ER Markers Research Articles

Expression of ER, PR, HER2, and Cadherin tumor markers in a series of Saudi patients with BC.

Breast cancer (BC) tumor markers have an important implication in the subsequent BC management and survival determinants. Thus, the present study aimed to formulate the expression of ER, PR, HER2, and E-cadherin tumor markers in a series of Saudi patients with BC. About 133 BC biopsies were retrieved from the Department of Pathology at King Salman Hospital, Hai'l, Northern Saudi Arabia, from November 2019 to November 2020. Out of the 133 biopsies, 50 (37.6%) were diagnosed with BC, including 46 ductal carcinoma, 2 lobular carcinomas, and 2 papillary carcinomas. ER was expressed in 30/44 (68.2%), 2/2 (100%), 2/2 (100%) of the cases of DC, LC, and PC, respectively. PR was expressed in 27/43 (63%), 2/2 (100%), 2/2 (100%) of the cases of DC, LC, and PC, correspondingly. HER2 was expressed in 13/31 (42%), 0%, and 0% of DC, LC, and PC cases, respectively. Correspondingly, E-cadherin was expressed in 11/21 (52.4%), 0%, 1/1 (100%) of the cases of DC, LC, and PC. Triple-negative BC and HER2+ve among Saudi women are among the higher globally reported ranges, associated with poorer response to treatment and prognosis. Luckily, only one patient was found with ER-ve PR+ve, the subtype usually associated with poorer survival outcomes. E-cadherin loss is lower among Saudi BC patients, which suggests a less rate of invasion in these patients. The current study's findings may help improve Saudi guidelines for the treatment of breast cancer.

Abstract P5-11-02: Humoral immune responses direct breast cancer towards a basal-like phenotype and increases stemness and invasive potential

Abstract Introduction: A large subset of triple negative breast cancers (TNBC) are characterized by a high degree of lymphocytic infiltration. Previous studies have shown that TNBC contain higher densities of B lymphocytes than other breast cancer subtypes. B lymphocytes infiltration occurs early in breast cancer and is associated with microinvasion. Although the exact role of B cell in growth of TNBC is unclear, B cells have been shown to induce chronic inflammatory responses in other types of cancer such as melanoma through the activation of NF-KB. Chronic inflammation is known to increase stem cell features and promote epithelial mesenchymal transition (EMT) in cancer. Therefore, we sought to examine interactions between B cells and TNBC and determine the effects on stemness and phenotype of breast cancer cells. Methods: CD20 expression was evaluated in tissue microarrays of ER- DCIS by immunohistochemistry. To mimic the B cell rich tumor microenvironment, co-culture studies were performed with the TNBC cell line SUM159 and EBV-transformed B cells using FalconTM Cell Culture Insert with a 0.4 um filter. Expression of stemness genes in tumor cells after co-culture was evaluated using Human pluripotent stem cell kit array (Qiagen). Expression of EMT and stemness genes and markers were determined by qPCR and western blotting. Self-renewal was assessed using mammosphere assays of co-cultured cells compared to controls. Invasion assays were performed in SUM159 and MCF-12A cell lines after co-culture using Corning® BiocoatTM cell culture inserts. Results: High densities of B cells were found in 9 of 36 DCIS (25%) and associated with microinvasion (p=.035). A 2-fold increase in invasion was observed in co-cultured cells compared to controls. RTqPCR showed increased expression of stemness genes OCT4 and Nanog (1.3- and 2.3-fold change respectively). Additionally, increased protein expression of stemness genes (Snail, SOX17, α-Fetoprotein AFP, Otx2, Nanog, Sox2, Oct4 and pdx-1) was observed in co-cultured cells compared to the control in the commercial array analysis. Likewise, we observed a 2- fold increase in ALDH1A3 gene expression which correlated with an increase in ALDH1A3 protein expression in co-cultured cells compared to controls. Additionally, co-cultured cells exhibited a two-fold increase in mammosphere formation compared to controls indicating an increase in self-renewal. A 7-fold increase in gene expression in the mesenchymal marker vimentin and a slight increase expression of snail was observed. This change correlated with a decrease in gene and protein expression of E-cadherin indicating an increase in EMT in co-cultured cells. Conclusion: Our study confirms the finding that B cell infiltration in stage 0 breast cancer is associated to microinvasion. Further, our in vitro studies indicate that interactions between B cells and TNBC may lead to increased invasion, stemness, and EMT. Thus, early B cell infiltration may be a driver of aggressive disease. Further study is needed to examine cross-talk between B cells and TNBC which may identify new targets for therapeutic intervention. Additionally, studies should examine CD20+ B cell infiltration as a prognostic marker for ER- DCIS progression. Citation Format: Jennifer Sims Mourtada, Steficah Maosa, Lynn Opdenaker, Nicole Toney. Humoral immune responses direct breast cancer towards a basal-like phenotype and increases stemness and invasive potential [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-11-02.

