The proteome of clear cell ovarian carcinoma.

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.

Reduction of Duration of Antibiotic Therapy for Suspected Early-Onset Sepsis in Late-Preterm and Term Newborns After Implementation of a Procalcitonin-Guided Algorithm: A Population-Based Study in Central Switzerland.

Background: Suspected early-onset sepsis (EOS) is the main reason for antibiotic therapy at the start of life. Prolonged antibiotic therapy for culture-negative sepsis is often reported. Antibiotic stewardship is mandatory due to the potential negative effects of unnecessary antibiotics. Procalcitonin (PCT)-guided therapy is one possible strategy with published evidence to shorten antibiotic therapy. The aim of this study is to analyze the feasibility and the performance of the published PCT-algorithm in the clinical setting without study support. Methods: This is a retrospective, population-based study regarding duration of antibiotic therapy for suspected EOS in Central Switzerland between 2014 and 2018. All neonates >34 0/7 weeks of gestational age started on antibiotic therapy for suspected EOS within the first 3 calendar days of life were included. The Procalcitonin-guided algorithm according to the NeoPInS study was used as strategy to determine duration of antibiotic therapy. Results: In a population-based cohort of 35,642 life born neonates, the duration of antibiotic therapy of 879 neonates (2.5% of the cohort) treated for suspected EOS was 4 calendar days (median, IQR 2-5). We observed a statistically significant reduction from 4 (median, IQR 3-6) to 3 calendar days (median, IQR 2-4) from 2014 to 2018. Duration of antibiotic therapy was independent of gestational age (late-preterm vs. term neonates), of the presence of risk factors or clinical signs, but dependent on the presence of abnormal laboratory measurements (C-reactive protein > 10 mg/l or leukocytopenia <5 Giga/l) before start of antibiotic therapy (p < 0.01). Conclusions: PCT-guided therapy using the NeoPInS algorithm is feasible and may lead to reduced duration of antibiotic therapy for suspected EOS as reported in the original study. We observed a learning curve to the new algorithm which may be explained as change process. The use of biomarker to guide duration of antibiotic therapy for suspected EOS may have unintended consequences with prolongation of antibiotic therapy in some cases.

Comparison of p53 immunohistochemical staining in differentiated vulvar intraepithelial neoplasia (dVIN) with that in inflammatory dermatoses and benign squamous lesions in the vulva.

AIMS: Differentiated vulvar intraepithelial neoplasia (dVIN), the precursor lesion to human papillomavirus-independent vulvar squamous cell carcinoma (VSCC), can be difficult to distinguish from vulvar inflammatory dermatoses. Our goal was to determine if p53 could be a useful biomarker for dVIN, by characterizing p53 percentage, intensity and patterns of staining in dVIN and its histological mimics. METHODS AND RESULTS: We studied p53 immunohistochemical staining patterns in 16 dVIN cases and 46 vulvar non-neoplastic squamous lesions [12 lichen sclerosus (LS); seven lichen simplex chronicus; three lichen planus (LP); six psoriasis; 13 spongiotic dermatitis (SPO); and five candidiasis]. dVIN cases were adjacent to a p16-negative invasive VSCC in resection specimens. All dVIN cases showed null-type or moderate to strong uniform p53 staining in >70% of basal cells, with moderate to strong continuous parabasal staining extending to two-thirds of the epidermis. This was in contrast to weak or weak to moderate patchy p53 staining in the majority of other lesions. Moderate to strong and increased basal p53 staining (≥70%) was also observed in a subset of LS cases (5/12, 42%), LP cases (1/3, 33%), and SPO cases (36%, 4/11); however, in all categories, this was limited to the basal layer, and any staining in the parabasal layers was patchy. CONCLUSION: Strong and uniform p53 staining of basal cells, extending into the parabasal layers, and a complete absence of staining (null type) is useful in distinguishing dVIN from other mimics in the vulva. p53 staining of lesser intensity or quantity, particularly basal overexpression only, overlaps with that in vulvar inflammatory lesions.

Arginine Depletion Therapy with ADI-PEG20 Limits Tumor Growth in Argininosuccinate Synthase-Deficient Ovarian Cancer, Including Small-Cell Carcinoma of the Ovary, Hypercalcemic Type.

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.

SWI/SNF Complex Mutations in Gynecologic Cancers: Molecular Mechanisms and Models.

