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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 High-grade squamous intraepithelial lesion (HSIL) is a known precursor for squamous cell carcinoma of uterine cervix. Although it is known that SILs are associated to infection by human papillomavirus, downstream biological mechanisms are still poorly described. In this study, we compared the microproteomic profile of HSIL to normal tissues: ectocervix (ectoC) and endocervix (endoC). Experimental design Tissue regions of endoC, ectoC, and HSlL were collected by laser microdissection (3500 cells each) from five patients. Samples were processed and analyzed using our recently developed laser microdissection-based microproteomic method. Tissues were compared in order to retrieve HSIL's proteomic profile. Potentially interesting proteins for pathology were stained by immunohistochemistry. Results We identified 3072 proteins among the fifteen samples and 2386 were quantified in at least four out of the five biological replicates of at least one tissue type. We found 236 proteins more abundant in HSIL. Gene ontology enrichments revealed mechanisms of DNA replication and RNA splicing. Despite the squamous nature of HSIL, a common signature between HSIL and endoC could be found. Finally, potential new markers could support diagnosis of dysplasia in SILs. Conclusion and clinical relevance This microproteomic investigation of HSIL gives insights into the biology of cervical precancerous lesions.

Matrix-assisted laser desorption/ionization (MALDI) imaging is an ideal tool to study intratumor heterogeneity (ITH) and its implication in prognostic stratification of patients. However, there are some drawbacks concerning protein identification. On the other hand, laser microdissection (LMD)-based microproteomics allows retrieving thousands of protein identifications from small tissue pieces. As a proof of concept, the authors combine these two complementary approaches to analyze heterogeneous regions in breast tumors. Invasive ductal breast cancer FFPE tissue sections from five patients are analyzed by MALDI imaging and the dataset is processed by segmentation. Heterogeneous regions within tumors are processed by LMD-based microproteomics, in duplicates. Liquid chromatography-tandem mass spectrometry data are classified by hierarchical clustering. Heterogeneous tissue regions are discriminated on the basis of their actual molecular heterogeneity. The dataset is correlated with MALDI imaging to identify m/z values discriminating heterogeneous regions. The molecular characterization of cell clones in tumors related to bad patient outcome could have great impact for pathology. A combined application of LMD-based microproteomics and MALDI imaging for ITH studies is presented.