Gene-Based Resistance to Erysiphe Species Causing Powdery Mildew Disease in Peas (Pisum sativum L.).

Globally powdery mildew (PM) is one of the major diseases of the pea caused by Erysiphe pisi. Besides, two other species viz. Erysiphe trifolii and Erysiphe baeumleri have also been identified to infect the pea plant. To date, three resistant genes, namely er1, er2 and Er3 located on linkage groups VI, III and IV respectively were identified. Studies have shown the er1 gene to be a Pisum sativum Mildew resistance Locus ‘O’ homologue and subsequent analysis has identified eleven alleles namely er1–1 to er1–11. Despite reports mentioning the breakdown of er1 gene-mediated PM resistance by E. pisi and E. trifolii, it is still the most widely deployed gene in PM resistance breeding programmes across the world. Several linked DNA markers have been reported in different mapping populations with varying linkage distances and effectiveness, which were used by breeders to develop PM-resistant pea cultivars through marker assisted selection. This review summarizes the genetics of PM resistance and its mechanism, allelic variations of the er gene, marker linkage and future strategies to exploit this information for targeted PM resistance breeding in Pisum.

Quantitative CK19 biomarker detection in breast cancer cell lines.

Cytokeratin19 (CK19) was detected as the most related marker for circulating tumor cells, which was assessed in specific cell lines. MCF7, SKBR3, T47D, and MDA-MB-231, and HeLa cell line as negative control were used. CK19 expression was confirmed by using mouse monoclonal anti-human CK19 antibody. CK19 detection in MDA-MB-231 was not observed. CK19 marker expression was compared in T47D, MCF7, and SKBR3 cell lines. T47D and MCF7 belonged to the luminal subtype of breast cancer (BC) that CK19 expression regulated with an ER marker. SKBR3 belonged to the HER2 positive subtype of BC. However, MDA-MB-231 belonged to the claudin-low subtype of BC that lack of CK19 expression strongly is related to negative ER, PR, and HER2. Therefore, there are not only quantitative differences in CK19 expression, but its expression could also link to the other markers of BC that should be considered in the molecular classification of breast carcinoma. Different expression levels related to cell classification could be useful in the prognosis and treatment of cancers with epithelial origins.

Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion.

Background The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen (p ≤ 0.05). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

SLC7A5 is linked to increased expression of genes related to proliferation and hypoxia in estrogen‑receptor‑positive breast cancer.

