Histopathological subtyping of non-small cell lung cancer (NSCLC) into adenocarcinoma (ADC), and squamous cell carcinoma (SqCC) is of utmost relevance for treatment stratification. However, current immunohistochemistry (IHC) based typing approaches on biopsies are imperfect, therefore novel analytical methods for reliable subtyping are needed. We analyzed formalin-fixed paraffin-embedded tissue cores of NSCLC by Matrix-assisted laser desorption/ionization (MALDI) imaging on tissue microarrays to identify and validate discriminating MALDI imaging profiles for NSCLC subtyping. 110 ADC and 98 SqCC were used to train a Linear Discriminant Analysis (LDA) model. Results were validated on a separate set of 58 ADC and 60 SqCC. Selected differentially expressed proteins were identified by tandem mass spectrometry and validated by IHC. The LDA classification model incorporated 339 m/z values. In the validation cohort, in 117 cases (99.1%) MALDI classification on tissue cores was in accordance with the pathological diagnosis made on resection specimen. Overall, three cases in the combined cohorts were discordant, after reevaluation two were initially misclassified by pathology whereas one was classified incorrectly by MALDI. Identification of differentially expressed peptides detected well-known IHC discriminators (CK5, CK7), but also less well known differentially expressed proteins (CK15, HSP27). In conclusion, MALDI imaging on NSCLC tissue cores as small biopsy equivalents is capable to discriminate lung ADC and SqCC with a very high accuracy. In addition, replacing multislide IHC by an one-slide MALDI approach may also save tissue for subsequent predictive molecular testing. We therefore advocate to pursue routine diagnostic implementation strategies for MALDI imaging in solid tumor typing.

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. Our aim was 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) were prepared for MALDI‐MSI. Samples were coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples were sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50μm spatial resolution. Data were analyzed using flexImaging, SCiLS and R. Results : Trypsin application and digestion were 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. This article is protected by copyright. All rights reserved