Improved early detection of ovarian cancer using longitudinal multimarker models

© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Background: Ovarian cancer has a poor survival rate due to late diagnosis and improved methods are needed for its early detection. Our primary objective was to identify and incorporate additional biomarkers into longitudinal models to improve on the performance of CA125 as a first-line screening test for ovarian cancer. Methods: This case–control study nested within UKCTOCS used 490 serial serum samples from 49 women later diagnosed with ovarian cancer and 31 control women who were cancer-free. Proteomics-based biomarker discovery was carried out using pooled samples and selected candidates, including those from the literature, assayed in all serial samples. Multimarker longitudinal models were derived and tested against CA125 for early detection of ovarian cancer. Results: The best performing models, incorporating CA125, HE4, CHI3L1, PEBP4 and/or AGR2, provided 85.7% sensitivity at 95.4% specificity up to 1 year before diagnosis, significantly improving on CA125 alone. For Type II cases (mostly high-grade serous), models achieved 95.5% sensitivity at 95.4% specificity. Predictive values were elevated earlier than CA125, showing the potential of models to improve lead time. Conclusions: We have identified candidate biomarkers and tested longitudinal multimarker models that significantly improve on CA125 for early detection of ovarian cancer. These models now warrant independent validation.Peer reviewe

Robust Identification of Target Genes and Outliers in Triple-negative Breast Cancer Data

Correct classification of breast cancer sub-types is of high importance as it directly affects the therapeutic options. We focus on triple-negative breast cancer (TNBC) which has the worst prognosis among breast cancer types. Using cutting edge methods from the field of robust statistics, we analyze Breast Invasive Carcinoma (BRCA) transcriptomic data publicly available from The Cancer Genome Atlas (TCGA) data portal. Our analysis identifies statistical outliers that may correspond to misdiagnosed patients. Furthermore, it is illustrated that classical statistical methods may fail in the presence of these outliers, prompting the need for robust statistics. Using robust sparse logistic regression we obtain 36 relevant genes, of which ca. 60\% have been previously reported as biologically relevant to TNBC, reinforcing the validity of the method. The remaining 14 genes identified are new potential biomarkers for TNBC. Out of these, JAM3, SFT2D2 and PAPSS1 were previously associated to breast tumors or other types of cancer. The relevance of these genes is confirmed by the new DetectDeviatingCells (DDC) outlier detection technique. A comparison of gene networks on the selected genes showed significant differences between TNBC and non-TNBC data. The individual role of FOXA1 in TNBC and non-TNBC, and the strong FOXA1-AGR2 connection in TNBC stand out. Not only will our results contribute to the breast cancer/TNBC understanding and ultimately its management, they also show that robust regression and outlier detection constitute key strategies to cope with high-dimensional clinical data such as omics data

Molecular Analysis of Precursor Lesions in Familial Pancreatic Cancer

PMCID: PMC3553106This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Poincar\'e's polyhedron theorem for cocompact groups in dimension 4

We prove a version of Poincar\'e's polyhedron theorem whose requirements are as local as possible. New techniques such as the use of discrete groupoids of isometries are introduced. The theorem may have a wide range of applications and can be generalized to the case of higher dimension and other geometric structures. It is planned as a first step in a program of constructing compact $\mathbb C$-surfaces of general type satisfying $c_1^2=3c_2$.Comment: 15 pages, 1 figure, 9 references. Introduction revised. Example 3.16 adde

The Impact Of Anterior Gradient 2 (AGR2) Expression In Models Of Arsenite And Cadmium Induced Breast And Bladder Cancers