The amino acid transporter named solute carrier family 7 member 5 (SLC7A5) is suggested to play a part in altered cell metabolism and proliferative signaling and has been reported to be overexpressed in various types of cancer, including breast cancer. Estrogen-receptor-positive (ER+) breast cancers constitute the most common type of breast malignancies and are often treated with anti-estrogenic therapies. In this group of patients, endocrine resistance is a challenging problem that could lead to recurrent disease. To overcome this, additional prognostic biomarkers are needed. The present study aimed therefore to determine whether SLC7A5 may be considered as a possible prognostic marker in ER+ breast cancer and to investigate its relation with certain cancer-related genes. We used a local breast cancer cohort (n=154) and immunohistochemistry to analyze the expression of SLC7A5 in association with clinicopathological characteristics and patient outcome. In addition, gene expression analysis was performed on 80 of these tumors. Furthermore, the METABRIC dataset was used for correlation analyses between expression of SLC7A5 and several genes related to breast cancer biology. The results demonstrated that overexpression of SLC7A5 was significantly associated with histopathological grade in patients with breast cancer, and that SLC7A5 mRNA expression was positively correlated with the expression of marker of proliferation Ki-67 and hypoxia inducible factor 1 subunit alpha. Overexpression of SLC7A5 may therefore play a role in the biology of endocrinologically-driven disease. However, when further assessing SLC7A5 using the METABRIC dataset, SLC7A5 mRNA expression level was more significantly increased in ER- subgroups compared with ER+ disease. All breast cancer subtypes included, SLC7A5 mRNA expression was correlated with a higher number of cancer-related genes than in estrogen receptor positive tumors alone. The present study suggested that SLC7A5 expression may be of importance for breast cancer cell proliferation and survival. In order to further establish the biological and clinical role of SLC7A5 in breast cancer, further investigation using different breast cancer subgroups is required.

Evaluation of ER, PR and HER2 markers by flow cytometry for breast cancer diagnosis and prognosis

Background and aimsLaboratory diagnosis of breast cancer (BC) is done by morphological analysis and immunohistochemistry (IHC). However, this methodology still has some limitations. The aim of this study is to validate flow cytometry (FC) immunophenotyping to investigate diagnostic and prognostic markers of BC. Methods: Tumor samples from surgical specimens of patients previously diagnosed with BC, were first sliced and then macerated together with PBS. Then, sample was filtered and the single cell suspension obtained was labeled with antibodies against estrogen (ERα), progesterone (PR) and HER2 receptors and CD45. The results were compared, in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), with reference methods. Results: Results obtained comparing FC with reference methods were: ERα detection (sensitivity: 75%; specificity: 90%; PPV: 96.7%; NPV: 47.4%); PR detection (sensitivity: 72%; specificity: 70%; PPV: 79.3%; NPV: 60.8%); HER2 detection (sensitivity: 80%; specificity: 90.2%; PPV: 66.7%; NPV: 94.9%). ConclusionThe results obtained show the capacity of this methodology on BC markers differentiation. FC, together with morphological analysis and IHC can overcome individual limitations of each methodology and provide reliable results on a faster and efficient manner, resulting in improvements on BC diagnosis and prognosis.

Hypolipidemia associated with inactivation of TM6SF2 is due to decreased VLDL-lipids secretion

Abstract Background A missense variant in Transmembrane 6 Superfamily Member 2 [TM6SF2 (E167K)] is associated with reduced plasma lipid levels and protection from coronary atherosclerosis. The substitution of lysine for glutamate at residue 167 is associated with a marked decrease in TM6SF2 protein expression, consistent with a loss-of-function mutation. However the biological role of TM6SF2 is not known, and the mechanism(s) responsible for the hypolipidemia associated with mutation gene has not been fully defined. To elucidate the pathological mechanism for the hypolipidemia associated with TM6SF2 deficiency, we inactivated Tm6sf2 in mice and rats. Methods Tm6sf2−/− mice were generated as described previously. Two lines of Tm6sf2−/− rats with different frameshift mutations in exon 1 were generated using CRISPR/Cas9 technology. Primary hepatocytes were isolated from WT and Tm6sf2−/− mice for microscopy. Rats were fasted 16 or 4 hours and tissues were collected on ice for cell fractionation, and in liquid nitrogen for biochemical analyses. Frozen samples were stored at −80°C for subsequent analyses. Result In both mice and rats, inactivation of Tm6sf2 recapitulated the phenotype of humans with the E167K substitution: steatosis, reduced plasma lipid levels, and transaminitis. The phenotype was readily apparent in animals fed chow diets. Both species had reduced secretion of VLDL-TG, as determined by TRITON WR1399 injection, with no decrease in secretion of ApoB. Experiments in isolated perfused livers from WT and Tm6sf2−/− mice confirmed that the decreased TG secretion observed in intact animals reflected reduced TG secretion from the liver. Lipidomic analysis of the liver perfusates by by LC-MS indicated that secretion of cholesteryl esters, and phospholipids was also decreased in the KO animals. Taken together, these findings are consistent with a role for TM6SF2 in lipidation of ApoB-containing lipoproteins. To further elucidate the function of TM6SF2, we used fluorescence microscopy and cell fractionation to determine the subcellular localization of the protein. Microscopic analysis showed that TM6SF2 co-localized with ER and Golgi markers, but cell fractionation studies indicated that the protein is located primarily in the smooth ER. The ratio of TG to ApoB was lower in Golgi fractions from TM6sf2−/− rats than in corresponding fractions from WT animals. Conclusions Since the sequela of TM6SF2 inactivation are already apparent in the Golgi, we speculate that TM6SF2 promotes lipidation of VLDL in a pre-Golgi compartment. We are currently performing additional studies to further define the specific mechanism whereby TM6SF2 promotes lipidation of ApoB-containing lipoproteins. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Institutes of Health

