industrialized-artificial-intelligence-guided-laser-microdissection

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

Uniformed Services University and Walter Reed National Military Medical Center

The Henry M. Jackson Foundation for the Advancement of Military Medicine

Uniformed Services University

The objective of this study was to identify molecular alterations associated with disease outcomes for white and black patients with endometrioid endometrial cancer (EEC).

Race-specific molecular alterations correlate with differential outcomes for black and white endometrioid endometrial cancer patients.

Solid tumour malignancies comprise a highly variable admixture of tumour and non-tumour cellular populations, forming a complex cellular ecosystem and tumour microenvironment. This tumour heterogeneity is not incidental, and is known to correlate with poor patient prognosis for many cancer types. Indeed, non-malignant cell populations, such as vascular endothelial and immune cells, are known to play key roles supporting and, in some cases, driving aggressive tumour biology, and represent targets of emerging therapeutics, such as antiangiogenesis and immune checkpoint inhibitors. The biochemical interplay between these cellular populations and how they contribute to molecular tumour heterogeneity remains enigmatic, particularly from the perspective of the tumour proteome. This review focuses on recent advances in proteomic methods, namely imaging mass spectrometry, single-cell proteomic techniques, and preanalytical sample processing, that are uniquely positioned to enable detailed analysis of discrete cellular populations within tumours to improve our understanding of tumour proteomic heterogeneity. This review further emphasizes the opportunity afforded by the application of these techniques to the analysis of tumour heterogeneity in formalin-fixed paraffin-embedded archival tumour tissues, as these represent an invaluable resource for retrospective analyses that is now routinely accessible, owing to recent technological and methodological advances in tumour tissue proteomics. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Recent advances and opportunities in proteomic analyses of tumour heterogeneity.

Efficient identification of neoantigen-specific T-cell responses in epithelial ovarian cancer (EOC) remains a challenge. Existing investigations of spontaneous T-cell response to tumor neoepitope in EOC have taken the approach of comprehensive screening all neoantigen candidates, with a validation rate of 0.5-2%.

Efficient identification of neoantigen-specific T-cell responses in advanced human ovarian cancer.

Preoperative use of metformin in obese women with endometrioid endometrial cancer (EEC) reduces tumor proliferation and inhibits the mammalian target of rapamycin pathway, though is only effective in select cases. This study sought to identify a predictive and/or pharmacodynamic proteomic signature of metformin response to tailor its pharmacologic use. Matched pre- and post-metformin-treated tumor tissues from a recently completed preoperative window trial of metformin in EEC patients (ClinicalTrials.gov: NCT01911247) were analyzed by mass spectrometry (MS)-based proteomic and immunohistochemical analyses. Jupiter microtubule-associated homolog 1 (JPT1) was significantly elevated in metformin responders (n = 13) vs nonresponders (n = 7), and found to decrease in abundance in metformin responders following treatment; observations that were verified by immunohistochemical staining for JPT1. Metformin response and loss of JPT1 were assessed in RL95-2 and ACI-181 endometrial cancer (EC) cell lines. We further identified that silencing of JPT1 abundance does not alter cellular response to metformin or basal cell proliferation, but that JPT1 abundance does decrease in response to metformin treatment in RL95-2 and ACI-181 EC cell lines. These data suggest that JPT1 represents a predictive and pharmacodynamic biomarker of metformin response that, if validated in larger patient populations, may enable preoperative EEC patient stratification to metformin treatment and the ability to monitor patient response.

Jupiter microtubule-associated homolog 1 (JPT1): A predictive and pharmacodynamic biomarker of metformin response in endometrial cancers.

To investigate racial disparities in uterine carcinosarcoma (UCS) and ovarian carcinosarcoma (OCS) in Commission on Cancer®-accredited facilities.

Racial disparities in uterine and ovarian carcinosarcoma: A population-based analysis of treatment and survival.

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 lower-energy IR laser methods.

The impact of ultraviolet- and infrared-based laser microdissection technology on phosphoprotein detection in the laser microdissection-reverse phase protein array workflow.

The diversity and heterogeneity within high-grade serous ovarian cancer (HGSC), which is the most lethal gynecologic malignancy, is not well understood. Here, we perform comprehensive multi-platform omics analyses, including integrated analysis, and immune monitoring on primary and metastatic sites from highly clinically annotated HGSC samples based on a laparoscopic triage algorithm from patients who underwent complete gross resection (R0) or received neoadjuvant chemotherapy (NACT) with excellent or poor response. We identify significant distinct molecular abnormalities and cellular changes and immune cell repertoire alterations between the groups, including a higher rate of NF1 copy number loss, and reduced chromothripsis-like patterns, higher levels of strong-binding neoantigens, and a higher number of infiltrated T cells in the R0 versus the NACT groups.

