Proteogenomic analysis of enriched HGSOC tumor epithelium identifies prognostic signatures and therapeutic vulnerabilities.

We performed a deep proteogenomic analysis of bulk tumor and laser microdissection enriched tumor cell populations from high-grade serous ovarian cancer (HGSOC) tissue specimens spanning a broad spectrum of purity. We identified patients with longer progression-free survival had increased immune-related signatures and validated proteins correlating with tumor-infiltrating lymphocytes in 65 tumors from an independent cohort of HGSOC patients, as well as with overall survival in an additional 126 HGSOC patient cohort. We identified that homologous recombination deficient (HRD) tumors are enriched in pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells are uniquely sensitive to BMI-1 inhibition.

Mapping three-dimensional intratumor proteomic heterogeneity in uterine serous carcinoma by multiregion microsampling.

BACKGROUND: Although uterine serous carcinoma (USC) represents a small proportion of all uterine cancer cases, patients with this aggressive subtype typically have high rates of chemotherapy resistance and disease recurrence that collectively result in a disproportionately high death rate. The goal of this study was to provide a deeper view of the tumor microenvironment of this poorly characterized uterine cancer variant through multi-region microsampling and quantitative proteomics. METHODS: Tumor epithelium, tumor-involved stroma, and whole "bulk" tissue were harvested by laser microdissection (LMD) from spatially resolved levels from nine USC patient tumor specimens and underwent proteomic analysis by mass spectrometry and reverse phase protein arrays, as well as transcriptomic analysis by RNA-sequencing for one patient's tumor. RESULTS: LMD enriched cell subpopulations demonstrated varying degrees of relatedness, indicating substantial intratumor heterogeneity emphasizing the necessity for enrichment of cellular subpopulations prior to molecular analysis. Known prognostic biomarkers were quantified with stable levels in both LMD enriched tumor and stroma, which were shown to be highly variable in bulk tissue. These USC data were further used in a comparative analysis with a data generated from another serous gynecologic malignancy, high grade serous ovarian carcinoma, and have been added to our publicly available data analysis tool, the Heterogeneity Analysis Portal ( https://lmdomics.org/ ). CONCLUSIONS: Here we identified extensive three-dimensional heterogeneity within the USC tumor microenvironment, with disease-relevant biomarkers present in both the tumor and the stroma. These data underscore the critical need for upfront enrichment of cellular subpopulations from tissue specimens for spatial proteogenomic analysis.

The murine metastatic microenvironment of experimental brain metastases of breast cancer differs by host age in vivo: a proteomic study.

Breast cancer in young patients is known to exhibit more aggressive biological behavior and is associated with a less favorable prognosis than the same disease in older patients, owing in part to an increased incidence of brain metastases. The mechanistic explanations behind these findings remain poorly understood. We recently reported that young mice, in comparison to older mice, developed significantly greater brain metastases in four mouse models of triple-negative and luminal B breast cancer. Here we have performed a quantitative mass spectrometry-based proteomic analysis to identify proteins potentially contributing to age-related disparities in the development of breast cancer brain metastases. Using a mouse hematogenous model of brain-tropic triple-negative breast cancer (MDA-MB-231BR), we harvested subpopulations of tumor metastases, the tumor-adjacent metastatic microenvironment, and uninvolved brain tissues via laser microdissection followed by quantitative proteomic analysis using high resolution mass spectrometry to characterize differentially abundant proteins potentially contributing to age-dependent rates of brain metastasis. Pathway analysis revealed significant alterations in signaling pathways, particularly in the metastatic microenvironment, modulating tumorigenesis, metabolic processes, inflammation, and neuronal signaling. Tenascin C (TNC) was significantly elevated in all laser microdissection (LMD) enriched compartments harvested from young mice relative to older hosts, which was validated and confirmed by immunoblot analysis of whole brain lysates. Additional in vitro studies including migration and wound-healing assays demonstrated TNC as a positive regulator of tumor cell migration. These results provide important new insights regarding microenvironmental factors, including TNC, as mechanisms contributing to the increased brain cancer metastatic phenotype observed in young breast cancer patients.

Brain proteomic atlas of alcohol use disorder in adult males.