The SWI/SNF (mating type SWItch/Sucrose NonFermentable) chromatin remodeling complexes interact with histones and transcription factors to modulate chromatin structure and control gene expression. These evolutionarily conserved multisubunit protein complexes are involved in regulating many biological functions, such as differentiation and cell proliferation. Genomic studies have revealed frequent mutations of genes encoding multiple subunits of the SWI/SNF complexes in a wide spectrum of cancer types, including gynecologic cancers. These SWI/SNF mutations occur at different stages of tumor development and are restricted to unique histologic types of gynecologic cancers. Thus, SWI/SNF mutations have to function in the appropriate tissue and cell context to promote gynecologic cancer initiation and progression. In this review, we summarize the current knowledge of SWI/SNF mutations in the development of gynecologic cancers to provide insights into both molecular pathogenesis and possible treatment implications for these diseases.

Napsin-A and AMACR are Superior to HNF-1ß in Distinguishing Between Mesonephric Carcinomas and Clear Cell Carcinomas of the Gynecologic Tract.

Mesonephric carcinoma is a rare gynecologic neoplasm commonly mistaken for clear cell carcinoma, because of their overlapping morphologic features. Both tumors are negative for estrogen receptor and p16, magnifying this diagnostic dilemma. Recently, hepatocyte nuclear factor-1 beta (HNF-1ß), a marker for clear cell carcinoma, has also been shown to be positive in mesonephric carcinomas. Other more recent markers for clear cell carcinoma, however, such as Napsin-A and alpha-methylacyl-CoA racemase (AMACR), have not yet been studied in mesonephric carcinomas. Here we examine HNF-1ß, AMACR, and Napsin-A immunohistochemistry in 18 mesonephric and 55 endometrial/cervical clear cell carcinomas. HNF-1ß was considered positive if nuclear staining was present in ≥70% of cells and at least moderate intensity; for Napsin-A and AMACR, any cytoplasmic staining was considered positive (≥1%). H-scores were determined by multiplying the intensity score by proportion score. HNF-1ß was positive in a substantial portion of mesonephric carcinomas (9/18, 50%; H-score 98) and clear cell carcinomas (34/55, 62%; H-score 163) and did not distinguish between the 2 entities (specificity, 50%; P-value of H-score=0.08). Napsin-A and AMACR expression was significantly higher in clear cell [43/55 (78%) and 41/55 (75%), respectively] than mesonephric carcinomas [4/18 (22%) and 4/18 (22%) respectively], and helpful in this differential (specificity: 78% and 78%; P<0.05 for both). When Napsin-A and AMACR staining were seen in mesonephric carcinomas, staining was focal (≤5%), whereas staining in clear cell carcinomas was patchy/diffuse. In summary, Napsin-A and AMACR are helpful in distinguishing mesonephric carcinomas from clear cell carcinomas of the female genital tract, but HNF-1ß is not.

Use of Immunohistochemical Markers (HNF-1ß, Napsin A, ER, CTH, and ASS1) to Distinguish Endometrial Clear Cell Carcinoma From Its Morphologic Mimics Including Arias-Stella Reaction.

The diagnosis of clear cell (CC) carcinoma of the endometrium can be challenging, especially when endometrioid (EC) and serous (SC) endometrial cancers exhibit nonspecific clear cell changes, in carcinomas with mixed histology and in the setting of Arias-Stella reaction (ASR). In this study, classic CC immunohistochemical markers (Napsin A, HNF-1ß, and ER) and 2 recent novel markers, cystathionine gamma-lyase (CTH) and arginosuccinate synthase (ASS1), are assessed for their utility in distinguishing CC from its morphologic mimics. Tissue microarrays containing 64 CC, 128 EC, 5 EC with clear cell change, 16 SC, 5 mixed carcinomas, and 11 whole ASR sections were stained, with 12 additional examples of ASR stained subsequently. A cutoff of 70% and moderate intensity were used for HNF-1ß, 80% of cells and strong intensity were used for CTH, and any staining was considered positive for the remaining markers. For differentiating CC from pure EC and SC, HNF-1ß, Napsin A, and CTH all performed well. HNF-1ß had higher specificity (99.3% vs. 95.1%) but lower sensitivity (55.8% vs. 73.1%) compared with Napsin A. CTH did not substantially outperform HNF- 1ß or Napsin A (sensitivity 51.9%, specificity 99.3%). ASS1 and ER were not helpful (specificities of 60.1% and 22.6%). For differentiating CC from ASR, HNF-1ß, Napsin A, and CTH stained a large proportion of ASR and were not useful. However, ER positivity and ASS1 negativity were helpful for identifying ASR (specificity 88.2% and 95.1%, respectively). EC with clear cell changes exhibited immunohistochemical patterns similar to pure EC (HNF-1ß-, ER+, and CTH-). No markers were useful in confirming the CC components in mixed carcinomas.

A novel microchip-based imaged CIEF-MS system for comprehensive characterization and identification of biopharmaceutical charge variants.

A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS-compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF-MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real-time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N-linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF-MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF-MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.

