Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells.

The primary site of metastasis for epithelial ovarian cancer (EOC) is the peritoneum, and it occurs through a multistep process that begins with adhesive contacts between cancer cells and mesothelial cells. Despite evidence that Notch signaling has a role in ovarian cancer, it is unclear how exactly it contributes to ovarian cancer omental metastasis, as well as the cellular dynamics and intrinsic pathways that drive this tropism. Here we show that tumor cells produced the Notch ligand Jagged2 is a clinically and functionally critical mediator of ovarian cancer omental metastasis by activating the Notch signaling in single-layered omental mesothelial cells. In turn, Jagged2 promotes tumor growth and therapeutic resistance by stimulating IL-6 release from mesothelial cells. Additionally, Jagged2 is a potent downstream mediator of the omental metastasis cytokine TGF-ß that is released during omental destruction. Importantly, therapeutic inhibition of Jagged2-mediated omental metastasis was significantly improved by directly disrupting the Notch pathway in omental mesothelial cells. These findings highlight the key role of Jagged2 to the functional interplay between the TGF-ß and the Notch signaling pathways during the metastatic process of ovarian cancer cells to the omentum and identify the Notch signaling molecule as a precision therapeutic target for ovarian cancer metastasis.

Long noncoding RNA DLEU2 and ROR1 pathway induces epithelial-to-mesenchymal transition and cancer stem cells in breast cancer.

Breast cancer (BC) patient who receives chemotherapy for an extended length of time may experience profound repercussions in terms of metastases and clinical outcomes due to the involvement of the epithelial-to-mesenchymal transition (EMT) mechanism and enriched cancer stem cells (CSCs). BC cells that express high levels of lncRNA deleted in lymphocytic leukemia-2 (lncRNA DLEU2) and type I tyrosine kinase-like orphan receptor ROR1 (ROR1) may play roles in the enhanced ability of the activation EMT and CSC induction. Here we find that lncRNA DLEU2 and ROR1 are specifically upregulated in tumor tissues compared to their normal counterparts in TCGA, PubMed GEO datasets, and samples from archived breast cancer tumor tissues. Following chemotherapy, lncRNA DLEU2 and ROR1 were enhanced in BC tumor cells, coupled with the expression of CSCs, EMT-related genes, and BMI1. Mechanistically, ROR1 and lncRNA DLEU2 overexpression led to enhanced tumor cell proliferation, inhibition of apoptosis, cell-cycle dysregulation, chemoresistance, as well as BC cell's abilities to invade, migrate, develop spheroids. These findings imply that the role of lncRNA DLEU2 and ROR1 in BC therapeutic failure is largely attributed to EMT, which is intricately linked to enriched CSCs. In conclusion, our findings indicate that a lncRNA DLEU2 and ROR1-based regulatory loop governs EMT and CSC self-renewal, implying that targeting this regulatory pathway may improve patients' responses to chemotherapy and survival.

KEAP1/NRF2 Mutations in Stem Cells Define an Aggressive Subset of Head and Neck Cancer Patients Who Have a Poor Prognosis, Lung Metastasis, and Therapeutic Failure.

Mutations in Keap1/Nrf2 in head and neck cancer result in abnormal cell growth. Progenitor cells, bulk tumor cells, and head and neck cancer stem cells (HN-CSCs) may all harbor these mutations. Nevertheless, whether Keap1/Nrf2 mutations in HN-CSCs have an impact on clinical outcomes is unknown. Cancerous HN-CSCs and benign stem cells were obtained from freshly resected head and neck cancer patients (n = 50) via flow cytometry cell sorting and tested for Keap1/Nrf2 mutations. The existence of Keap1/Nrf2 mutations in HN-CSCs, as well as their correlations with tumor mutations, pathologic tumor stage, tumor histologic grades, lung metastasis, treatment outcomes, and the patient's age and conditions, are assessed at the last follow-up visit. Thirteen tumors were found to have Keap1/Nrf2 mutations in their HN-CSCs. More than half of the lung metastases and disease progression occurred in HN-CSCs with mutations. Patients whose tumors carried Keap1/Nrf2 mutations in their HN-CSCs had significantly shorter progression-free survival, overall survival, and time of treatment failure than their non-HN-CSC counterparts. These associations were partly driven by HN-CSCs, in which Keap1/Nrf2 mutations were overrepresented in fast progressors and associated with an increased risk of disease progression. Our findings suggest that molecular genotyping of HN-CSCs may facilitate personalized treatment strategies and assist in identifying patients who are likely to benefit from chemotherapy.

