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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.

PDF - 8738K, Figure S1 H&E staining from three high-grade serous (A, B, C) and three endometrioid carcinomas (D, E, F)

Supplemental material and methods

PDF - 59K, Legend for Supplementary Figure 1.

PDF - 100K, Table S1 Demographics from the OTTA cohorts representing the test set and excluded cases Table S2 Antibodies, details of immunohistochemical protocols and scoring cut-off Table S3 Training set revision of the COSP model - Areas under the curve (AUC) by histology and model Table S4 Test for heterogeneity for marker expression between training and testing set Table S5 Pairwise agreement of histological types in the testing set by classification method for A_COSP Table S6 Univariate Cox model in the testing set comparing A_COPS and TB_COSPv2. Table S7 Five-year survival estimates for four different type assignments as defined in the text within histological types Table S8 Demographics of endometrioid carcinomas in the test set classified by different methods Table S9 Prediction of type in test set among cases with unclear original diagnosis (N=71).

The impact of liver metastases on immune checkpoint-inhibitor effectiveness in patients with solid-tumor malignancies has been the focus of several recent clinical and translational studies. We review the literature describing the immune functions of the liver and particularly the mechanistic observations in these studies. The initial clinical observation was that pembrolizumab appeared to be much less effective in melanoma and non–small cell lung cancer (NSCLC) patients with liver metastasis. Subsequently other clinical studies have extended and reported similar findings with programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) inhibitors in many cancers. Two recent translational studies in animal models have dissected the mechanism of this systemic immune suppression. In both studies CD11b+ suppressive macrophages generated by liver metastasis in a two-site MC38 model appear to delete CD8+ T cells in a FasL-dependent manner. In addition, regulatory T-cell (Treg) activation was observed and contributed to the distal immunosuppression. Finally, we discuss some of the interventions reported to address liver immune suppression, such as radiation therapy, combination checkpoint blockade, and Treg depletion.

Introduction Position dependent obstructive sleep apnea (PdOSA) is highly prevalent, but the implication of PdOSA in cardiovascular (CV) outcomes has not been studied. We hypothesized that PdOSA is associated with lower risk of future CV events than those without PdOSA. Methods We included patients who underwent diagnostic polysomnography (PSG) at a single academic center. The study included patients with ≥2 hours of total sleep time, 30 minutes in both supine and non-supine sleep, and OSA (apnea hypopnea index [AHI]≥5/hr). We defined PdOSA as either POSA (overall AHI≥5/hr accompanied by supine AHI (sAHI) at least twice as high as non-supine AHI (ns-AHI)), or ePOSA (POSA criteria + ns-AHI < 5/hr (i.e., normalized)). Blood pressure (BP) was derived by averaging multiple measures of clinic BP within 1 year of PSG. All patients were followed up to 11 years to identify incident CV events (including acute coronary syndrome, stroke, and new heart failure). We examined the association of PdOSA with CV events with a logistic regression, adjusting for age, sex, race, BMI, systolic BP, and OSA severity (mild [AHI 5-15] vs. moderate to severe [≥15]). Results A total of 1750 patients (mean age: 54 years, female 60%) were included. POSA and ePOSA were present in 47% and 23% of the cohort. Patients with either POSA or ePOSA were less obese and had less severe OSA compared with those without POSA and ePOSA. Patients with ePOSA had lower BP (systolic BP 128.8 vs 131.2 mmHg, p=0.003) than those without ePOSA. Over a median 8.3 years of follow-up, a total of 340 CV events occurred. In multivariable analysis, both POSA and ePOSA were associated with lower odds of future CV events (OR 0.8, 95% CI [0.64-0.99], p=0.044; OR 0.69 [0.52-0.89], p=0.0061, respectively). Analysis stratified by OSA severity showed that the ePOSA association was present only in those with mild OSA (OR 0.67 [0.48-0.94], p=0.021). Conclusion Among patients with OSA, PdOSA is associated with more favorable CV outcomes than those without PdOSA. The benefit appears to be limited to those with mild OSA. Future studies should compare the CV risks between PdOSA patients and those without OSA. Support (if any)

