrometry imaging (MSI) combines the excellence in molecular characterization of mass spectrometry with microscopic imaging capabilities of hematoxylin- and eosin-stained samples, enabling the precise location of several analytes in the tissue. Especially in the field of pathology, MSI may have an impactful role in tumor diagnosis, biomarker identification, prognostic prediction, and characterization of tumor margins during tumor resection procedures. This article discusses the recent developments in the field that are paving the way for this technology to become accepted as an analytical tool in the clinical setting, its current limitations, and future directions.

3D histopathology of human tumours by fast clearing and ultramicroscopy.

Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid. (b) Ultrafast active chemical dehydration with 2,2-dimethoxypropane and (c) refractive index matching with dibenzyl ether at up to 56 °C. After clearing, the tumour resectates are imaged. The images are computationally post-processed for contrast enhancement and artefact removal and then 3D reconstructed. Importantly, the sequence a-c is fully reversible, allowing the morphological correlation of one and the same histological structures, once visualized with our novel technique and once visualized by standard H&E- and IHC-staining. After reverting the clearing procedure followed by standard H&E processing, the hallmarks of ductal carcinoma in situ (DCIS) found in the cleared samples could be successfully correlated with the corresponding structures present in H&E and IHC staining. Since the imaging of several thousands of optical sections is a fast process, it is possible to analyse a larger part of the tumour than by mechanical slicing. As this also adds further information about the 3D structure of malignancies, we expect that our technology will become a valuable addition for histological diagnosis in clinical pathology.

Vertebrae, Vertebral End Plates, and Disks: Concepts and Specific Pathologies.

Vertebral end plates cover the osseous vertebral body. The integrity of the cartilaginous end plates is of great importance for the entire vertebral segment because the vascularized end plate provides the nutrition for the avascular disk. Yet several pathologies may occur at these end plates at the embryonic stage, in childhood to adolescence (e.g., ossification and segmentation disorders of the spine, persistent notochord, slippage of the growth plate), as well as in the mature spine of an adult (degenerative disk disease), that may impact the integrity of the cartilaginous end plate and therefore lead to severe diseases of the spine. This article reviews specific congenital, developmental, and degenerative disorders of the vertebral end plate as well as both established and newly introduced imaging techniques, such as ultrashort echo time imaging based on magnetic resonance imaging, that are suitable for imaging of the end plate.

Novel prognostic histopathological grading system in oral squamous cell carcinoma based on tumour budding and cell nest size shows high interobserver and intraobserver concordance.

AIMS: Squamous cell carcinoma of the oral cavity (OSCC) is a common tumour entity with a variable, partially highly aggressive clinical course. Recently, we proposed a novel (three-tiered) clinically useful grading scheme strongly associated with patient outcome in OSCC, consisting of a sum score of the histomorphological patterns tumour budding and cell nest size which outperforms WHO based grading algorithms currently in use. The aim of our study was to probe for interobserver and intraobserver reliability of this novel grading system. METHODS: 108 OSCC were retrospectively scored according to the proposed grading scheme by three independent pathologists-two experienced head and neck pathologists and one pathologist in training-blinded to each other's scoring results. RESULTS: The Cohen's Kappa (κ) values for concordance rates between experienced pathologists were κ=0.97 for the overall grade, κ=0.97 for budding activity and κ=0.91 for cell nest size, indicating a strong interobserver reliability of our proposed grading system. Initial interobserver agreement was markedly lower with the pathologist in training (κ=0.55 for overall grade) but improved significantly after a training session (κ=0.87 for overall grade). Intraobserver concordance was high (κ=0.95 for overall grade), indicating a high reproducibility of the algorithm. CONCLUSIONS: In conclusion, our study indicates that OSCC grading based on our proposed novel scheme yields an excellent inter-reader and intrareader agreement, further supporting the suitability of this grading system for routine pathological practice.

Identification of MALDI Imaging Proteolytic Peptides Using LC-MS/MS-Based Biomarker Discovery Data: A Proof of Concept.

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.

Site-to-Site Reproducibility and Spatial Resolution in MALDI-MSI of Peptides from Formalin-Fixed Paraffin-Embedded Samples.

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.