Structural analysis of a water insoluble polysaccharide from pearl millet and evaluating its prebiotic activity.

Epidemiological studies have shown an inverse correlation between dietary intake of prebiotics and the risk of chronic diseases. Pearl millet is a potential economic source to develop a new class of prebiotics in the form of its polysaccharide. In the present study, the chemical structure of a water insoluble homopolysaccharide (PMG), and its prebiotic properties were investigated. The structure of PMG was elucidated on the basis of total hydrolysis, methylation analysis, and 1D/2D NMR (1H, 13C, DEPT-135, HSQC, DQF-COSY, NOESY and ROESY) experiments. The results indicated that PMG was a glucan with an average molecular weight ~ 361 kDa having a backbone of (1 â†’ 3) α-d-glucopyranosyl residues. Hydrolysis of PMG by salivary and pancreatic α amylase was 1.75 % ± 0.34 and 1.99 % ± 0.18 respectively. A positive prebiotic score of PMG with both L. acidophilus and L. brevis (0.446 ± 0.031 & 0.427 ± 0.016) hints towards its prebiotic potential. These observations suggest that PMG might be used as a potential prebiotic component in the food and pharmaceutical applications.

Impact of admission viral load on respiratory outcomes in hospitalized SARS-CoV-2 infected patients with cancer and without cancer: A 2-, 4- and 6-months follow-up prospective study.

BACKGROUND: This prospective follow-up study aimed to determine the temporal changes in respiratory outcomes over 6 months period in patients with and without cancer hospitalized for severe COVID-19 and to determine the associated risk factors based on admission viral load. METHODS: All adult patients hospitalized with a confirmed diagnosis of severe SARS-CoV-2 infection were investigated using rRT-PCR on nasopharyngeal swab specimens. Patients were divided into three arbitrary groups according to their cycle threshold (CT) values obtained at admission as high (CT<25.0), medium (CT between 25.0 and 30.0), and low (CT>30.0) viral load. Patients had pulmonary function tests, chest high-resolution computed tomography (HRCT), and a 6-minute walking time distance measured at each follow-up visit. RESULTS: This follow-up study had a total of 112 participants, of which 75 were cancer-free and 37 had active cancer. Overall, 29.5% had a low viral load, compared to 48.2% who had a high viral load, and 22.3% had a medium viral load. For patients who did not have cancer, the mean age was 57.3 (SD 15.4) and for those who had cancer, it was 62.3 (SD 18.4). Most patients had overall better temporal changes in pulmonary function and tolerance, as well as exercise capacity, even though severe and chronic respiratory abnormalities persisted in a fraction of the patients. In patients without cancer who had a high viral load, we have seen a substantial reduction in diffusion capacity of the lungs for carbon monoxide (DLCO) predicted value with a median of 65 (IQR 63-70) while in patients with cancer, it was 60 (IQR 56-67) at 2 months. At 4 and 6 months, the predicted DLCO values for patients without cancer were 65 (IQR 61-70), whereas the predicted DLCO values for patients with active cancer were 62 (IQR 60-67) and 67 (59-73). Importantly, radiological abnormalities persisted in 22 (29%) non-cancer patients and 16 (43%) cancer patients. Multivariate regression analysis showed an increased odds ratio of impaired HRCT associated with a high viral load of 3.04 (95% CI:1.68-6.14; p < 0.001) for patients without cancer and 5.07 (95% CI: 4.04-10.8; p < 0.0001) for patients with cancer. The CT pneumonia score at hospitalization was 2.25 (95% CI:1.76-3.08; p = 0.041) and 2.85 (95% CI:1.89-5.14; p = 0.031) for non-cancer and cancer patients respectively. CONCLUSIONS: The evidence of persistent pulmonary abnormalities and radiographic changes was found in both patient groups who had high viral load at hospital admission and suggesting that SARS-CoV-2 viral load might serve as a useful indicator to predict the development of respiratory complications in patients with COVID-19.

Activated breast stromal fibroblasts exhibit myoepithelial and mammary stem cells features.

