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IC50 curves for ADI-PEG20 in ovarian cancer cell lines. A, ASS1-deficient ovarian cancer cell lines and B, ASS1-proficient ovarian cancer cell lines.

Table containing proteins differentially expressed in at least two rarer ovarian cancer subtypes when compared to HGSC.

Purpose:Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.Experimental Design:We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT.Results:Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo.Conclusions:Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

Supplemental material and methods

There is a need for an animal model that closely parallels human cochlea gestational development. This study aims to document porcine inner ear anatomy, and in vitro porcine derived inner ear cell culture characteristics. Twenty-four temporal bone were harvested from 12 adult pigs (Sus scrofa). Six were formalin fixed and their maximal diameters were measured. The cochlea duct length was determined by the insertion length of a Nucleus 22 cochlear implant in two bones. Four formalin fixed bones were sectioned for histology. Cochlear and vestibular tissues were harvested from non-fixed bones, cultured and characterized at different passages (P). Gene and protein expression of multipotent stem/progenitor (Nestin and Sox2), inner ear hair (Myosin VIIa, Prestin) and supporting (Cytokeratin 18 and Vimentin) cell markers were determined. The porcine cochlea was a 3.5 turn spiral. There was a separate vestibular compartment. The cochlear mean maximal diameter and height was 7.99 and 3.77 mm, respectively. Sphere forming cells were identified on phase-contrast microscopy. The relative mRNA expression levels of KRT18, MYO7A and SLC26A5 were significantly positively correlated in cochlear cultures; and MYO7A and SLC26A5; SOX2 and KRT18; NES and SLC26A5 genes were positively correlated in vestibular cultures (p = .037, Spearman correlation [τ] = .900). Inner ear sensory and stem cell characteristics persist in passaged porcine inner ear cells. Further work is required to establish the usefulness of porcine inner ear cell cultures to the study of human inner ear disorders.

Purpose: Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes. Experimental Design: We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT. Results: Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo. Conclusions: Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell-cell adhesion molecule E-cadherin. Loss of E-cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single-cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage-specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation-dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The human inner ear compared with that of other mammalian species is very complex. Although the mouse’s cochlea is frequently studied the mouse’s inner ear continues to develop postnatally whilst the human inner ear is fully developed by the third month of gestation which leads one to question the applicability of findings based on research on mice to human regenerative therapies. Here, we report a novel in vitro culture of adult porcine (Sus scrofa) inner ear cells developed from post-mortem labyrinth specimens. Anatomical findings based on maximal transverse and vertical axial diameters and the length of the cochlear duct suggest that the pig’s cochlea is similar to the human cochlea. In vitro cultures of porcine cochlear and vestibular cells showed the persistence of both inner ear hair cell (HC), supporting cell (SC) and stem/progenitor cell characteristics across passages up to 6 based on scanning electron microscopy, fluorescence immunocytochemistry and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings showed that porcine cochlear and vestibular epithelia maintained multipotent stem/progenitor cell populations into adulthood although their regenerative capacities differed across the passages. The development of a viable and reproducible method to culture porcine inner ear cells provides an important investigative tool that can be utilized to study and evaluate the pathophysiological causes and cellular consequences of human inner ear disorders.

The associations between airway eosinophilia, measured in sputum or peripheral blood, and acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are inconsistent. We therefore aimed to determine the association between eosinophilia in bronchoalveolar lavage (BAL) fluid and AECOPD in a clinical cohort. We analyzed differential cell counts from baseline BAL fluid in participants in the DISARM clinical trial (Clinicaltrials.gov #NCT02833480) and classified participants by the presence or absence of BAL eosinophilia (>1% of total leukocytes). We determined the association between BAL eosinophilia and AECOPD over 1 year of follow-up using negative binomial regression and Cox proportional hazards test. N = 63 participants were randomized, and N = 57 had BAL differential cell counts available. Participants with BAL eosinophilia (N = 21) had a significantly increased rate of acute exacerbations (unadjusted incidence rate ratio (IRR) 2.0, p = 0.048; adjusted IRR 2.24, p = 0.04) and a trend toward greater probability of acute exacerbation (unadjusted hazard ratio (HR) 1.74, p = 0.13; adjusted HR 2.3, p = 0.1) in the year of follow-up compared to participants without BAL eosinophilia (N = 36). These associations were not observed for BAL neutrophilia (N = 41 participants), BAL lymphocytosis (N = 27 participants) or peripheral blood eosinophilia at various threshold definitions (2%, N = 37; 3%, N = 27; 4%, N = 16). BAL may therefore be a sensitive marker of eosinophilic inflammation in the distal lung and may be of benefit for risk stratification or biomarker-guided therapy in COPD.