Critical questions in ovarian cancer research and treatment: Report of an American Association for Cancer Research Special Conference.

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease.

Clear cell carcinomas of the ovary and kidney: clarity through genomics.

Clear cell ovarian carcinoma (CCOC) and clear cell renal cell carcinoma (ccRCC) both feature clear cytoplasm, owing to the accumulation of cytoplasmic glycogen. Genomic studies have demonstrated several mutational similarities between these two diseases, including frequent alterations in the chromatin remodelling SWI-SNF and cellular proliferation phosphoinositide 3-kinase-mammalian target of rapamycin pathways, as well as a shared hypoxia-like mRNA expression signature. Although many targeted treatment options have been approved for advanced-stage ccRCC, CCOC patients are still treated with conventional platinum and taxane chemotherapy, to which they are resistant. To determine the extent of similarity between these malignancies, we performed unsupervised clustering of mRNA expression data from these cancers. This review highlights the similarities and differences between these two clear cell carcinomas to facilitate knowledge translation within future research efforts. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Characteristics and outcomes of Canadian MD/PhD program graduates: a cross-sectional survey.

BACKGROUND: Combined MD/PhD programs provide a structured path for physician-scientist training, but assessment of their success within Canada is limited by a lack of quantitative data. We collected outcomes data for graduates of Canadian MD/PhD programs. METHODS: We developed and implemented a Web-based survey consisting of 41 questions designed to collect outcomes data for Canadian MD/PhD program alumni from 8 Canadian universities who had graduated before September 2015. Respondents were categorized into 2 groups according to whether they had or had not completed all training. RESULTS: Of the 186 eligible alumni of MD/PhD programs, 139 (74.7%) completed the survey. A total of 136/138 respondents (98.6%) had completed or were currently completing residency training, and 66/80 (82%) had completed at least 1 postgraduate fellowship. Most (58 [83%]) of the 70 respondents who had completed all training were appointed as faculty at academic institutions, and 37 (53%) had been principal investigators on at least 1 recent funded project. Among the 58 respondents appointed at academic institutions, 44/57 (77%) dedicated at least 20% of their time to research, and 25/57 (44%) dedicated at least 50% to research. During their combined degree, 102/136 respondents (75.0%) published 3 or more first-author papers, and 133/136 (97.8%) matched with their first choice of specialty. The median length of physician-scientist training was 13.5 years. Most respondents graduated with debt despite having been supported by Canadian Institutes of Health Research MD/PhD studentships. INTERPRETATION: Most Canadian MD/PhD program alumni pursued careers consistent with their physician-scientist training, which indicates that these programs are meeting their primary objective. Nevertheless, our findings highlight that a minority of these positions are research intensive; this finding warrants further study. Our data provide a baseline for future monitoring of the output of Canadian MD/PhD programs.

Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay.

Conventional cytotoxic therapies for synovial sarcoma provide limited benefit. Drugs specifically targeting the product of its driver translocation are currently unavailable, in part because the SS18-SSX oncoprotein functions via aberrant interactions within multiprotein complexes. Proximity ligation assay is a recently-developed method that assesses protein-protein interactions in situ. Here we report use of the proximity ligation assay to confirm the oncogenic association of SS18-SSX with its co-factor TLE1 in multiple human synovial sarcoma cell lines and in surgically-excised human tumor tissue. SS18-SSX/TLE1 interactions are disrupted by class I HDAC inhibitors and novel small molecule inhibitors. This assay can be applied in a high-throughput format for drug discovery in fusion-oncoprotein associated cancers where key effector partners are known.

The Canadian clinician-scientist training program must be reinstated.

Clinical investigators within the Canadian and international communities were shocked when the Canadian Institutes of Health Research (CIHR) announced that their funding for the MD/PhD program would be terminated after the 2015-2016 academic year. The program has trained Canadian clinician-scientists for more than two decades. The cancellation of the program is at odds with the CIHR's mandate, which stresses the translation of new knowledge into improved health for Canadians, as well as with a series of internal reports that have recommended expanding the program. Although substantial evidence supports the analogous Medical Scientist Training Program in the United States, no parallel analysis of the MD/PhD program has been performed in Canada. Here, we highlight the long-term consequences of the program's cancellation in the context of increased emphasis on translational research. We argue that alternative funding sources cannot ensure continuous support for students in clinician-scientist training programs and that platform funding of the MD/PhD program is necessary to ensure leadership in translational research.

Differential Effects of Sodium Butyrate and Lithium Chloride on Rhesus Monkey Trophoblast Differentiation.

Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O2) and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/ß-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of ß-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of ß-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/ß-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNFα production. The results provide valuable tools to manipulate trophoblast differentiation in vitro and to better understand the differentiation pathways that occur during early gestation.