BACKGROUND: Active breast cancer-associated fibroblasts (CAFs) promote tumor growth and spread, and like tumor cells they are also heterogeneous with various molecular sub-types and different pro-tumorigenic capacities. METHODS: We have used immunoblotting as well as quantitative RT-PCR to assess the expression of various epithelial/mesenchymal as well as stemness markers in breast stromal fibroblasts. Immunofluorescence was utilized to assess the level of different myoepithelial and luminal markers at the cellular level. Flow cytometry allowed to determine the proportion of CD44- and ALDH1-positive breast fibroblasts, while sphere formation assay was used to test the ability of these cells to form mammospheres. RESULTS: We have shown here that IL-6-dependent activation of breast and skin fibroblasts promotes mesenchymal-to-epithelial transition and stemness in a STAT3- and p16-dependent manner. Interestingly, most primary CAFs isolated from breast cancer patients exhibited such transition and expressed lower levels of the mesenchymal markers N-cadherin and vimentin as compared to their adjacent normal fibroblasts (TCFs) isolated from the same patients. We have also shown that some CAFs and IL-6-activated fibroblasts express high levels of the myoepithelial markers cytokeratin 14 and CD10. Interestingly, 12 CAFs isolated from breast tumors showed higher proportions of CD24low/CD44high and ALDHhigh cells, compared to their corresponding TCF cells. These CD44high cells have higher abilities to form mammospheres and to enhance cell proliferation of breast cancer cells in a paracrine manner relative to their corresponding CD44low cells. CONCLUSION: Together, the present findings show novel characteristics of active breast stromal fibroblasts, which exhibit additional myoepithelial/progenitor features.

Fascin is essential for mammary gland lactogenesis.

Fascin expression has commonly been observed in certain subtypes of breast cancer, where its expression is associated with poor clinical outcome. However, its role in normal mammary gland development has not been elucidated. Here, we used a fascin knockout mouse model to assess its role in normal mammary gland morphogenesis and lactation. Fascin knockout was not embryonically lethal, and its effect on the litter size or condition at birth was minimal. However, litter survival until the weaning stage significantly depended on fascin expression solely in the nursing dams. Accordingly, pups that nursed from fascin-/- dams had smaller milk spots in their abdomen, suggesting a lactation defect in the nursing dams. Mammary gland whole-mounts of pregnant and lactating fascin-/- mice showed significantly reduced side branching and alveologenesis. Despite a typical composition of basal, luminal, and stromal subsets of mammary cells and normal ductal architecture of myoepithelial and luminal layers, the percentage of alveolar progenitors (ALDH+) in fascin-/- epithelial fraction was significantly reduced. Further in-depth analyses of fascin-/- mammary glands showed a significant reduction in the expression of Elf5, the master regulator of alveologenesis, and a decrease in the activity of its downstream target p-STAT5. In agreement, there was a significant reduction in the expression of the milk proteins, whey acidic protein (WAP), and ß-casein in fascin-/- mammary glands. Collectively, our data demonstrate, for the first time, the physiological role of fascin in normal mammary gland lactogenesis, an addition that could reveal its contribution to breast cancer initiation and progression.

Genetic alterations of Keap1 confers chemotherapeutic resistance through functional activation of Nrf2 and Notch pathway in head and neck squamous cell carcinoma.

Keap1 mutations regulate Nrf2 activity and lead to chemoresistance in cancers. Yet the underlying molecular mechanisms of chemoresistance are poorly explored. By focusing and genotyping head and neck squamous cell carcinoma (HNSCC) that had available pathologic and clinical data, we provide evidence that Keap1 displays frequent alterations (17%) in HNSCC. Functional loss of Keap1 results in significant activation of Nrf2 and promotes cancer cell growth, proliferation, and elevated cancer stem cell (CSCs) self-renewal efficiency and resistance to oxidative stress. Furthermore, decreased Keap1 activity in these cells increased nuclear accumulation of Nrf2 and activation of the Notch pathway, causing enhanced transcriptional alterations of antioxidants, xenobiotic metabolism enzymes, and resistance to chemotherapeutic treatment. Limiting the Nrf2 activity by either Keap1 complementation or by Nrf2 silencing increased the sensitivity to chemotherapy in Keap1-mutated cells and repressed the CSC self-renewal activity. Our findings suggest that Keap1 mutations define a distinct disease phenotype and the Keap1-Nrf2 pathway is one of the leading molecular mechanisms for clinical chemotherapeutic resistance. Targeting this pathway may provide a potential and attractive personalized treatment strategy for overcoming chemotherapeutic resistance conferred by Keap1 mutations.