Ovarian carcinoma histotypes are distinct diseases with variable clinical outcomes and response to treatment. There is a need for new subtype‐specific treatment modalities, especially for women with widespread and chemo‐resistant disease. Stimulator of interferon genes (STING) is a part of the cGAS–STING pathway that mediates innate immune defence against infectious DNA‐containing pathogens and also detects tumour‐derived DNA and generates intrinsic antitumour immunity. The STING signalling pathway is suppressed by several mechanisms in a variety of malignant diseases and, in some cancers that may be a requirement for cellular transformation. The aim of this study was to use immunohistochemistry to evaluate STING protein expression across normal tissue, paratubal and ovarian cysts, and ovarian tumour histotypes including ovarian carcinomas. Herein, we show that the fallopian tube ciliated cells express STING protein, whereas the secretory cells are negative. STING expression differs among ovarian cancer histotypes; low‐grade serous ovarian carcinomas and serous borderline tumours have uniform high STING expression, while high‐grade serous and endometrioid carcinomas have heterogeneous expression, and clear cell and mucinous carcinomas show low expression. As low‐grade serous carcinomas are known to be genomically stable and typically lack a prominent host immune response, the consistently high STING expression is unexpected. High STING expression may reflect pathway activation or histogenesis and the mechanisms may be different in different ovarian carcinoma histotypes. Further studies are needed to determine whether the STING signalling pathway is active and whether these tumours would be candidates for therapeutic interventions that trigger innate immunity activation.

Clear cell ovarian carcinoma (CCOC) is the second most common subtype of epithelial ovarian carcinoma. Late-stage CCOC is not responsive to gold-standard chemotherapy and results in suboptimal outcomes for patients. In-depth molecular insight is urgently needed to stratify the disease and drive therapeutic development. We conducted global proteomics for 192 cases of CCOC and compared these with other epithelial ovarian carcinoma subtypes. Our results showed distinct proteomic differences in CCOC compared with other epithelial ovarian cancer subtypes including alterations in lipid and purine metabolism pathways. Furthermore, we report potential clinically significant proteomic subgroups within CCOC, suggesting the biologic plausibility of stratified treatment for this cancer. Taken together, our results provide a comprehensive understanding of the CCOC proteomic landscape to facilitate future understanding and research of this disease. © 2022 The Pathological Society of Great Britain and Ireland.

Multiple newer medications benefit patients with heart failure with reduced ejection fraction (HFrEF). While these therapies benefit the broad population with HFrEF, the efficacy and safety of these therapies have been less well characterized in patients with significant comorbidities. Common comorbidities of high interest in heart failure (HF) include diabetes mellitus, chronic kidney disease (CKD), atrial fibrillation, and obesity, and each has potential implications for clinical management. As the burden of comorbidities increases in HF populations, risk-benefit assessments of HF therapies in the context of different comorbidities are increasingly relevant for clinical practice. This review summarizes data regarding the core HFrEF therapies in the context of comorbidities, with specific attention to sodium-glucose cotransporter 2 inhibitors, sacubitril/valsartan, mineralocorticoid receptor antagonists (MRAs), and beta-blockers. In general, studies support consistent treatment effects with regard to clinical outcome benefits in the presence of comorbidities. Likewise, safety profiles are relatively consistent irrespective of comorbidities, with the exception of heightened risk of hyperkalemia with MRA therapy in patients with severe CKD. In conclusion, while HF management is complex in the context of multiple comorbidities, the totality of evidence strongly supports guideline-directed medical therapies as foundational for improving outcomes in these high-risk patients.

Endometrial carcinoma (EC) is the most common gynecologic cancer in North America. A clinically molecular classifier called ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) was developed based on the TCGA classification. p53abn EC is the most aggressive of the four ProMisE prognostic molecular subtypes, responsible for 50-70% of EC mortality. Therapeutic advances are urgently needed to improve outcomes for these patients. This research focuses on determining the significance of HER2 and CCNE1 amplifications in p53abn EC to identify therapeutic opportunities for this aggressive cancer. Tumor DNA extracted from cores of 203 formalin-fixed paraffin-embedded (FFPE) p53abn EC were Shallow Whole Genome (sWGS) sequenced. sWGS is a high-throughput technology designed to achieve genome-wide genetic variation accurately and cost-effectively. Raw data were aligned and treated to correct CG content and discard low-quality reads. Copy number alterations were called considering 5 to 9 copies as amplification and more than 9 as high-level amplification. Amplification data was later compared to HER2 and CCNE1 whole-section immunohistochemistry (IHC). Gene amplification is one of the major molecular pathways that can drive tumorigenesis, and many amplified genes have emerged as biological markers and therapeutic targets. HER2 amplification has an important prognostic and therapeutic implications in breast and gastric cancer. It is also seen in EC, arising as a possible targeted treatment opportunity for p53abn EC using anti-HER2 drugs. CCNE1 amplifications have been reported in platinum resistance aggressive ovarian and endometrial cancers and is known to be a biomarker to predict responsive to low-dose WEE1i-ATRi. Amplifications in CCNE1 were identified in 13% (26/203) of the cohort while the IHC overexpression (2/3+) was recorded in 64% of cases. HER2 amplifications were observed in 11% (22/203) of p53abn ECs compared to the IHC overexpression (2/3+) detected in 21%. Significant intratumor heterogeneity was identified. We found discrepancy between the techniques, with IHC detecting a significantly greater proportion of p53abn ECs overexpressing HER2 and CCNE1 than amplification found on sWGS. This phenomenon may be explained by the intratumor heterogeneity that was identified during the IHC scoring. The IHC was performed in whole sections while cores were used to extract DNA for the sWGS, which may have missed the gene amplification areas. Although there are some limitations, sWGS is a relatively inexpensive assay that can be performed on FFPE, and it is able to obtain whole genome sequence at a very low coverage (between 0.4x and 1x) with over 99% accuracy for copy number calls. In recent years sWGS have been used efficiently for copy number profiling tumors and for amplification detection thus having the potential to be implemented in clinical practice to guide targeted therapy. Citation Format: Juliana Sobral de Barros, Amy Jamieson, Dawn Cochrane, Maxwell Douglas, Janine Senz, Blake Gilks, Christine Chow, Shelby Thornton, Jessica N. McAlpine, David G. Huntsman. Identifying gene amplifications in p53 abnormal endometrial carcinomas by shallow whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 223.