Oncogene-regulated release of extracellular vesicles.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

The release of viral particle from hepatitis B virus infected hepatocytes is inhibited by recombinant hepatitis B virus immunoglobulin (lenvervimab)

Potent and relatively low cost hepatitis B immunoglobulin (HBIG) could be good alternatives by overcoming the drawbacks of recent regimen and recombinant monoclonal HBIG (lenvervimab) has been reported the consistent avidity of variety to cloned S antigens including the immune escape mutant G145R and it did not interfere with the antibody binding in hepatitis B virus (HBV) with mutations of the S gene sequence which makes drug resistance to NA. We investigated the action of intracellularly located lenvervimab in HBV infected cells which is incompletely understood yet. We used five human hepatoma cell lines; Huh7, HepG2 as HBV negative and PLC/PRF/5, HepG2.2.15, and Hep3B expressing HBsAg. and various intracellular orgarnells markers; endosome (Rab5), autophagy marker (LC3) , anti-calnexin (ER), giantin (golgi), multivesicular body (Rab7). Immunofluorescence images were used to investigate the intracellular locations of lenvervimab and HBsAg. Western blot and PEG down, ELISA were used for protein analysis. Lenvervimab was co-localized with Rab5 (early endosome marker) and and induces autophagosome but not autolysis. HBsAg and Lenvervimab were co-localized with making precipitations in cytoplasm and these precipitations were not co-localized with ER and golgi marker. Rab7 (multivesicular body marker) and lenvervimab, HBsAg were co-localized in PLC/PRF/5. The release of viral particle is suppressed by lenvervimab with antigen-antibody reaction in multivesicular body.

Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella ‐containing vacuole (Cellular Microbiology 05/2021)

Cellular MicrobiologyVolume 23, Issue 5 e13333 COVER IMAGEFree Access Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole (Cellular Microbiology 05/2021) First published: 12 April 2021 https://doi.org/10.1111/cmi.13333AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Confocal fluorescence micrograph of Dictyostelium discoideum Δsey1 (atlastin) mutant amoeba producing the ER marker calnexin-GFP (green) and the pathogen vacuole/PtdIns(4)P marker P4C-mCherry (orange) infected with mCerulean-producing Legionella pneumophila (blue). Volume23, Issue5May 2021e13333 RelatedInformation

Mesalazine Suppresses Proinflammatory Cytokines in Patients with Acute Anterior Uveitis Independently of HLA-B27