A Rare Presentation of a True Intra-articular Lipoma: A Case Report and Review of Imaging Findings.

This report describes the unique case of an intra-articular lipoma in a 34-year-old male. The patient presented with a history of chronic knee pain associated with an intermittent sensation of the knee giving way. Physical examination and initial radiographic imaging were unremarkable. Magnetic resonance imaging (MRI) revealed a 9.2 x 6.7 mm ovoid mass posterior to the posterior cruciate ligament (PCL) exhibiting hyperintense signals on T1-weighted images and intermediate-to-high intensity signals on T2. On subsequent proton density fat suppression sequences, the mass demonstrated homogenous signal suppression and was confirmed as being a lipoma. To the best of our knowledge, this is only the second reported case of an intra-articular lipoma arising posterior to the PCL. Intra-articular lipomas, albeit rare, should be considered in the differential diagnosis for chronic knee pain with associated joint motion abnormalities. MRI remains the gold standard in imaging intra-articular soft tissue pathology and should be the study of choice in differentiating intra-articular lipomas from similar conditions such as pigmented villonodular synovitis and lipoma arborescens.

Decline in Respiratory Functions in Hospitalized SARS-CoV-2 Infected Cancer Patients Following Cytotoxic Chemotherapy-An Additional Risk for Post-chemotherapy Complications.

Background: Patients recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection demonstrate impaired lung function and those requiring chemotherapy after recovering from SARS-CoV-2 infection have yet to be explored. In this study, we sought to investigate the possible pulmonary functional changes during and after administering chemotherapy in patients with prior SARS-CoV-2 infection. Methods: In this study, a total of 37 SARS-CoV-2 infected patients with cancer who were discharged from hospital and received subsequent cytotoxic chemotherapy were enrolled and prospectively followed-up. The following parameters were prospectively measured before (P1), after first chemotherapy cycle (P2), and 10 weeks after the end of chemotherapy (P3), to assess their impact on respiratory complications in terms of diffusion capacity of the lungs for carbon monoxide (DLCO), forced expiratory volume in 1-s (FEV1), forced vital capacity (FVC), 6-min walking distance (6MWD) test and levels of key inflammatory markers. Results: All patients completed at least 2 cycles of chemotherapy without showing overt respiratory complications. Six patients (16%) complained about dyspnea during chemotherapy or at follow-up period. DLCO was significantly impaired during follow-up period [from P1 78 to P3 60% of predicted values; interquartile range (IQR) 55-89] and in 32 of 37 (86% of patients) from P1 to P2 (65% of predictive value; IQR 58-70; p < 0.001). Several patients experienced post-chemotherapy respiratory complications. As expected, all patients from control groups showed persistent improved pulmonary functions. Conclusion: The risk of pulmonary impairments due to cytotoxic chemotherapy in prior SARS-CoV-2 infected patients is linked to the loss of DLCO. Accordingly, we recommend that for patients with cancer requiring chemotherapy after recovering from prior SARS-CoV-2 infection, pulmonary tests to be performed routinely before and during chemotherapy treatment to monitor the pulmonary performance.

SARS-CoV-2 Viral Load Is Correlated With the Disease Severity and Mortality in Patients With Cancer.

The correlation between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral load and risk of disease severity in cancer patients is poorly understood. Given the fact that cancer patients are at increased risk of severe coronavirus disease 2019 (COVID-19), analysis of viral load and disease outcome in COVID-19-infected cancer patients is needed. Here, we measured the SARS-CoV-2 viral load using qPCR cycle threshold (Ct) values collected from 120 noncancer and 64 cancer patients' nasopharyngeal swab samples who are admitted to hospitals. Our results showed that the in-hospital mortality for high viral load cancer patients was 41.38%, 23.81% for medium viral load and 14.29% for low viral load patients (p < -0.01). On the other hand, the mortality rate for noncancer patients was lower: 22.22% among patients with high viral load, 5.13% among patients with medium viral load, and 1.85% among patients with low viral load (p < 0.05). In addition, patients with lung and hematologic cancer showed higher possibilities of severe events in proportion to high viral load. Higher attributable mortality and severity were directly proportional to high viral load particularly in patients who are receiving anticancer treatment. Importantly, we found that the incubation period and serial interval time is shorter in cancer patients compared with noncancer cases. Our report suggests that high SARS-CoV-2 viral loads may play a significant role in the overall mortality and severity of COVID-19-positive cancer patients, and this warrants further study to explore the disease pathogenesis and their use as prognostic tools.