Objective. The development of effective cancer treatments depends on the availability of cell lines that faithfully recapitulate the cancer in question. This study definitively re-assigns the histologic identities of two ovarian cancer cell lines, COV434 (originally described as a granulosa cell tumour) and TOV-112D (originally described as grade 3 endometrioid carcinoma), both of which were recently suggested to represent small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), based on their shared gene expression profiles and sensitivity to EZH2 inhibitors. Methods. For COV434 and TOV-112D, we re-reviewed the original pathology slides and obtained clinical followup on the patients, when available, and performed immunohistochemistry for SMARCA4, SMARCA2 and additional diagnostic markers on the original formalin-fixed, paraffin-embedded (FFPE) clinical material, when available. For COV434, we further performed whole exome sequencing and validated SMARCA4 mutations by Sanger sequencing. We studied the growth of the cell lines at baseline and upon re-expression of SMARCA4 in vitro for both cell lines and evaluated the serum calcium levels in vivo upon injection into immunodeficient mice for COV434 cells.Results. The available morphological, immunohistochemical, genetic, and clinical features indicate COV434 is derived from SCCOHT, and TOV-112D is a dedifferentiated carcinoma. Transplantation of COV434 into mice leads to increased serum calcium level. Re-expression of SMARCA4 in either COV434 and TOV-112D cells suppressed their growth dramatically. Conclusions. COV434 represents a bona fide SCCOHT cell line. TOV-112D is a dedifferentiated ovarian carcinoma cell line.(c) 2020 Elsevier Inc. All rights reserved.

Introduction: Multiplex biomarker analysis of cytological body fluid specimens is often used to assist cytologists in distiguishing metastatic cancer cells from reactive mesothelial cells. However, evaluating biomarker expression visually may be challenging, especially when the cells of interest are scant. Deep-learning algorithms (DLAs) may be able to assist cytologists in analyzing multiple biomarker expression at the single cell level in the multiplex fluorescence imaging (MFI) setting. This preliminary study was performed to test the feasibility of using DLAs to identify immunofluorescence-stained metastatic adenocarcinoma cells in body fluid cytology samples. Methods: A DLA was developed to analyze MFI-stained cells in body fluid cytological samples. A total of 41 pleural fluid samples, comprising of 20 positives and 21 negatives, were retrospectively collected. Multiplex immunofluorescence labeling for MOC31, BerEP4, and calretinin, were performed on cell block sections, and results were analyzed by manual analysis (manual MFI) and DLA analysis (MFI-DLA) independently. Results: All cases with positive original cytological diagnoses showed positive results either by manual MFI or MFI-DLA, but 2 of the 14 (14.3%) original cytologically negative cases had rare cells with positive MOC31 and/or BerEP4 staining in addition to calretinin. Manual MFI analysis and MFI-DLA showed 100% concordance. Conclusion: MFI combined with DLA provides a potential tool to assist in cytological diagnosis of metastatic malignancy in body fluid samples. Larger studies are warranted to test the clinical validity of the approach.

The mixture of epithelial and stromal components in pancreatic ductal adenocarcinoma (PDAC) may confound sequencing-based studies of tumor gene expression. Virtual microdissection has been suggested as a bioinformatics approach to segment the aforementioned components, and subsequent prognostic gene sets have emerged from this research. We examined the prognostic signature from the epithelial gene set of one such study using laser capture microdissected (LCM) epithelial samples. We also examined this gene set in matched stromal samples to determine whether prognostic findings were specific to the epithelium. LCM samples from 48 long-term and 48 short-term PDAC survivors were obtained. The resultant epithelial and stromal components were subjected to direct mRNA quantification using a 49 gene published PDAC classifier. Component-specific unsupervised hierarchical clustering was used to derive groups and survival differences were quantified. Immunohistochemical validation of particular genes was performed in an independent cohort. Clustering in the epithelial component yielded prognostic differences in univariable analysis (P = .02), but those differences were not significant when controlled for other clinicopathologic covariates (P = .06). Clustering in the stromal component yielded prognostic differences that persisted in the presence of other clinicopathologic covariates (P = .0005). Validation of selected genes in the epithelium (KRT6A-negative prognostic [P = .004]) and stroma (LY6D-improved prognostic [P = .01] and CTSV-negative prognostic [P = .0002]) demonstrated statistical independence in multivariable analysis. Although the genes used in this study were originally identified as being representative of the epithelial component of PDAC, their expression in the stroma appears to provide additional information that may aid in improved prognostication.

