HighlightsLMD was used to investigate 3-D molecular heterogeneity in ovarian cancer tissue Diverse molecular profiles were identified from 3-D spatially separated samples Molecular heterogeneity impacts HGSOC prognostic sub-type assignment Proteomic heterogeneity analysis web portal deployed at www. lmdomics.org
An ocular toxic reaction presenting as conjunctivitis or keratitis develops in a significant number of patients who are treated with high‐dose cytosine arabinoside (ara‐C). Although eye drops containing glucocorticoid reportedly decrease the incidence, they do not totally eliminate this side effect. In comparing this technique with artificial tears, both were found to be equally effective. The primary mechanism by which eye drops decrease ocular toxic reactions associated with high‐dose ara‐C is presumably due to dilution of intraocular concentrations of ara‐C.
Reversible protein phosphorylation represents a key mechanism by which signals are transduced in eukaryotic cells. Dysregulated phosphorylation is also a hallmark of carcinogenesis and represents key drug targets in the precision medicine space. Thus, methods that preserve phosphoprotein integrity in the context of clinical tissue analyses are crucially important in cancer research. Here we investigated the impact of UV laser microdissection (UV LMD) and IR laser capture microdissection (IR LCM) on phosphoprotein abundance of key cancer signaling protein targets assessed by reverse-phase protein microarray (RPPA). Tumor epithelial cells from consecutive thin sections obtained from four high-grade serous ovarian cancers were harvested using either UV LMD or IR LCM methods. Phosphoprotein abundances for ten phosphoproteins that represent important drug targets were assessed by RPPA and revealed no significant differences in phosphoprotein integrity from those obtained using higher-energy UV versus the lowerenergy IR laser methods.
A series of 200 consecutive thin sections were generated from a high-grade serous ovarian tumor and laser microdissected four spatially separated "core" regions of tumor epithelium, along with tumor epithelium, stroma or whole tissue harvests at 200 µm intervals. These distinct tissue collections were analyzed by quantitative proteomics and RNA-Seq. Unsupervised analyses revealed co-clustering of tumor cores with enriched tumor epithelium, which were distinct from the enriched stroma and whole tumor collections. Strong correlations in protein and transcript abundance in tumor epithelium and stromal collections from neighboring thin sections were decreased in samples harvested just hundreds of microns apart. Stroma (mesenchymal) and tumor epithelium (differentiated) displayed a distinct association with ovarian cancer prognostic molecular sub-types with a 2-year difference in median survival. These data reveal substantial tumor microenvironment protein and transcript expression heterogeneity that directly bear on prognostic signatures and underscore the need to enrich cellular subpopulations for expression profiling.
Inflammatory myofibroblastic tumors (IMTs) are intermediate-grade mesenchymal neoplasms commonly characterized by chromosomal rearrangements causing constitutive activation of anaplastic lymphoma kinase (ALK) and/or ALK mutations causing reduced sensitivity to ALK tyrosine kinase inhibitors (TKI). We present a patient with an IMT who initially responded to first-line alectinib, but who later suffered disease relapse and presently survives with moderate residual disease after receiving second-line lorlatinib. Biopsy specimens were analyzed using next generation sequencing (DNA-seq and RNA-seq) and reverse phase protein microarray (RPPA) as part of an institutional Molecular Tumor Board (MTB) study. An EML4-ALK rearrangement and EGFR activation (pEGFRY1068) were present in both the primary and recurrent tumors, while a secondary ALK I1171N mutation was exclusive to the latter. EGFR signaling in the background of a secondary ALK mutation is correlated with reduced ALK TKI sensitivity in vitro, implicating an important mechanism of drug resistance development in this patient. The RPPA results also critically demonstrate that ALK signaling (ALKY1604) was not activated in the recurrent tumor, thereby indicating that standard-of-care use of third- or fourth-line ALK TKI would not likely be efficacious or durable. These results underscore the importance of real-time clinical integration of functional protein drug target activation data with NGS in the MTB setting for improving selection of patient-tailored therapy.
The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations.To characterize proteome-level variation and tumor heterogeneity at scale, highthroughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.