Chemotherapeutic resistance of head and neck squamous cell carcinoma is mediated by EpCAM induction driven by IL-6/p62 associated Nrf2-antioxidant pathway activation.

Overexpression of epithelial cell adhesion molecule (EpCAM) has been associated with chemotherapeutic resistance, leads to aggressive tumor behavior, and results in an adverse clinical outcome. The molecular mechanism by which EpCAM enrichment is linked to therapeutic resistance via Nrf2, a key regulator of antioxidant genes is unknown. We have investigated the link between EpCAM and the Nrf2 pathway in light of therapeutic resistance using head and neck squamous cell carcinoma (HNSCC) patient tumor samples and cell lines. We report that EpCAM was highly expressed in Nrf2-positive and HPV-negative HNSCC cells. In addition, cisplatin-resistant tumor cells consisted of a higher proportion of EpCAMhigh cells compared to the cisplatin sensitive counterpart. EpCAMhigh populations exhibited resistance to cisplatin, a higher efficiency in colony formation, sphere growth and invasion capacity, and demonstrated reduced reactive oxygen species (ROS) activity. Furthermore, Nrf2 expression was significantly higher in EpCAMhigh populations. Mechanistically, expression of Nrf2 and its target genes were most prominently observed in EpCAMhigh populations. Silencing of EpCAM expression resulted in the attenuation of expressions of Nrf2 and SOD1 concomitant with a reduction of Sox2 expression. On the other hand, silencing of Nrf2 expression rendered EpCAMhigh populations sensitive to cisplatin treatment accompanied by the inhibition of colony formation, sphere formation, and invasion efficiency and increased ROS activity. The molecular mechanistic link between EpCAM expression and activation of Nrf2 was found to be a concerted interaction of interleukin-6 (IL-6) and p62. Silencing of p62 expression in EpCAMhigh populations resulted in the attenuation of Nrf2 pathway activation suggesting that Nrf2 pathway activation promoted resistance to cisplatin in EpCAMhigh populations. We propose that therapeutic targeting the Nrf2-EpCAM axis might be an excellent approach to modulate stress resistance and thereby survival of HNSCC patients enriched in EpCAMhigh populations.

Widespread expression of Sonic hedgehog (Shh) and Nrf2 in patients treated with cisplatin predicts outcome in resected tumors and are potential therapeutic targets for HPV-negative head and neck cancer.

BACKGROUND: Sonic hedgehog (Shh) and Nrf2 play a critical role in chemotherapeutic resistance. These two genes have been found to be dysregulated in head and neck squamous cell carcinomas (HNSCC). The purpose of this study was to analyze the expression, function and clinical prognostic relationship of Shh and Nrf2 in HNSCC in the context of therapeutic resistance and cancer stem cells (CSCs). METHODS: We analyzed a cohort of patients with HNSCC to identify potential therapeutic biomarkers correlating with overall survival (OS) as well as disease-free survival (DFS) from our own data and validated these results using The Cancer Genome Atlas dataset. Expression of Shh and Nrf2 was knocked down by siRNA and cell growth, sphere growth and chemotherapeutic resistance were evaluated. RESULTS: Widespread abundant expression of Shh and Nrf2 proteins were associated with shorter OS and DFS. The combination of Shh and Nrf2 expression levels was found to be a significant predictor of patient DFS. The tumor stromal index was correlated with Shh expression and inversely associated with shorter OS and DFS. Inhibition of Shh by siRNA or cyclopamine resulted in the attenuation of resistant CSC self-renewal, invasion, clonogenic growth and re-sensitization to the chemotherapeutic agents. Concomitant upregulation of Shh and Nrf2 proved to be an independent predictor of poor OS and DFS in patients with HNSCC. CONCLUSIONS: These findings suggest that Shh and Nrf2 could serve as therapeutic targets as well as promising dual prognostic therapeutic biomarkers for HNSCC.