We have recently encountered p53 immunohistochemical (IHC) patterns in human papillomavirus (HPV)-associated carcinomas of the gynecologic tract, which were confused with absent (null) or overexpression TP53 mutational staining. We therefore evaluated p53 and p16 IHC in 25 squamous cell carcinomas (SCC) (16 vulva, 4 Bartholin's gland, and 5 cervix), 20 endocervical adenocarcinomas (EDAC), 14 high-grade squamous intraepithelial lesions (HSIL), 2 adenocarcinoma in situ (AIS), all of which exhibited morphologic features of HPV. Only cases showing diffuse/strong block-like p16 staining were included for further study. All EDACs underwent TP53 sequencing and HPV in situ hybridization (ISH) was performed in selected cases. p53 IHC staining fell into two main patterns. The most common was designated as "markedly reduced (null-like)" (absence or significantly attenuated staining in >70% of cells), which could be confused with true null mutational pattern. This was present in 14/25 (56%) SCCs, 7/14 (50%) HSILs, and 18/20 (90%) EDACs. The second notable pattern was "mid-epithelial (basal sparing)" (distinct absence of staining in basal cells juxtaposed with strong staining in parabasal cells), seen in 10/25 (40%) SCC, 7/14 (50%) HSIL, and none of the EDACs. There was scattered weak to moderate p53 staining (conventional wild type) in 1/25 (4%) SCC and 2/20 (10%) EDAC. No cases showed strong/diffuse overexpression. One EDAC had a TP53 missense mutation and exhibited "markedly reduced (null-like)" staining. HPV ISH revealed an inverse relationship with p53, cells positive for HPV mRNA were negative for p53. Knowledge of these patterns can help pathologists avoid misinterpreting p53 status in the setting of HPVA cancers.

Endometrial carcinoma, the most common gynaecological cancer, develops from endometrial epithelium which is composed of secretory and ciliated cells. Pathologic classification is unreliable and there is a need for prognostic tools. We used single cell sequencing to study organoid model systems derived from normal endometrial endometrium to discover novel markers specific for endometrial ciliated or secretory cells. A marker of secretory cells (MPST) and several markers of ciliated cells (FAM92B, WDR16, and DYDC2) were validated by immunohistochemistry on organoids and tissue sections. We performed single cell sequencing on endometrial and ovarian tumours and found both secretory-like and ciliated-like tumour cells. We found that ciliated cell markers (DYDC2, CTH, FOXJ1, and p73) and the secretory cell marker MPST were expressed in endometrial tumours and positively correlated with disease-specific and overall survival of endometrial cancer patients. These findings suggest that expression of differentiation markers in tumours correlates with less aggressive disease, as would be expected for tumours that retain differentiation capacity, albeit cryptic in the case of ciliated cells. These markers could be used to improve the risk stratification of endometrial cancer patients, thereby improving their management. We further assessed whether consideration of MPST expression could refine the ProMiSE molecular classification system for endometrial tumours. We found that higher expression levels of MPST could be used to refine stratification of three of the four ProMiSE molecular subgroups, and that any level of MPST expression was able to significantly refine risk stratification of the copy number high subgroup which has the worst prognosis. Taken together, this shows that single cell sequencing of putative cells of origin has the potential to uncover novel biomarkers that could be used to guide management of cancers. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Decades of advancements in immuno-oncology have enabled the development of current immunotherapies, which provide long-term treatment responses in certain metastatic cancer patients. However, cures remain infrequent, and most patients ultimately succumb to treatment-refractory metastatic disease. Recent insights suggest that tumors at certain organ sites exhibit distinctive response patterns to immunotherapy and can even reduce antitumor immunity within anatomically distant tumors, suggesting the activation of tissue-specific immune tolerogenic mechanisms in some cases of therapy resistance. Specialized immune cells known as regulatory T cells (Tregs) are present within all tissues in the body and coordinate the suppression of excessive immune activation to curb autoimmunity and maintain immune homeostasis. Despite the high volume of research on Tregs, the findings have failed to reconcile tissue-specific Treg functions in organs, such as tolerance, tissue repair, and regeneration, with their suppression of local and systemic tumor immunity in the context of immunotherapy resistance. To improve the understanding of how the tissue-specific functions of Tregs impact cancer immunotherapy, we review the specialized role of Tregs in clinically common and challenging organ sites of cancer metastasis, highlight research that describes Treg impacts on tissue-specific and systemic immune regulation in the context of immunotherapy, and summarize ongoing work reporting clinically feasible strategies that combine the specific targeting of Tregs with systemic cancer immunotherapy. Improved knowledge of Tregs in the framework of their tissue-specific biology and clinical sites of organ metastasis will enable more precise targeting of immunotherapy and have profound implications for treating patients with metastatic cancer.