ABSTRACT Purpose: The aim is to unravel the mechanism of mesalazine (5-ASA) on proinflammatory cytokines in PBMCs of patients with HLA-B27 +and HLA-B27 -acute anterior uveitis (AAU), and whether this may explain the different effects of 5-ASA in both disoders. Methods: PBMCs from 12 HLA-B27+ and 4 HLA-B27- AAU patients were preincubated with 5-ASA and stimulated with LPS. As mesalazine (5-ASA) could be involved in ER stress, proinflammatory and ER stress-associated cytokines and markers were measured. Results: Mesalazine (5-ASA) suppressed IL-6 mRNA in healthy donors and in HLA-B27+ and HLA-B27- patients but did not lead to induction and secretion of IL-1β. In HLA-B27 + or – patients the ER stress-associated markers CHOP (DDIT3) and ATF6 were suppressed. Conclusions: Here we show that mesalazine (5-ASA) inhibits the transcription of proinflammatory and (ER) stress associated cytokines and markers, independently of the HLA-B27 status. Results show the similarities of both AAU types but do not decipher the mechanism why the HLA-B27 status determines the therapeutic response to mesalazine in AAU.

High SLC20A1 Expression Is Associated With Poor Prognoses in Claudin-low and Basal-like Breast Cancers.

SLC20A1 has been identified as a prognostic marker in ER+ breast cancer. However, the role of SLC20A1 expression in breast cancer subtypes other than the ER+ types remains unclear. Genomics datasets were downloaded and analyzed, and the effect of SLC20A1 knockdown using targeted siRNA on cell viability and tumor-sphere formation was assessed. SLC20A1high patients with ER+, claudin-low or basal-like breast cancers showed poor prognoses. SLC20A1high patients treated with radiotherapy had poor clinical outcomes. SLC20A1 knockdown suppressed the viability of MDA-MB 231 (claudin-low), MDA-MB 468 (basal-like) and MCF-7 (ER+) cells, and tumor-sphere formation by ALDH1high cells. These results suggest that SLC20A1 is involved in cancer progression and contributes to clinical outcomes in patients with ER+, claudin-low and basal-like breast cancers. SLC20A1 is a potential prognostic marker and therapeutic target in ER+, claudin-low and basal-like breast cancers.

Proteomic analysis of subcellular compartments containing disseminated alpha-synuclein seeds

The pathological form of a-synuclein (a-syn) is transmitted through neural circuits in the brains of Parkinson disease (PD) patients and amplifies misfolded a-syn, further forming intracellular deposits. However, the details of a-syn pre-formed fibrils (PFFs) transmission in vivo have not been fully elucidated. By inoculating Quantum dots (QD)-labeled a-syn PFFs (QD-a-syn PFFs) into the unilateral striatum, we detected QD-a-syn PFFs in brain homogenates obtained from the ipsilateral and contralateral sides of the inoculated site and further obtained QD-a-syn PFFs enriched-particles with fluorescence-activated organelle sorting. Proteomic analysis suggested that QD-a-syn PFFs-enriched particles in the contralateral side were associated with component proteins of synapse. In contrast, QD-a-syn PFFs-enriched particles in the ipsilateral side were associated with proteins belonging to ER components. Immunostaining of brain sections confirmed that QD-a-syn PFFs in the contralateral side were co-localized with synaptic vesicle marker proteins in the cortex and striatum. Additionally, QD-a-syn PFFs in the ipsilateral side were more co-localized with ER marker proteins compared to the contralateral side. These results correspond to proteomic analysis. This study provides potential candidates for the subcellular localization of a-syn PFFs in vivo during the dissemination phase of seeds. These subcellular compartments could be involved in the transmission of seeds.

The relationship between endoplasmic reticulum stress and liver function, insulin resistance and vascular endothelial function in patients with non-alcoholic fatty liver disease.

The aim of the study was to investigate the relationship between ER stress and liver function, insulin resistance and vascular endothelial function in patients with non-alcoholic fatty liver disease. A total of 95 patients with non-alcoholic fatty liver disease were selected. They were admitted to our hospital from November 2016 to January 2019. A total of 90 cases of obese patients without fatty liver were selected as control group during the same period. The levels of ER stress marker protein were compared between the two groups, and the relationship between ER stress and liver function, insulin resistance, and vascular endothelial function was analyzed. The protein level of ER stress markers in the test group was significantly higher than that in the control group (p<0.05). The liver function index and insulin resistance level were significantly higher than those in the control group (p<0.05). The level of vascular endothelial function was significantly lower than that of the control group (p<0.05). Pearson correlation analysis showed that ER stress marker protein was positively correlated with liver function and insulin resistance (p<0.05), while ER marker protein was negatively correlated with vascular endothelial function (p<0.05). Liver function and insulin resistance are closely related to ER stress in patients with non-alcoholic fatty liver disease. Insulin resistance is one of the factors inducing and aggravating endothelial dysfunction.