Structural characterization of a heteroglycan from an edible mushroom Termitomyces heimii.

A water soluble heteroglycan (THPS) of an average molecular weight ~1.98 × 105 Da was isolated from the aqueous extract of the fruit bodies of an edible mushroom Termitomyces heimii. Structural characterization of THPS was carried out using acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation and 1D/2D NMR studies. Sugar analysis indicated the presence of glucose, mannose, galactose, and fucose in a molar ratio of nearly 6:2:2:1. The repeating unit of the THPS had a backbone consisting of four (1 â†’ 3)-ß-d-glucopyranosyl, one (1 â†’ 6)-ß-d-glucopyranosyl, two (1 â†’ 3)-α-D-manopyranosyl, and two (1 â†’ 6)-α-D-galactopyranosyl residues, out of which one (1 â†’ 3)-ß-d-glucopyranosyl residue was branched at O-6 position with terminal ß-d-glucopyranosyl residue and one (1 â†’ 6)-α-D-galactopyranosyl residue was branched at O-2 position with terminal α-L-fucopyranosyl residue.

Longterm, Real-world Safety of Adalimumab in Rheumatoid Arthritis: Analysis of a Prospective US-based Registry.

OBJECTIVE: To assess longterm safety in a US cohort of patients with rheumatoid arthritis (RA) treated with adalimumab (ADA) in real-world clinical care settings. METHODS: This observational study analyzed the longterm incidence of safety outcomes among patients with RA initiating ADA, using data from the Corrona RA registry. Patients were adults (≥ 18 yrs) who initiated ADA treatment between January 2008 and June 2017, and who had at least 1 followup visit. RESULTS: In total, 2798 ADA initiators were available for analysis, with a mean age of 54.5 years, 77% female, and mean disease duration of 8.3 years. Nearly half (48%) were biologic-naive, and 9% were using prednisone ≥ 10 mg at ADA initiation. The incidence rates per 100 person-years for serious infections, congestive heart failure requiring hospitalization, malignancy (excluding nonmelanoma skin cancer), and all-cause mortality were 1.86, 0.15, 0.64, and 0.33, respectively. The incidence of serious infections was higher in the first year of therapy (3.44, 95% CI 2.45-4.84) than in subsequent years, while other measured adverse effects did not vary substantially by duration of exposure. The median time to ADA discontinuation was 11 months, while the median time to first serious infection among those experiencing a serious infection event was 12 months. CONCLUSION: Analysis of longterm data from this prospective real-world registry demonstrated a safety profile consistent with previous studies in patients with RA. This analysis did not identify any new safety signals associated with ADA treatment and provides guidance for physicians prescribing ADA for extended periods.

Incidence of inpatient venous thromboembolism in treated patients with rheumatoid arthritis and the association with switching biologic or targeted synthetic disease-modifying antirheumatic drugs (DMARDs) in the real-world setting.

Objectives: To assess incidence rates (IRs) of VTE in patients with rheumatoid arthritis (RA) on different DMARDs and DMARD switchers. Methods: Adults with RA on a DMARD between 2007 and 2017 were studied in a US claims database. Conventional synthetic DMARD (csDMARD) users, first biologic/targeted synthetic DMARD (b/tsDMARD) users and b/tsDMARD switchers (from a b/tsDMARD to another b/tsDMARD) were followed for inpatient VTE (pulmonary embolism (PE)/deep vein thrombosis (DVT)). Crude and adjusted IR and 95% CIs of VTE were estimated. HRs for VTE were estimated via Cox regression. VTE risk was also evaluated by number of switches between b/tsDMARDs and in patients without a VTE history. Results: The age and sex standardised IR (95% CI) of VTE (per 100 person-years) was 0.86 (0.70 to 1.03), 0.60 (0.52 to 0.68) and 0.58 (0.51 to 0.65) for b/tsDMARD switchers, first b/tsDMARD users and csDMARD users, respectively. After adjustment, b/tsDMARD switchers had an increased risk of VTE, compared with csDMARD users, HRadj (95% CI) being 1.36 (1.16 to 1.58), 1.36 (1.13 to 1.63) and 1.47 (1.18 to 1.83) for VTE, DVT and PE, respectively. Compared with first b/tsDMARD users, the HRadj (95% CI) for VTE was 1.35 (1.15 to 1.60) for first b/tsDMARD switchers and 1.48 (1.19 to 1.85) for second b/tsDMARD switchers. Conclusions: In RA, b/tsDMARD switchers have a higher VTE risk compared with csDMARD users and first b/tsDMARD users. Switching b/tsDMARDs may be a proxy for higher disease severity or poorly controlled RA and an important confounder to consider in obtaining unbiased estimates of VTE risk in observational RA safety studies.