Bladder cancer constitutes one of the most prevalent cancers and is among the leading causes of cancer-related deaths in the United States. This work sought to better characterize and understand the role of the proto-oncogene anterior gradient 2 (AGR2) in bladder cancers. Immunohistochemical analysis of AGR2 expression in a range of bladder cancer specimens indicated that the expression of AGR2 decreases with an increase in the stage and grade of bladder cancers. The environmental carcinogens arsenic and cadmium (Cd2+) have been implicated in various cancers. This laboratory has demonstrated that arsenite (As3+) and Cd2+ can malignantly transform the bladder epithelium cell line UROtsa. This study therefore sought to determine AGR2 expression in six As3+ and seven Cd2+-transformed UROtsa cell lines in which real-time PCR and western blotting data indicated that AGR2 expression was increased in several of the transformed cell lines. Next AGR2 expression was evaluated in mouse heterotransplant tumors arising from the injection of the previously transformed UROtsa cell lines in athymic nude (Foxn1nu) mice. Results from real-time PCR analysis and immunohistochemistry on tumor samples demonstrated that AGR2 expression remained increased in most of the Cd2+ heterotransplants but was significantly increased in only a couple of the As3+ heterotransplants. UROtsa cells were further exposed to 2, 4, or 6 μM As3+ or to 1, 2, or 4 μM Cd2+ for up to 72 hours and real-time PCR analysis was performed to evaluate AGR2 expression. While AGR2 exhibited increased expression in response to 48 hours As3+ exposure, expression was not induced to the same extent in Cd2+ exposed UROtsa cells. Spheroids containing cancer initiating cells (CICs) were generated from the transformed and the parent UROtsa cells and the expression of AGR2 was determined. The results obtained suggested that there was an increase in the expression of AGR2 in all of the spheroids isolated from the As3+-transformed cells whereas some of the spheroids isolated from the Cd2+-transformed cells expressed high levels of AGR2 when compared to the spheroids isolated from the parent UROtsa cells. This laboratory has also shown that As3+ and Cd2+ can cause malignant transformation of a breast epithelial cell line, MCF-10A. Previous studies have shown that the expression of AGR2 promotes breast tumorigenesis in mice. This gene is known to play a role in promoting cellular transformation, tumor growth, and metastasis in various cancers. This study was interested in determining the expression level of AGR2 in As3+ and Cd2+-transformed MCF-10A cells. Real-time PCR and Western analysis indicated that the expression of AGR2 was significantly increased in the MCF-10A cells transformed with As3+ when compared to the Cd2+-transformed cells. Exposure of the parent MCF-10A cells to 4, 8, and 16 μM As3+ for 48 hours resulted in a significant increase in the expression of AGR2 whereas exposure to Cd2+ did not increase the expression of AGR2, suggesting that As3+ has the potential to induce AGR2 in MCF-10A cells. In order to further investigate the effects of AGR2 expression on breast epithelial cells, MCF-10A cells were transfected with the AGR2 gene. Overexpression of AGR2 in the MCF-10A cells increased the ability of the cells to migrate faster in the wound scratch assay when compared to the blank vector transfected cells. In addition, MTT and scratch assays revealed increased growth and migration in the As3+-transformed cells when compared to the parent MCF-10A cells and the Cd2+-transformed MCF-10A cells further implicating AGR2 in enhancing cell growth and migration. Treatment of the MCF-10A cells with the histone deacetylase inhibitor MS-275 and the demethylating agent, 5-Aza-2\u27-deoxycytidine (5-AZC) increased the expression of AGR2 suggesting that epigenetic modifications may be involved in regulating the expression of AGR2 in MCF-10A cells. These data also suggest that the expression of AGR2 in the MCF-10A cells may be regulated independently of the estrogen receptor status of the cells. Seeking to gain a better understanding of the relationship between AGR2 expression and breast cancer grade and stage, immunohistochemical staining was performed on a range of breast cancer specimens. Results showed elevated AGR2 expression with increasing grade of breast carcinoma. In conclusion, this study demonstrated the usefulness of AGR2 expression as a biomarker for bladder and breast cancers, further characterized the effect of AGR2 on the growth and migration of MCF-10A cells, and began to evaluate possible mechanisms by which AGR2 expression may be induced