MUC1 Is Expressed by Human Skin Fibroblasts and Plays a Role in Cell Adhesion and Migration.

The mucin MUC1 is expressed by normal and cancerous epithelial cells and some nonepithelial cells in which it plays roles in regulating adhesion, migration, and cell signaling. In the present studies we found that MUC1 is expressed by normal human neonatal and adult skin fibroblasts. Fibroblasts are usually considered negative for MUC1 expression. Reverse-transcription polymerase chain reaction and Western blot analyses indicate the presence of full-length MUC1, and immunofluorescence and subcellular fractionation studies show that the protein is expressed on the plasma membrane. Immunohistochemical analyses confirmed the expression of MUC1 by fibroblasts in cryosections of normal human skin. Silencing MUC1 expression in fibroblasts using MUC1 shRNA increased the adhesion of cells to collagen and laminin. Transfection with MUC1 shRNA also increased fibroblast migration on collagen as measured in a wound-healing assay. The expression of α2-integrin was increased in MUC1 shRNA-transfected fibroblasts in which it was localized to membrane ruffles, providing a possible explanation for the increased cell migration on collagen. These results extend the range of expression of MUC1 to skin fibroblasts and suggest a functional role for MUC1 in fibroblast adhesion and motility.

The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes.

MUC1 is a large transmembrane glycoprotein and oncogene expressed by epithelial cells and overexpressed and underglycosylated in cancer cells. The MUC1 cytoplasmic subunit (MUC1-C) can translocate to the nucleus and regulate gene expression. It is frequently assumed that the MUC1 extracellular subunit (MUC1-N) does not enter the nucleus. Based on an unexpected observation that MUC1 extracellular domain antibody produced an apparently nucleus-associated staining pattern in trophoblasts, we have tested the hypothesis that MUC1-N is expressed inside the nucleus. Three different antibodies were used to identify MUC1-N in normal epithelial cells and tissues as well as in several cancer cell lines. The results of immunofluorescence and confocal microscopy analyses as well as subcellular fractionation, Western blotting, and siRNA/shRNA studies, confirm that MUC1-N is found within nuclei of all cell types examined. More detailed examination of its intranuclear distribution using a proximity ligation assay, subcellular fractionation, and immunoprecipitation suggests that MUC1-N is located in nuclear speckles (interchromatin granule clusters) and closely associates with the spliceosome protein U2AF65. Nuclear localization of MUC1-N was abolished when cells were treated with RNase A and nuclear localization was altered when cells were incubated with the transcription inhibitor 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole (DRB). While MUC1-N predominantly associated with speckles, MUC1-C was present in the nuclear matrix, nucleoli, and the nuclear periphery. In some nuclei, confocal microscopic analysis suggest that MUC1-C staining is located close to, but only partially overlaps, MUC1-N in speckles. However, only MUC1-N was found in isolated speckles by Western blotting. Also, MUC1-C and MUC1-N distributed differently during mitosis. These results suggest that MUC1-N translocates to the nucleus where it is expressed in nuclear speckles and that MUC1-N and MUC1-C have dissimilar intranuclear distribution patterns.

Isolating the effects of vection and optokinetic nystagmus on optokinetic rotation-induced motion sickness.

OBJECTIVE: This study investigates isolated effects of vection and optokinetic nystagmus (OKN) on visually induced motion sickness (VIMS) provoked by rotating optokinetic drum patterns. BACKGROUND: VIMS was the subject of recent standardization activities, but the effects of OKN have not been studied in the absence ofvection. METHOD: Experiment 1 suppressed OKN by eye fixation and examined VIMS severity (both ordinal and ratio scale) and time spent in saturated vection at four pattern rotating velocities of 0, 2, 14, and 34 degrees per second (dps). Experiment 2 suppressed vection by adding a peripheral visual field rotating in the opposite direction to the rotating patterns. VIMS severity and OKN slow-phase velocity were studied at four rotating velocities of 0, 30, 60, and 90 dps. RESULTS: Results from Experiment 1 indicated that VIMS severity increased as the pattern velocity increased from 0 dps to 34 dps. Results from Experiment 2 indicated that as the velocity of the rotating pattern increased, the slow-phase velocity of OKN and the severity of VIMS increased and peaked in the 60-dps condition. In both experiments, ratio-scaled nausea data significantly correlated with ordinal-scaled nausea ratings. CONCLUSION: VIMS can still occur in the absence of either vection or OKN. Interestingly, the profile of the summed results of the two experiments matches nicely with the profile reported by Hu et al. in which neither OKN nor vection were controlled. APPLICATION: Potential applications include modeling and reduction of VIMS in computer gaming environments.