Cell-free DNA levels of twins and sibling pairs indicate individuality and possible use as a personalized biomarker.

Cell-free DNA (cfDNA) in the human blood circulation has been under investigation since its initial observation in 1948. Plasma cfDNA is known to be significantly elevated in diseased people. Due to possible variation in the population, evaluating cfDNA as a non-invasive biomarker at disease onset alone may not be sensitive enough to accurately diagnose diseases, particularly early stage cancers on a personal level. To understand the factors that define the cfDNA levels on the personal level and for better use as a non-invasive biomarker, we isolated cfDNA from the plasma of healthy individuals with varying degrees of genetic and/or environmental similarities (monozygotic twins, dizygotic twins, sibling pairs, and unrelated individuals) as well as from patients with varying stages of breast and ovarian cancer undergoing treatment. Cell-free DNA levels were quantified by a fluorometer (ng/ml) and/or real-time PCR (copies/ml). The associations between individuals with various degrees of genetic and/or environmental similarities and their plasma cfDNA levels were evaluated. The ACE model (A = additive genetic, C = common environment, and E = specific environmental factors) was used to determine the proportion of each factor on the cfDNA levels. We found a high correlation (r = 0.77; p < 0.0001) in plasma cfDNA levels between monozygotic twins (n = 39). However, the correlation was gradually reduced to moderate (r = 0.47; p = 0.016) between dizygotic twins (n = 13) and low correlation (r = 0.28; p = 0.043) between sibling pairs (n = 26). The ACE model analysis showed that the plasma cfDNA level of a given healthy individual is influenced both by genetic and the environmental components in similar proportions (53% and 47%, respectively; A = 53%, C = 22.5%, E = 24.5%). Moreover, while age had no effect, gender significantly influenced the individual's plasma cfDNA level. As expected, cfDNA levels were significantly higher in both breast (n = 26) (p<0.0001) and ovarian (n = 64) (p<0.0001) cancer patients compared to the healthy individuals. Our study demonstrated that both genome and environmental factors modulate the individual's cfDNA level suggesting that its diagnostic sensitivity may be improved only if the person's cfDNA level is known prior to disease presentation.

Novel mutations in actionable breast cancer genes by targeted sequencing in an ethnically homogenous cohort.

BACKGROUND: Genetic testing is becoming an essential tool for breast cancer (BC) diagnosis and treatment pathway, and particularly important for early detection and cancer prevention. The purpose of this study was to explore the diagnostic yield of targeted sequencing of the high priority BC genes. METHODS: We have utilized a cost-effective targeted sequencing approach of high priority actionable BC genes (BRCA1, BRCA2, ERBB2 and TP53) in a homogeneous patient cohort from Bangladesh (n = 52) by using tumor and blood samples. RESULTS: Blood derived targeted sequencing revealed 25.58% (11/43) clinically relevant mutations (both pathogenic and variants of uncertain significance (VUS)), with 13.95% (6/43) of samples carrying a pathogenic mutations. We have identified and validated five novel pathogenic germline mutations in this cohort, comprising of two frameshift deletions in BRCA2, and missense mutations in BRCA1, BRCA2 and ERBB2 gene respectively. Furthermore, we have identified three pathogenic mutations and a VUS within three tumor samples, including a sample carrying pathogenic mutations impacting both TP53 (c.322dupG; a novel frameshift insertion) and BRCA1 genes (c.116G > A). 22% of tissue samples had a clinically relevant TP53 mutation. Although the cohort is small, we have found pathogenic mutations to be enriched in BRCA2 (9.30%, 4/43) compare to BRCA1 (4.65%, 2/43). The frequency of germline VUS mutations found to be similar in both BRCA1 (4.65%; 2/43) and BRCA2 (4.65%; 2/43) compared to ERBB2 (2.32%; 1/43). CONCLUSIONS: This is the first genetic study of BC predisposition genes in this population, implies that genetic screening through targeted sequencing can detect clinically significant and actionable BC-relevant mutations.