AGR2, an Endoplasmic Reticulum Protein, Is Secreted into the Gastrointestinal Mucus

The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2(-/-) mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine

Investigating a potential oncogenic role for AGR2 in pro-survival autophagy

Members of the Protein Disulphide Isomerase (PDI) family have essential roles in mediating the oxidation, reduction and isomerisation of disulphide bonds during protein maturation in the endoplasmic reticulum. PDI family members are characterised by the presence of a thioredoxin motif (CXXC) with dual cysteines that permit proteins their oxidoreductase activity. However, PDI proteins that harbour evolutionary divergent thioredoxin motifs have also been identified; the Anterior Gradient-2 (AGR2) protein that possesses a single cysteine residue in its thioredoxin-like domain (CPHS) is one such protein. AGR2 is known to be required for the correct folding and secretion of mucins, the primary gel-forming proteins within mucus. Although its expression is normally restricted to certain secretory and reproductive organs, AGR2 is found derepressed in various cancers. We have previously shown that AGR2 forms a disulphide-dependent interaction with the autophagy receptor Sequestosome 1 (SQSTM1). The oxidation of SQSTM1 is required for the stimulation of autophagy to promote cell survival under conditions of oxidative and proteotoxic stress. The disulphide-dependent interaction between AGR2 and SQSTM1 links AGR2 to autophagy for the first time. Tumours are exposed to high levels of oxidative stress, and thus autophagy can prevent cytotoxicity by removing the cellular components damaged by oxidative stress that may otherwise be toxic to the cell. It is plausible, therefore, that the upregulation of SQSTM1 and AGR2 in human cancers could be involved in the induction of pro-survival autophagy

Anterior gradient protein 2 promotes survival, migration and invasion of papillary thyroid carcinoma cells

Through a transcriptome microarray analysis, we have isolated Anterior gradient protein 2 (AGR2) as a gene up-regulated in papillary thyroid carcinoma (PTC). AGR2 is a disulfide isomerase over-expressed in several human carcinomas and recently linked to endoplasmic reticulum (ER) stress. Here, we analyzed the expression of AGR2 in PTC and its functional role

Evaluation of the Adenocarcinoma-Associated Gene AGR2 and the Intestinal Stem Cell Marker LGR5 as Biomarkers in Colorectal Cancer

We aim to estimate the diagnostic performances of anterior gradient homolog-2 (AGR2) and Leucine-rich repeat-containing-G-protein-coupled receptor 5 (LGR5) in peripheral blood (PB) as mRNA biomarkers in colorectal cancer (CRC) and to explore their prognostic significance. Real-time PCR was used to analyze AGR2 and LGR5 in 54 stages I-IV CRC patients and 19 controls. Both mRNAs were significantly increased in PB from CRC patients compared to controls. The area under the receiver-operating characteristic curves were 0.722 (p = 0.006), 0.376 (p = 0.123) and 0.767 (p = 0.001) for AGR2, LGR5 and combined AGR2/LGR5, respectively. The AGR2/LGR5 assay resulted in 67.4% sensitivity and 94.7% specificity. AGR2 correlated with pT3–pT4 and high-grade tumors. LGR5 correlated with metastasis, R2 resections and high-grade. The progression-free survival (PFS) of patients with high AGR2 was reduced (p = 0.037; HR, 2.32), also in the stage I-III subgroup (p = 0.046). LGR5 indicated a poor prognosis regarding both PFS (p = 0.007; HR, 1.013) and overall survival (p = 0.045; HR, 1.01). High AGR2/LGR5 was associated with poor PFS (p = 0.014; HR, 2.8) by multivariate analysis. Our findings indicate that the assessment of AGR2 and LGR5 in PB might reflect the presence of circulating tumor cells (CTC) and stem cell like CTC in CRC. Increased AGR2 and LGR5 are associated with poor outcomes