Tubo-ovarian transitional cell carcinoma (TCC) is grouped with high-grade serous carcinoma (HGSC) in the current World Health Organization classification. TCC is associated with BRCA mutations and a better prognosis compared to HGSC. Previous papers examining the immunohistochemical features of TCC have studied limited numbers of samples. No marker reflecting the biological difference between TCC and HGSC is known. We collected a large cohort of TCC to determine whether TCC and HGSC could be distinguished by immunohistochemistry. A tissue microarray was built from 89 TCC and a control cohort of 232 conventional HGSC. Immunohistochemistry was performed, scored and statistically analyzed for routine markers of HGSC and urothelial tumors: PAX8, WT1, p53, p16, ER, p63 and GATA3. Using scoring cutoffs commonly employed in clinical practice, the immunohistochemical profile of TCC was indistinguishable from HGSC for all markers. However, more detailed scoring criteria revealed statistically significant differences between the two groups of tumors with respect to ER, PAX8 and WT1. HGSC showed more diffuse and intense staining for PAX8 (P=0.004 and P=0.001, respectively) and WT1 (P=0.002 and P=0.002, respectively); conversely, TCC showed more intense staining for ER (P=0.007). TCC and HGSC therefore show subtle differences in their immunohistochemical profiles which might reflect underlying (epi)genetic differences. Further studies using proteomic analysis will focus on the identification of differentially expressed proteins that might serve as markers of transitional cell carcinoma-like differentiation, which could help explain biological differences between TCC and HGSC and might identify other cases of HGSC with a better prognosis.

Room temperature Suzuki cross‐coupling polymerization of aryl dibromides/diiodides with aryldiboronic acids/acid esters with t‐Bu₃P‐coordinated 2‐phenylaniline‐based palladacycle complex, [2′‐(amino‐kN)[1,1′‐biphenyl]‐2‐yl‐kC]chloro(tri‐t‐butylphosphine)palladium, as a general precatalyst is described. Such room temperature Suzuki cross‐coupling polymerization is achieved by employing six equivalents or more of the base and affords polymers within an hour, with the yields and the molecular weights in general comparable to or higher than reported results that required higher reaction temperature and/or longer polymerization time. Our study provides a general catalyst system for the room temperature Suzuki cross‐coupling polymerization of aryl dibromides/diiodides with aryldiboronic acids/acid esters and paves the road for the investigation of employing other monodentate ligand‐coordinated palladacycle complexes including other electron‐rich monophosphine‐coordinated ones for room temperature cross‐coupling polymerizations. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 1606–1611

Architects, engineers, developers, contractors, building facility managers, and relevant governmental officers are the key players in developing and managing low-carbon buildings and communities, whose decisions and actions largely determine the level of carbon emission from them. This study is intended to investigate how these key players perform in current practices and to identify any areas for improvement. This paper presents a case study conducted in Ontario, Canada's province that can best represent the leading development industry within a developed country. A significant number of key players were surveyed through questionnaires and interviews. Key findings include: (1) Although low-carbon emission is a target for the Ontario construction industry, design considerations are still highly dependent on financial limitations and more attention is required towards technical factors and (2) knowledge, skills and tools are not sufficient to support improvements to low-carbon design and development. This paper also discusses ongoing research activities and anticipated outcomes.

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.