Aberrant Diffuse Intense CD10 Expression In P16 Positive Undifferentiated Endometrial Carcinoma: A Diagnostic Dilemma

Abstract Introduction/Objective High-grade endometrial neoplasms frequently pose a diagnostic challenge on purely histologic evaluation due to their ambiguous morphologic features. High grade endometrioid adenocarcinoma, serous carcinoma, undifferentiated carcinoma, and a minority of sarcoma cases can present with a solid growth pattern. Methods We present a 59-year-old patient who underwent a hysterectomy and bilateral salpingo-oophorectomy for a biopsy proven FIGO grade 2 endometrial carcinoma. Results Grossly, the entire endometrial cavity was involved by a hemorrhagic tumor. Microscopic examination showed the entire specimen to be replaced by predominantly non-cohesive diffuse sheets of mitotically active, pleomorphic cells without any glandular, papillary, serous or clear cell features.The differential diagnosis included undifferentiated carcinoma, serous carcinoma, carcinosarcoma, stromal sarcoma and other tumors including high- grade lymphoma. By immunohistochemistry, the tumor cells showed variable positivity for CK7, EMA, CK19, CK18, CK8, AE1/AE3, pan-keratin, CAM5.2, CD56, synaptophysin, p53, cyclinD1 and with a high Ki-67 proliferation index of 80%. The tumor was diffusely/intensely positive for CD10 and P16 while being negative for ER, PR, PAX8, HPV, mesenchymal and lymphoid markers. While diffuse/intense CD10 positivity and ER/PR negativity raised a concern for stromal sarcoma component, the above phenotype confirmed an aggressive/high grade carcinoma. Diffuse/intense p53 and p16 expression raised a consideration of serous carcinoma, but morphology and absence of PAX-8 failed to support this diagnosis. Undifferentiated carcinoma of the endometrium is a high-grade carcinoma which has been recognized as a distinct entity with diffuse p16 and p53 positivity in the absence of PAX-8 and hormone receptors. Positive vimentin, along with a negative HPV, confirmed the endometrial rather than cervical origin of this neoplasm. Conclusion By reporting this case, we draw attention to the previously unreported diffuse intense CD10 expression in undifferentiated carcinoma of endometrium to avoid misdiagnosis with considerations that include stromal sarcoma.

Plasmodium DEH is ER-localized and crucial for oocyst mitotic division during malaria transmission.

Cells use fatty acids (FAs) for membrane biosynthesis, energy storage, and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase-DEH-is a key component of very long chain fatty acid synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics, and ultrastructure analysis using the mouse malaria model Plasmodium berghei DEH is evolutionarily conserved across eukaryotes, with a single DEH in Plasmodium spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. Δdeh mutants showed a small but significant reduction in oocyst size compared with WT controls from day 10 postinfection onwards, and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of Δdeh oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER, and plays a crucial role in sporogony.

Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons.

SummaryMisfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.