Molecular Analysis of Clinically Defined Subsets of High-Grade Serous Ovarian Cancer.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality.

Standardization and harmonization of distributed multi-center proteotype analysis supporting precision medicine studies.

Enriched tumor epithelium, tumor-associated stroma, and whole tissue were collected by laser microdissection from thin sections across spatially separated levels of ten high-grade serous ovarian carcinomas (HGSOCs) and analyzed by mass spectrometry, reverse phase protein arrays, and RNA sequencing. Unsupervised analyses of protein abundance data revealed independent clustering of an enriched stroma and enriched tumor epithelium, with whole tumor tissue clustering driven by overall tumor "purity." Comparing these data to previously defined prognostic HGSOC molecular subtypes revealed protein and transcript expression from tumor epithelium correlated with the differentiated subtype, whereas stromal proteins (and transcripts) correlated with the mesenchymal subtype. Protein and transcript abundance in the tumor epithelium and stroma exhibited decreased correlation in samples collected just hundreds of microns apart. These data reveal substantial tumor microenvironment protein heterogeneity that directly bears on prognostic signatures, biomarker discovery, and cancer pathophysiology and underscore the need to enrich cellular subpopulations for expression profiling.

Extensive three-dimensional intratumor proteomic heterogeneity revealed by multiregion sampling in high-grade serous ovarian tumor specimens.

Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with disease pathogenesis. Nonsynonymous single-nucleotide polymorphisms encoding missense substitutions within tryptic peptides exhibiting high allele frequencies in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs), were prioritized from 1000 genomes.  analysis identified that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R = 0.99). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues combined resulted in the quantitation of 62 pAIMs that correlate with patient race and genotype-confirmed ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. pAIMs will support generalized proteoancestry assessment as well as efforts investigating the impact of ancestry on the human proteome and how this relates to the pathogenesis of uterine neoplasms.

Peptide ancestry informative markers in uterine neoplasms from women of European, African, and Asian ancestry.

While  and  mutations are known to confer the largest risk of breast cancer and ovarian cancer, the incomplete penetrance of the mutations and the substantial variability in age at cancer onset among carriers suggest additional factors modifying the risk of cancer in  mutation carriers. To identify genetic modifiers of , we carried out a whole-genome sequencing study of 66 ovarian cancer patients that were enriched with  carriers, followed by validation using data from the Pan-Cancer Analysis of Whole Genomes Consortium. We found , a master regulator of mitochondrial biogenesis and function, to be highly mutated in  carriers, and patients with both  and  mutations were diagnosed with breast or ovarian cancer at significantly younger ages, while the mutation status of each gene alone did not significantly associate with age of onset. Our study suggests  as a possible  modifier gene. Upon further validation, this finding can help improve cancer risk prediction and provide personalized preventive care for  carriers.

Article	Year
Race-specific molecular alterations correlate with differential outcomes for black and white endometrioid endometrial cancer patients. Cancer\| PubMed ID: 28654152	2017
Recent advances and opportunities in proteomic analyses of tumour heterogeneity. The Journal of pathology\| PubMed ID: 29344964	2018
Efficient identification of neoantigen-specific T-cell responses in advanced human ovarian cancer. Journal for immunotherapy of cancer\| PubMed ID: 31221207	2019
Jupiter microtubule-associated homolog 1 (JPT1): A predictive and pharmacodynamic biomarker of metformin response in endometrial cancers. Cancer medicine\| PubMed ID: 31808620	2020
Racial disparities in uterine and ovarian carcinosarcoma: A population-based analysis of treatment and survival. Gynecologic oncology\| PubMed ID: 32029291	2020
The impact of ultraviolet- and infrared-based laser microdissection technology on phosphoprotein detection in the laser microdissection-reverse phase protein array workflow. Clinical proteomics\| PubMed ID: 32165870	2020
Molecular Analysis of Clinically Defined Subsets of High-Grade Serous Ovarian Cancer. Cell reports\| PubMed ID: 32294438	2020
Standardization and harmonization of distributed multi-center proteotype analysis supporting precision medicine studies. Nature communications\| PubMed ID: 33067419	2020
Extensive three-dimensional intratumor proteomic heterogeneity revealed by multiregion sampling in high-grade serous ovarian tumor specimens. iScience\| PubMed ID: 34278265	2021
Peptide ancestry informative markers in uterine neoplasms from women of European, African, and Asian ancestry. iScience\| PubMed ID: 35036865	2022
Whole-Genome Sequencing Identifies as a Putative Modifier of Cancer Risk in Mutation Carriers. Cancers\| PubMed ID: 35625955	2022

Nicholas W. Bateman

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