Background: Drones have great potential to speed the delivery of AEDs in the critical first few minutes of OHCA. However, it is unclear whether bystanders can balance high-quality cardiopulmonary resuscitation (CPR) with AED deployment. The 2015 AHA CPR guidelines recommend a chest compression (cc) rate of 100-120/minute, cc depth of 50-60mm, and cc fraction of >60%. Method: We performed mock cardiac arrest simulations using bystander volunteers, including simulated 911 call, telephone-assisted dispatcher CPR instructions, bystander CPR, drone-delivered AED, and AED application. CPR performance was recorded by a Laerdal Resusci Anne Quality Feedback System and compared between two groups of participants: recent CPR training (2 years) or no CPR training. Prior data had shown CPR skill degradation after 2 years. Chi-squared tests compared demographics; T-tests compared age and CPR performance data. Results: Between 9/2019-3/2020, 5 simulations were conducted with 51 participants. The mean age was 39.7 years, 56.9% were female, and 78.4% had a college or graduate degree. Racial/ethnic makeup consisted of 64.7% White, 15.7% African-American, 15.7% Asian, and 11.8% Hispanic. 41.2% had recent CPR training (n=21); 58.8% had remote CPR training (n=19) or no CPR training (n=11). There were no differences in demographics by CPR training groups. Participants with recent CPR training had shorter time from CPR initiation to AED shock delivery (3:45 vs. 4:14 [min:sec], p=0.01) and a trend toward higher percent of time with cc depth (77.4% vs 50.4%, p=0.11) and higher cc fraction (46.8% vs 42.9 %, p=0.12). There were no differences for percent of time with cc rate or CPR recoil. Conclusion: Overall, CPR quality was low regardless of prior CPR training status. Those recently trained had shorter resuscitation time and appeared to have better CPR performance. Realization of a drone AED networks may require novel CPR programs focused on high-quality CPR.

Mesonephric carcinomas (MEs) and female adnexal tumors of probable Wolffian origin (FATWO) are derived from embryologic remnants of Wolffian/mesonephric ducts. Mesonephric-like carcinomas (MLCs) show identical morphology to ME of the cervix but occur in the uterus and ovary without convincing mesonephric remnants. ME, MLC, and FATWO are challenging to diagnose due to their morphologic similarities to Müllerian/paramesonephric tumors, contributing to a lack of evidence-based and tumor-specific treatments. We performed whole-proteomic analysis on 9 ME/MLC and 56 endometrial carcinomas (ECs) to identify potential diagnostic biomarkers. Although there were no convincing differences between ME and MLC, 543 proteins showed increased expression in ME/MLC relative to EC. From these proteins, euchromatic histone lysine methyltransferase 2 (EHMT2), glutathione S-transferase Mu 3 (GSTM3), eukaryotic translation elongation factor 1 alpha 2 (EEF1A2), and glycogen synthase kinase 3 beta were identified as putative biomarkers. Immunohistochemistry was performed on these candidates and GATA3 in 14 ME/MLC, 8 FATWO, 155 EC, and normal tissues. Of the candidates, only GATA3 and EHMT2 were highly expressed in mesonephric remnants and mesonephric-derived male tissues. GATA3 had the highest sensitivity and specificity for ME/MLC versus EC (93% and 99%) but was absent in FATWO. EHMT2 was 100% sensitive for ME/MLCFATWO but was not specific (65%). Similarly, EEF1A2 was reasonably sensitive to ME/MLC (92%) and FATWO (88%) but was the least specific (38%). GSTM3 performed intermediately (sensitivity for ME/MLC and FATWO: 83% and 38%, respectively; specificity 67%). Although GATA3 remained the best diagnostic biomarker for ME/MLC, we have identified EHMT2, EEF1A2, and GSTM3 as proteins of interest in these cancers. FATWO's cell of origin is uncertain and remains an area for future research.

Background PTEN loss is a putative driver in histotypes of ovarian cancer (high-grade serous (HGSOC), endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous (LGSOC)). We aimed to characterise PTEN expression as a biomarker in epithelial ovarian cancer in a large population-based study. Methods Tumours from 5400 patients from a multicentre observational, prospective cohort study of the Ovarian Tumour Tissue Analysis Consortium were used to evaluate associations between immunohistochemical PTEN patterns and overall survival time, age, stage, grade, residual tumour, CD8+ tumour-infiltrating lymphocytes (TIL) counts, expression of oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) by means of Cox proportional hazard models and generalised Cochran–Mantel–Haenszel tests. Results Downregulation of cytoplasmic PTEN expression was most frequent in ENOC (most frequently in younger patients; p value = 0.0001) and CCOC and was associated with longer overall survival in HGSOC (hazard ratio: 0.78, 95% CI: 0.65–0.94, p value = 0.022). PTEN expression was associated with ER, PR and AR expression (p values: 0.0008, 0.062 and 0.0002, respectively) in HGSOC and with lower CD8 counts in CCOC (p value p value = 0.019) and associated with higher CD8 counts (p value = 0.0016). Conclusions PTEN loss is a frequent driver in ovarian carcinoma associating distinctly with expression of hormonal receptors and CD8+ TIL counts in HGSOC and CCOC histotypes.