Plasma thymidine kinase 1 activity and outcome of ER+ HER2\u2212 metastatic breast cancer patients treated with palbociclib and endocrine therapy

PurposePrevious cohort studies have reported plasma TK1 activity (pTKa) as a potential prognostic biomarker in estrogen receptor-positive (ER+) HER2-negative (HER2−) metastatic breast cancer (MBC). In this prospective study, we report here the prognostic impact of pTKa in ER+/HER2− MBC patients treated with endocrine therapy and CDK4/6 inhibitor.Experimental designPatients were included into the prospective, ethics committee-approved ALCINA study (NCT02866149). Eligibility criteria were patients with ER+/HER2− MBC treated at Institut Curie with endocrine therapy and palbociclib. Plasma samples were obtained at baseline and after 4 weeks of treatment. pTKa was quantified by the DiviTum® assay (Biovica, Sweden).ResultsFrom May 2016 to August 2018, 103 patients treated with endocrine therapy and palbociclib were included. Patients had received a median of two prior systemic therapies for MBC (range 0–14). Median follow-up was 13.8 months (range 6–31), with median PFS and OS of 9.6 months (95%CI [7.0–11.3]) and 28 months (95%CI [23–not reached]), respectively. Median baseline pTKa was 292 Du/L (range 20–27,312 Du/L, IQR [89–853]). After adjusting for other prognostic factors, baseline pTKa remained an independent prognostic factor for both PFS (HR = 1.3 95%CI [1.1–1.4], p = 0.0005) and OS (HR = 1.3 95%CI [1.2–1.6], p < 0.0001), and 4-week pTKa was associated with OS (HR = 1.6 95%CI [1.3–2], p < 0.0001). That survival prediction was significantly improved by the addition of baseline pTKa to clinicopathological characteristics. Adding pTKa changes at 4 weeks to baseline pTKa did not further increase survival prediction.ConclusionThis study demonstrates the clinical validity of pTKa as a new circulating prognostic marker in ER+/HER2− MBC patients treated with endocrine therapy and palbociclib.

Abstract 5045: Predictive factors for successful growth of patient derived xenograft (PDX)

Abstract Background: PDXs have become a core component of translational cancer research. Developing these models can become challenging since little is known about which factors influence engraftment rates. We sought to determine which clinical, pathological or molecular factors may predict a higher chance of success for PDX development. Methods: Between March 2017 and August 2019, biopsies obtained from patients with primary or metastatic cancer were implanted into athymic nude mice. Statistical analyses were performed to identify factors that could correlate with final engraftment defined as tumor volume of 150 mm3 in at least three consecutive measurements. We focus on clinical (patient factors) pathological (tumor sample) and molecular characteristics (tumor sample). Information regarding sampling process was also analyzed. Results: 440 tumor samples were collected and implanted. 15 failed due lack of tumor cells. Of 425 tumor-positive samples, 143 PDXs achieved successful growth at time of analysis. The following clinical characteristics were correlated with engraftment success: systemic steroid administration within 2 weeks of sampling (45.5% (30/66) within the steroids-receiving group achieved growth vs 31% (113/359) no-steroids) (p: 0.027). High neutrophils/lymphocytes (NLR) ratio (&amp;gt;5) (42.3% (47/111) of tumors with NLR&amp;gt;5 achieved growth vs 30% (96/313) in the NLR&amp;lt;5 group; P:0,018). Regarding pathological tumor characteristics, higher ki67 levels were correlated with better engraftment rates (Low (Ki67&amp;lt;15%): 10% (5/46) vs. Medium (Ki67:15-30%): 25% (7/28) and high (Ki67&amp;gt;30%): 34% (16/47) (p:0.029). The presence of mucinous cells (signet ring cells) was also correlated with higher chance of tumor engraftment (50% (19) vs. 32% (124/387) (p: 0.022). Negativity for estrogen receptors could also be a positive predictive marker (14% (8/57) of ER+ achieved growth vs 46.7% (7/15) ER negative (p: 0.011). Lastly, sampling source (either primary of metastatic tumors) was not correlated with successful growth. Conclusions: tumors with higher Ki67, mucinous features and ER expression negative have higher chance of PDX development. Some clinical characteristics can also interfere with PDXs development such as use of steroids or NLR. Citation Format: Tatiana Hernandez, Natalia Baños, Victoria Bonilla, Laura del Puerto, Bernard Doger, Jesus Garcia-Foncillas, Michael Wick, Victor Moreno. Predictive factors for successful growth of patient derived xenograft (PDX) [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 5045.