Metaplastic breast carcinoma (MBC) is a highly aggressive form of triple-negative cancer (TNBC), defined by the presence of metaplastic components of spindle, squamous, or sarcomatoid histology. The protein profiles underpinning the pathological subtypes and metastatic behavior of MBC are unknown. Using multiplex quantitative tandem mass tag-based proteomics we quantify 5798 proteins in MBC, TNBC, and normal breast from 27 patients. Comparing MBC and TNBC protein profiles we show MBC-specific increases related to epithelial-to-mesenchymal transition and extracellular matrix, and reduced metabolic pathways. MBC subtypes exhibit distinct upregulated profiles, including translation and ribosomal events in spindle, inflammation-and apical junction-related proteins in squamous, and extracellular matrix proteins in sarcomatoid subtypes. Comparison of the proteomes of human spindle MBC with mouse spindle (CCN6 knockout) MBC tumors reveals a shared spindle-specific signature of 17 upregulated proteins involved in translation and 19 downregulated proteins with roles in cell metabolism. These data identify potential subtype specific MBC biomarkers and therapeutic targets.

BACKGROUND. Although aberrant glycosylation is recognized as a hallmark of cancer, glycosylation in clinical breast cancer (BC) metastasis has not yet been studied. While preclinical studies show that the glycocalyx coating of cancer cells is involved in adhesion, migration, and metastasis, glycosylation changes from primary tumor (PT) to various metastatic sites remain unknown in patients. METHODS. We investigated N-glycosylation profiles in 17 metastatic BC patients from our rapid autopsy program. Primary breast tumor, lymph node metastases, multiple systemic metastases, and various normal tissue cores from each patient were arranged on unique single-patient tissue microarrays (TMAs). We performed mass spectrometry imaging (MSI) combined with extensive pathology annotation of these TMAs, and this process enabled spatially differentiated cell-based analysis of N-glycosylation patterns in metastatic BC. RESULTS. N-glycan abundance increased during metastatic progression independently of BC subtype and treatment regimen, with high-mannose glycans most frequently elevated in BC metastases, followed by fucosylated and complex glycans. Bone metastasis, however, displayed increased core-fucosylation and decreased high-mannose glycans. Consistently, N-glycosylated proteins and N-glycan biosynthesis genes were differentially expressed during metastatic BC progression, with reduced expression of mannose-trimming enzymes and with elevated EpCAM, N-glycan branching, and sialyation enzymes in BC metastases versus PT. CONCLUSION. We show in patients that N-glycosylation of breast cancer cells undergoing metastasis occurs in a metastatic site–specific manner, supporting the clinical importance of high-mannose, fucosylated, and complex N-glycans as future diagnostic markers and therapeutic targets in metastatic BC. FUNDING. NIH grants R01CA213428, R01CA213492, R01CA264901, T32CA193145, Dutch Province Limburg “LINK”, European Union ERA-NET TRANSCAN2-643638.

Context: In the early months of the response to the coronavirus disease 2019 (COVID-19) pandemic, the Johns Hopkins University School of Medicine (JHUSOM) (Baltimore, Maryland) leadership reached out to faculty to develop and implement virtual clinical clerkships after all in-person medical student clinical experiences were suspended. Objective: To develop and implement a digital slide-based virtual surgical pathology clinical elective to meet the demand for meaningful and robust virtual clinical electives in response to the temporary suspension of in-person clinical rotations at JHUSOM. Design: The Virtual Surgical Pathology (VSP) elective was modeled after the in-person surgical pathology elective, to include virtual previewing and sign-out with standardized cases supplemented by synchronous and asynchronous pathology educational content. Results: Validation of existing web communications technology and slide scanning systems was performed by feasibility testing. Curriculum development included drafting of course objectives and syllabus, Blackboard course site design, electronic-lecture creation, communications with JHUSOM leadership, scheduling, and slide curation. Subjectively, the weekly schedule averaged 35–40 hours to asynchronous, synchronous, and independent content, approximately 10–11 hours of which were synchronous. As of February 2021, VSP has hosted 35 JHUSOM and eight non-JHUSOM students, who have provided positive subjective and objective course feedback. Conclusions: The Johns Hopkins VSP elective provided meaningful clinical experience to 43 students in a time of immense online education need. Added benefits of implementing VSP included increased medical student exposure to pathology as a medical specialty and demonstration of how digital slides have the potential to improve standardization of the pathology clerkship curriculum.

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