We have recently encountered p53 immunohistochemical (IHC) patterns in human papillomavirus (HPV)-associated carcinomas of the gynecologic tract, which were confused with absent (null) or overexpression TP53 mutational staining. We therefore evaluated p53 and p16 IHC in 25 squamous cell carcinomas (SCC) (16 vulva, 4 Bartholin's gland, and 5 cervix), 20 endocervical adenocarcinomas (EDAC), 14 high-grade squamous intraepithelial lesions (HSIL), 2 adenocarcinoma in situ (AIS), all of which exhibited morphologic features of HPV. Only cases showing diffuse/strong block-like p16 staining were included for further study. All EDACs underwent TP53 sequencing and HPV in situ hybridization (ISH) was performed in selected cases. p53 IHC staining fell into two main patterns. The most common was designated as "markedly reduced (null-like)" (absence or significantly attenuated staining in70% of cells), which could be confused with true null mutational pattern. This was present in 14/25 (56%) SCCs, 7/14 (50%) HSILs, and 18/20 (90%) EDACs. The second notable pattern was "mid-epithelial (basal sparing)" (distinct absence of staining in basal cells juxtaposed with strong staining in parabasal cells), seen in 10/25 (40%) SCC, 7/14 (50%) HSIL, and none of the EDACs. There was scattered weak to moderate p53 staining (conventional wild type) in 1/25 (4%) SCC and 2/20 (10%) EDAC. No cases showed strong/diffuse overexpression. One EDAC had a TP53 missense mutation and exhibited "markedly reduced (null-like)" staining. HPV ISH revealed an inverse relationship with p53, cells positive for HPV mRNA were negative for p53. Knowledge of these patterns can help pathologists avoid misinterpreting p53 status in the setting of HPVA cancers.

Cancer cell mechanotype changes are newly recognized cancer phenotypic events, whereas metastatic cancer cells show decreased cell stiffness and increased deformability relative to normal cells. To further examine how cell mechanotype changes in early stages of cancer transformation and progression, anin vitromulti-step human urothelial cell carcinogenic model was used to measure cellular Young’s modulus, deformability, and transit time using single-cell atomic force microscopy, microfluidic-based deformability cytometry, and quantitative deformability cytometry, respectively. Measurable cell mechanotype changes of stiffness, deformability, and cell transit time occur early in the transformation process. As cells progress from normal, to preinvasive, to invasive cells, Young’s modulus of stiffness decreases and deformability increases gradually. These changes were confirmed in three-dimensional cultured microtumor masses and urine exfoliated cells directly from patients. Using gene screening and proteomics approaches, we found that the main molecular pathway implicated in cell mechanotype changes appears to be epithelial to mesenchymal transition.

Dedifferentiated endometrial carcinoma (DDEC) is a rare but highly aggressive type of endometrial cancer, in which an undifferentiated carcinoma arises from a low-grade endometrioid endometrial carcinoma. The low-grade component is often eclipsed, likely due to an outgrowth of the undifferentiated component, and the tumor may appear as a pure undifferentiated endometrial carcinoma (UEC). We and others have recently identified inactivating mutations of SMARCA4, SMARCB1 or ARID1B, subunits of the SWI/SNF chromatin-remodeling complex, that are unique to the undifferentiated component and are present in a large portion of DDEC and UEC. However, the understanding of whether and how these mutations drive cancer progression and histologic dedifferentiation is hindered by lack of cell line models of DDEC or UEC. Here, we established the first UEC cell line, VOA1066, which is highly tumorigenic in vivo. This cell line has a stable genome with very few somatic mutations, which do include inactivating mutations of ARID1A and ARID1B (2 mutations each), and a heterozygous hotspot DICER1 mutation in its RNase IIIb domain. Immunohistochemistry staining confirmed the loss of ARID1B, but ARID1A staining was retained due to the presence of a truncating non-functional ARID1A protein. The heterozygous DICER1 hotspot mutation has little effect on microRNA biogenesis. No additional DICER1 hotspot mutations have been identified in a cohort of 33 primary tumors. Therefore, we have established the first UEC cell line with dual inactivation of both ARID1A and ARID1B as the main genomic feature. This cell line will be useful for studying the roles of ARID1A and ARID1B mutations in the development of UEC.

Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)–neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)–based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor–mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.

Pancreatic neuroendocrine neoplasms (PNENs) are biologically and clinically heterogeneous. Here, we use a multi-omics approach to uncover the molecular factors underlying this heterogeneity. Transcriptomic analysis of 84 PNEN specimens, drawn from two cohorts, is substantiated with proteomic profiling and identifies four subgroups: Proliferative, PDX1-high, Alpha cell-like and Stromal/Mesenchymal. The Proliferative subgroup, consisting of both well- and poorly differentiated specimens, is associated with inferior overall survival probability. The PDX1-high and Alpha cell-like subgroups partially resemble previously described subtypes, and we further uncover distinctive metabolism-related features in the Alpha cell-like subgroup. The Stromal/Mesenchymal subgroup exhibits molecular characteristics of YAP1/WWTR1(TAZ) activation suggestive of Hippo signaling pathway involvement in PNENs. Whole-exome sequencing reveals subgroup-enriched mutational differences, supported by activity inference analysis, and identifies hypermorphic proto-oncogene variants in 14.3% of sequenced PNENs. Our study reveals differences in cellular signaling axes that provide potential directions for PNEN patient stratification and treatment strategies.