Sequential combination of cisplatin with eugenol targets ovarian cancer stem cells through the Notch-Hes1 signalling pathway.

BACKGROUND: Ovarian carcinomas are the deadliest gynecological malignancies owing to their high rate of recurrence and high resistance to platinum-based chemotherapy. Recent studies have shown platinum-dependent enrichment of ovarian tumors with side population as well as cancer stem cells, which are highly resistant to the treatment. To overcome this treatment-limiting factor, we sought to combine cisplatin with eugenol, a natural substance known to have anti-cancer effects. METHODS: The efficiency of combining cisplatin with eugenol was first tested in vitro on two ovarian cancer cell lines SKOV3 and OV2774 using the WST1 and the flow cytometry techniques. The effect of this combination on ovarian cancer stem cells was determined by the tumorsphere formation assay, while the implication of the Notch pathway was evaluated post-ectopic expression of the Hes1 gene. The resulting changes in the expression of several markers was assessed by immunoblotting, immunofluorescence as well as quantitative RT-PCR. Cell sorting was also used to isolate specific ovarian cancer sub-population of cells. Furthermore, tumor-bearing mouse models were utilized to prove the potential therapeutic value of the cisplatin/eugenol combination treatment in vivo. RESULTS: We have shown that adding eugenol to cisplatin-treated ovarian cancer cells synergistically inhibited their growth and survival through induction of apoptosis. Importantly, this sequential inhibition strongly reduced the proportion of side population cells and suppressed cisplatin-dependent enrichment in ovarian cancer stem cells. Additionally, while increase in the level of Hes1 promoted stemness and enhanced resistance to cisplatin, cisplatin/eugenol cotreatment inhibited the Notch-Hes1 pathway and strongly downregulated the drug resistance ABC transporter genes. These findings were confirmed in vivo by showing that cisplatin/eugenol cotherapy inhibited tumor growth in animals, reduced the proportion and self-renewal capacities of cancer stem cells and significantly improved disease-free survival of tumor-bearing animals compared with either therapy alone. CONCLUSIONS: These results indicate that cisplatin/eugenol sequential combination could be of great therapeutic value for ovarian cancer patients through targeting the Notch-Hes1 pathway and the consequent elimination of the resistant cancer stem cells.

Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1.

BACKGROUND: A newly developed drug trastuzumab emtansine (T-DM1) has improved the survival of breast cancer (BC) patients. Despite an impressive initial clinical response, a subgroup of patient develop resistance and present therapeutic challenges. The underlying resistance mechanisms are not fully investigated. We report that T-DM1 treatment modulates the expression of ROR1 (type 1 receptor tyrosine kinase-like orphan receptor) and induces self-renewal of cancer stem cells (CSCs) leading to therapeutic resistance. METHODS: Using BC patient tumor samples, and BC cell lines we gained insight into the T-DM1 treatment induced ROR1 overexpression and resistance. In vitro sphere forming assays and in vivo extreme dilution assays were employed to analyze the stemness and self-renewal capacity of the cells. A series of molecular expression and protein assays including qRT-PCR, FACS-sorting, ELISA, immunostaining, Western blotting were used to provide evidence. FINDINGS: Exposure of cells to T-DM1 shifted ROR1 expression from low to high, enriched within the CSC subpopulation, coincident with increased Bmi1 and stemness factors. T-DM1 induced ROR1 cells showed high spheroid and tumor forming efficiency in vitro and in an animal model exhibiting shorter tumor-free time. Mechanistically, the overexpression of ROR1 is partly induced by the activation of YAP1 and its target genes. Silencing of ROR1 and YAP1 by pharmacologic inhibitors and/or sh/siRNA inhibited spheroid formation, the initiation of tumors and the capacity for self-renewal and ROR1 overexpression. INTERPRETATIONS: The results presented here indicate that simultaneous targeting of ROR1 and YAP1 may suppress CSC self-renewal efficacy and inhibit tumor progression in BC. In this manner such treatments may overcome the T-DM1 mediated therapeutic resistance and improve clinical outcome. FUND: This study was supported by Neurogen Technologies for interdisciplinary research.