Background: Ovarian cancers are the most common gynecologic malignancies. Low grade serous ovarian carcinoma (LGSOC) is a rare tumor, accounting for ~2000 cases diagnosed every year in North America. Most of LGSOCs are characterized by high fatality rates over the long term, with only 20% of women surviving 10 years after diagnosis, due suboptimal response to current chemotherapies. Understanding the molecular events is crucial for developing better early detection strategies and more informed therapeutic options. LGSOC harbors a relatively stable genome, with common activating mutations in BRAF, KRAS and NRAS. Recently, NRAS mutations (Q61R) were found to co-exist with EIF1AX mutations (G8E) in LGOSC, and the two mutated proteins functionally cooperate. Increasing histological and gene expression evidence suggest that the cell of origin of LGSOC is in the Fallopian tube. Low incidence of this disease means it is poorly understood, and the resulting lack of available models further limits the study of underlying mechanisms. We therefore propose to use organoid cultures. These consist of 3D multicellular units that resemble in vitro a tissue or organ of body, both structurally and functionally. Objective: to elucidate molecular events underpinning LGSOC, specifically how NRAS(Q61R) and EIF1AX (G8E) mutations co-operate to drive early stages of tumorigenesis, with organoid system and single-cell RNA sequencing (scRNA-seq) technologies. Method: To reflect genetic background and cell of origin of LGSOC, NRAS Q61R and EIF1AX G8E mutant proteins were overexpressed via lentiviral transduction in organoid cultures of normal human Fallopian tubes. After allowing organoids to establish, 2 weeks after transduction gene expression alterations were resolved with scRNA-seq. Histology of organoids were assessed for histomorphological signs of transformation. Patient-derived tumor organoids (PDTOs) were also cultured to assess how well our LGSOC-modelling organoids (LMOs) recapitulate the histological features of patient tumours. Result: LMOs showed cytologic signs of transformation such as increased nuclear/cytoplasmic ratio, prominent nucleoli, and cellular pleomorphism. Papillary structures, a major histologic characteristic of LGSOC tumor were also observed in LMOs. PDTOs showed similar cytological features and organization as LMOs. From scRNA-seq, we identified genes up-regulated in double-mutant compared to single-mutant organoids such as CA125 and TACSTD2. CA125 is one of the earliest identified biomarkers for ovarian cancer and has remained to be the most useful serum marker despite limited sensitivity and specificity; whereas TACSTD2 overexpression has been found to correlate with a chemo-resistant, aggressive malignant phenotype. Conclusion and future directions: Organoid culture and scRNA-seq is a powerful duo in studying early tumorigenesis events. We established a novel model system of LGSOC by introducing common co-occurring mutations into normal Fallopian tube tissues. Our model recapitulates to a large extent of LGSOC histology. Genes upregulated in double mutants included well-characterized biomarker (CA125) and a potential biomarker or therapeutic target (TACSTD2). Our work will be crucial for developing early detection strategies and targeted treatment options. Citation Format: Joyce Yu Han Zhang, Dawn Cochrane, Kieran Campbell, Minh Bui, Germain Ho, Cindy Shen, Winnie Yang, Clara Salamanca, Genny Trigo, David G. Huntsman. Investigating mutation co-operativity in early tumorigenesis of low-grade serous ovarian carcinoma with organoid model system and single-cell RNA sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4899.

Both clear cell ovarian carcinoma (CCOC) and endometrioid ovarian carcinoma (ENOC) are associated with ovarian endometriotic cysts, which is believed to be their precursor lesion. However, genomic evidence is lacking which could explain how these two clinically distinct histotypes of ovarian cancer arise from the same precursor lesion. We therefore hypothesized that these cancers arise from distinct cells of origin within endometrial tissue. Global proteomic analysis of ovarian cancer histotypes identified CTH as a marker for CCOC. We further found that CTH is highly expressed in the ciliated cells of endometrium (both ectopic endometrium and endometriosis), and of the fallopian tube, with little expression in the secretory cells. We also find that other ciliated cell markers are expressed in CCOC, whereas endometrial secretory cell markers are expressed in ENOC. We propose a new model of CCOC and ENOC histogenesis wherein ENOC is derived from cells of secretory cell lineage whereas CCOC is derived from cells of ciliated cell lineage. However, it remains unclear how external factors in the endometriotic cyst cooperate with cell of origin and mutation to promote cancer formation. To study normal tissue biology, we are using organoid cultures of normal endometrium. As ciliated cells of the endometrium are rare, and we have a particular interest in determining whether they have other features that may link them to CCOC, we used a Notch inhibitor, DBZ, to force ciliated cell differentiation in the organoids. We observed a dramatic shift in the cellular content with DBZ treamtment towards ciliated cells. We performed single cell RNA sequencing (scRNAseq) on these endometrial organoids. In the normal endometrial organoids, cells were predominantly a secretory phenotype, characterized by high ESR1 expression, with a minor ciliated cell population. The ciliated cell population expressed several known ciliated markers (FOXJ1 and DNAH12). Upon treatment with DBZ, the number of secretory cells decreases dramatically and two populations of cells emerge which have ciliated cell markers. The larger ciliated cell population is similar to the ciliated cells in the untreated organoids. The smaller ciliated cell population in the DBZ treated organoids express some ciliated cell markers, but clusters separately from normal ciliated cells. We believe this population may reprepsent an intermediary population, which has not fully differentiated. Interestingly, this population expresses the cytokine IL6, while the normal ciliated cell population does not. This is of note because CCOCs express more IL6 compared to the other histotypes. Therefore, we can speculate that this intermediary ciliated cell population may represent cells from which CCOC arise, however more testing is needed. In the future, the scRNAseq data from organoids will be compared to CCOC and ENOC tumors to determine whether the tumors resemble more closely one population of normal cells. We will use viral transduction to introduce mutations into the organoid cultures to determine whether specific mutation leads to transformation towards a CCOC or ENOC-like phenotype. These studies will enable us to tease apart the relative contribution of mutation, microenvironment and the cell of origin to promote tumor formation. Citation Format: Dawn R Cochrane, Basile Tessier-Cloutier, Germain Ho, Kieran Campbell, Evan Gibbard, Katherine M Lawrence, Tayyebeh Nazeran, Anthony N. Karnezis, Clara Salamanca, Angela S Cheng, Jessica N McAlpine, Sohrab Shah, Lien N Hoang, C Blake Gilks and David G Huntsman. CELL OF ORIGIN, MUTATION AND MICROENVIRONMENT: MODELING EARLY EVENTS OF ENDOMETRIOSIS ASSOCIATED CANCERS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-020.