Molecular subclassification of endometrial carcinoma (EC) with Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) identifies four subtypes (POLE mutant, mismatch repair deficient (MMR-D), p53 wild-type (wt), and p53 abnormal). The aim of this study was to evaluate additional EC biomarkers in the context of these subtypes. Tissue microarrays encompassing 460 previously characterized ECs were assessed for L1-cell adhesion molecule (L1CAM), progesterone receptor (PR), estrogen receptor alpha (ER), stathmin and PTEN, by immunohistochemistry (IHC). Associations with clinicopathological parameters, molecular subtype, and outcomes were determined. 413 ECs (75% endometrioid, >15% serous) had complete data. L1CAM overexpression was found in 16%, associated with older age, lower body mass index (BMI), advanced stage, grade 3 (97%), non-endometrioid histology (84%), deep myometrial invasion, lymphovascular space invasion (LVSI), and ER-negative, PR-negative status. Tumours overexpressing L1CAM were associated with poor outcomes [hazard ratio (HR) (95% confidence interval (CI)) 3.35 (2.10-5.23) for disease-specific survival (DSS), p

Primary ovarian mucinous tumors can be difficult to distinguish from metastatic gastrointestinal neoplasms by histology alone. The expected immunoprofile of a suspected metastatic lower gastrointestinal tumor is CK7-/CK20+/CDX2+/PAX8-. This study assesses the addition of a novel marker SATB2, to improve the diagnostic algorithm. A test cohort included 155 ovarian mucinous tumors (105 carcinomas and 50 borderline tumors) and 230 primary lower gastrointestinal neoplasms (123 colorectal adenocarcinomas and 107 appendiceal neoplasms). All cases were assessed for SATB2, PAX8 CK7, CK20, and CDX2 expression on tissue microarrays. Expression was scored in a 3-tier system as absent, focal (1-50% of tumor cells) and diffuse ( >50% of tumor cells) and then categorized into either absent/present or nondiffuse/diffuse. SATB2 and PAX8 expression was further evaluated in ovarian tumors from an international cohort of 2876 patients (expansion cohort, including 159 mucinous carcinomas and 46 borderline mucinous tumors). The highest accuracy of an individual marker in distinguishing lower gastrointestinal from ovarian mucinous tumors was CK7 (91.7%, nondiffuse/diffuse cut-off) followed by SATB2 (88.8%, present/absent cut-off). The most effective combination was CK7 and SATB2 with accuracy of 95.3% using the 3-tier interpretation, absent/focal/diffuse. This combination outperformed the standard clinical set of CK7, CK20 and CDX2 (87.5%). Re-evaluation of outlier cases confirmed ovarian origin for all but one case. The accuracy of SATB2 was confirmed in the expansion cohort (91.5%). SATB2 expression was also detected in 15% of ovarian endometrioid carcinoma but less than 5% of other ovarian histotypes. A simple two marker combination of CK7 and SATB2 can distinguish lower gastrointestinal from ovarian primary mucinous tumors with greater than 95% accuracy. PAX8 and CDX2 have value as second-line markers. The utility of CK20 in this setting is low and this warrants replacement of this marker with SATB2 in clinical practice.

The estrogen receptor (ER) is a key predictive biomarker in the treatment of breast cancer. There is uncertainty regarding the use of hormonal therapy in the setting of weakly positive ER by immunohistochemistry (IHC). We report intrinsic subtype classification on a cohort of ER weakly positive early-stage breast cancers. Consecutive cases of breast cancer treated by primary surgical resection were retrospectively identified from 4 centers that engage in routine external proficiency testing for breast biomarkers. ER-negative (Allred 0 and 2) and ER weakly positive (Allred 3-5) cases were included. Gene expression profiling was performed using qRT-PCR. Intrinsic subtype prediction was made based upon the PAM50 gene expression signature. 148 cases were included in the series: 60 cases originally diagnosed as ER weakly positive and 88 ER negative. Of the cases originally assessed as ER weakly positive, only 6 (10 %) were confirmed to be of luminal subtype by gene expression profiling; the remaining 90 % of cases were classified as basal-like or HER2-enriched subtypes. This was not significantly different than the fraction of luminal cases identified in the IHC ER-negative cohort (5 (5 %) luminal, 83(95 %) non-luminal). Recurrence-free, and overall, survival rates were similar in both groups (p = 0.4 and 0.5, respectively) despite adjuvant hormonal therapy prescribed in the majority (59 %) of weakly positive ER cases. Weak ER expression by IHC is a poor correlate of luminal subtype in invasive breast cancer. In the setting of highly sensitive and robust IHC methodology, cutoffs for ER status determination and subsequent systemic therapy should be revisited.