Alcohol use disorder (AUD) affects transcriptomic, epigenetic and proteomic expression in several organs, including the brain. There has not been a comprehensive analysis of altered protein abundance focusing on the multiple brain regions that undergo neuroadaptations occurring in AUD. We performed a quantitative proteomic analysis using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of human postmortem tissue from brain regions that play key roles in the development and maintenance of AUD, the amygdala (AMG), hippocampus (HIPP), hypothalamus (HYP), nucleus accumbens (NAc), prefrontal cortex (PFC) and ventral tegmental area (VTA). Brain tissues were from adult males with AUD (n = 11) and matched controls (n = 16). Across the two groups, there were >6000 proteins quantified with differential protein abundance in AUD compared to controls in each of the six brain regions. The region with the greatest number of differentially expressed proteins was the AMG, followed by the HYP. Pathways associated with differentially expressed proteins between groups (fold change > 1.5 and LIMMA p < 0.01) were analyzed by Ingenuity Pathway Analysis (IPA). In the AMG, adrenergic, opioid, oxytocin, GABA receptor and cytokine pathways were among the most enriched. In the HYP, dopaminergic signaling pathways were the most enriched. Proteins with differential abundance in AUD highlight potential therapeutic targets such as oxytocin, CSNK1D (PF-670462), GABAB receptor and opioid receptors and may lead to the identification of other potential targets. These results improve our understanding of the molecular alterations of AUD across brain regions that are associated with the development and maintenance of AUD. Proteomic data from this study is publicly available at www.lmdomics.org/AUDBrainProteomeAtlas/ .

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

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.

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

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.

Relative Effects of Age, Race, and Stage on Mortality in Gestational Choriocarcinoma.

OBJECTIVE: Gestational choriocarcinoma is a malignant form of gestational trophoblastic disease that usually arises after a molar pregnancy, but may follow any antecedent pregnancy. Investigations in this rare cancer are limited. We evaluated the prognostic effects of age, race, and stage in choriocarcinomas diagnosed for 4 decades. METHODS: Patients diagnosed as having gestational choriocarcinoma between 1973 and 2014 from the Surveillance, Epidemiology, and End Results program were eligible. Relationships with overall survival and cancer-specific survival were evaluated using log-rank testing and Cox modeling. Multivariate analyses included adjustments for age, race, and stage. RESULTS: There were 947 patients with choriocarcinoma including 403 non-Hispanic white (NHW) patients, 473 with distant stage, and 142 who died. Median age at diagnosis was 25 years for non-Hispanic black (NHB) patients and 35 years for Asian/Pacific Islanders (API) compared with 29 years for NHW patients (P = 0.0001). Five-year overall survival varied between 82% and 92% when diagnosed at the age of at least 40 years compared with less than 20 years (P < 0.0001), and from 85% to 95% in patients with distant vs local disease (P < 0.0001), respectively. Multivariate analysis demonstrated that age, race, and stage were independent predictors of mortality. Risk of death increased incrementally in patients diagnosed at 20 to 39 years of age (adjusted hazard ratio [aHR], 3.87; 95% confidence interval [CI], 1.69-8.86; P = 0.001) and at least 40 years of age (aHR, 7.18; 95% CI, 2.95-17.49; P < 0.0001) compared with 20 years or younger. Non-Hispanic black patients were the only racial group at higher risk of death compared with NHW patients (aHR, 1.86; 95% CI, 1.22-2.82; P < 0.004). Distant vs local disease added an additional risk of death (aHR, 2.43; 95% CI, 1.57-3.75; P < 0.0001) over that attributable to age at diagnosis and NHB race. Similar relationships to cancer-specific survival were also observed (P < 0.05). CONCLUSIONS: Most patients with choriocarcinoma have excellent prognosis. However, NHB patients and patients who are diagnosed at the age of at least 20 years or have distant stage have significantly worse mortality.

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

BACKGROUND: The objective of this study was to identify molecular alterations associated with disease outcomes for white and black patients with endometrioid endometrial cancer (EEC). METHODS: EEC samples from black (n = 17) and white patients (n = 13) were analyzed by proteomics (liquid chromatography-tandem mass spectrometry) and transcriptomics (RNA-seq). Coordinate alterations were validated with RNA-seq data from black (n = 49) and white patients (n = 216). Concordantly altered candidates were further tested for associations with race-specific progression-free survival (PFS) in black (n = 64) or white patients (n = 267) via univariate and multivariate Cox regression modeling and log-rank testing. RESULTS: Discovery analyses revealed significantly altered candidate proteins and transcripts between black and white patients, suggesting modulation of tumor cell viability in black patients and cell death signaling in black and white patients. Eighty-nine candidates were validated as altered between these patient cohorts, and a subset significantly correlated with differential PFS. White-specific PFS candidates included serpin family A member 4 (SERPINA4; hazard ratio [HR], 0.89; Wald P value = .02), integrin subunit α3 (ITGA3; HR, 0.76; P = .03), and Bet1 Golgi vesicular membrane trafficking protein like (BET1L; HR, 0.48; P = .04). Black-specific PFS candidates included family with sequence similarity 228 member B (FAM228B; HR, 0.13; P = .001) and HEAT repeat containing 6 (HEATR6; HR, 4.94; P = .047). Several candidates were also associated with overall survival (SERPINA4 and ITGA3) as well as PFS independent of disease stage, grade and myometrial invasion (SERPINA4, BET1L and FAM228B). CONCLUSIONS: This study has identified and validated molecular alterations in tumors from black and white EEC patients, including candidates significantly associated with altered disease outcomes within these patient cohorts. Cancer 2017;123:4004-12. © 2017 American Cancer Society.