Global proteomic characterization of microdissected estrogen receptor positive breast tumors

We here describe two proteomic datasets deposited in ProteomeXchange via PRIDE partner repository [1] with dataset identifiers PXD000484 (defined as "training") and PXD000485 (defined as "test") that have been used for the development of a tamoxifen outcome predictive signature [2]. Both datasets comprised 56 fresh frozen estrogen receptor (ER) positive primary breast tumor specimens derived from patients who received tamoxifen as first line therapy for recurrent disease. Patient groups were defined based on time to progression (TTP) after start of tamoxifen therapy (6 months cutoff): 32 good and 24 poor treatment outcome patients were comprised in the training set, respectively. The test set included 41 good and 15 poor treatment outcome patients. All specimens were subjected to laser capture microdissection (LCM) to enrich for epithelial tumor cells prior to high resolution mass spectrometric (MS) analysis. Protein identificat

Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer

In a previous study, we detected a significant association between phosphoserine aminotransferase 1 (PSAT1) hyper-methylation and mRNA levels to outcome to tamoxifen treatment in recurrent disease. We here aimed to study the association of PSAT1 protein levels to outcome upon tamoxifen treatment and to obtain more insight in its role in tamoxifen resistance. A cohort of ER positive, hormonal therapy naïve primary breast carcinomas was immunohistochemically (IHC) stained for PSAT1. Staining was analyzed for association with patient's time to progression (TTP) and overall response on first-line tamoxifen for recurrent disease. PSAT1 mRNA levels were also assessed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR; n = 161) and Affymetrix GeneChip (n = 155). Association of PSAT1 to biological pathways on tamoxifen outcome were assessed by global test. PSAT1 protein and mRNA levels were significantly associated to poor outcome to tamoxifen treatment. When comparing PSAT1

4-protein signature predicting tamoxifen treatment outcome in recurrent breast cancer

Estrogen receptor (ER) positive tumors represent the majority of breast malignancies, and are effectively treated with hormonal therapies, such as tamoxifen. However, in the recurrent disease resistance to tamoxifen therapy is common and a major cause of death. In recent years, in-depth proteome analyses have enabled identification of clinically useful biomarkers, particularly, when heterogeneity in complex tumor tissue was reduced using laser capture microdissection (LCM). In the current study, we performed high resolution proteomic analysis on two cohorts of ER positive breast tumors derived from patients who either manifested good or poor outcome to tamoxifen treatment upon recurrence. A total of 112 fresh frozen tumors were collected from multiple medical centers and divided into two sets: an in-house training and a multi-center test set. Epithelial tumor cells were enriched with LCM and analyzed by nano-LC Orbitrap mass spectrometry (MS), which yielded >3000 and >4000 quantified proteins in the training and test sets, respectively. Raw data are available via ProteomeXchange with identifiers PXD000484 and PXD000485. Statistical analysis showed differential abundance of 99 proteins, of which a subset of 4 proteins was selected through a multivariate step-down to develop a predictor for tamoxifen treatment outcome. The 4-protein signature significantly predicted poor outcome patients in the test set, independent of predictive histopathological characteristics (hazard ratio [HR] = 2.17; 95% confidence interval [CI] = 1.15 to 4.17; multivariate Cox regression p value = 0.017). Immunohistochemical (IHC) staining of PDCD4, one of the signature proteins, on an independent set of formalin-fixed paraffin-embedded tumor tissues provided and independent technical validation (HR = 0.72; 95% CI = 0.57 to 0.92; multivariate Cox regression p value = 0.009). We hereby report the first validated protein predictor for tamoxifen treatment outcome in recurrent ER-positive breast cancer. IHC further showed that PDCD4 is an independent marker

Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients

We have previously identified UMP-CMP kinase (CMPK1) as a prognostic marker for triple negative breast cancer (TNBC) by mass spectrometry (MS). In this study we evaluated CMPK1 association to prognosis in an independent set of samples by immunohistochemistry (IHC) and assessed biological pathways associated to its expression through gene set enrichment analysis (GSEA). A total of 461 TNBC paraffin-embedded tissues were collected from different academic hospitals in Europe, incorporated into tissue micro-arrays (TMA), and stained for CMPK1 expression. We also collected gene expression data of 60 samples, which were also present in the TMA, for GSEA correlation analysis. CMPK1 IHC staining showed both cytoplasmic and nuclear components. While cytoplasmic CMPK1 did not show any association to metastasis free survival (MFS), nuclear CMPK1 was associated to poor prognosis independently from other prognostic factors in stratified Cox regression analyses. GSEA correlation analysis of the nuclear CMPK1-stratified gene expression dataset showed a significant enrichment of extracellular matrix (ECM; positive correlation) and cell cycle (negative correlation) associated genes. We have shown here that nuclear CMPK1 is indicative of poor prognosis in TNBCs and that its expression may be related to dysregulation of ECM and cell cycle molecules

Development of a questionnaire specifically for patients with Ileal Orthotopic Neobladder (IONB)

The ileal orthotopic neobladder (IONB) is often used in patients undergoing radical cystectomy. The IONB allows to void avoiding the disadvantages of the external urinary diversion.In IONB patients the quality of life (QoL) appears compromised by the need to urinate voluntarily. The patients need to wake up at night interrupting the sleep-wake rhythm with consequences on social and emotional life.At present the QoL in IONB patients is evaluated by generic questionnaires. These are useful when IONB patients are compared with patients with different urinary diversions but they are less effective when only IONB patients are evaluated. To address this problem a specific questionnaire-the IONB-PRO-was developed. METHODS: A) Based on a conceptual framework, narrative-based interviews were conducted on 35 IONB patients. A basic pool of 43 items was produced and organized throughout two clinical and four QoL dimensions. An additional 15 IONB patients were interviewed for face validity testing.B) Psychometric testing was conducted on 145 IONB patients. Both classic test strategy and Rasch analysis were applied. Psychometric properties of the resulting scales were comparatively tested against other QoL-validated scales. RESULTS: The IONB-PRO questionnaire includes two sections: one on the QoL and a second section on the capability of the patient to manage the IONB. For evaluation of the QoL, three versions were delivered: 1) a basic 23-item QoL version (3 domains 23-items; alpha 0.86÷ 9.69), 2) a short-form 12-item QoL scale (alpha = 0.947), and 3) a short-form 15-item Rasch QoL scale (alpha = 0.967). Correlations of the long version scales with the corresponding dimensions of the EORTC-QLQ C30 and the EORTC-BLM30 were significant. The short forms exhibited significant correlations with the global health dimension of the EORTC-QLQ and with the urinary subscales of the EORTC-BLM30. The effect size was approximately 1.00 between patients at the 1-year follow-up period and those with 3, 5, and > 5-year follow-up periods for all scales. No relevant differences were observed between the 12-item short-form and the Rasch scale. CONCLUSIONS: The IONB-PRO long and short-forms demonstrated a high level of internal consistency and reliability with an excellent discriminanting validity

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Annexin-A1 and caldesmon are associated with resistance to tamoxifen in estrogen receptor positive recurrent breast cancer

Tamoxifen therapy resistance constitutes a major cause of death in patients with recurrent estrogen receptor (ER) positive breast cancer. Through high resolution mass spectrometry (MS), we previously generated a 4-protein predictive signature for tamoxifen therapy outcome in recurrent breast cancer. ANXA1 and CALD1, which were not included in the classifier, were however the most differentially expressed proteins. We first evaluated the clinical relevance of these markers in our MS cohort, followed by immunohistochemical (IHC) staining on an independent set of tumors incorporated in a tissue microarray (TMA) and regression analysis in relation to time to progression (TTP), clinical benefit and objective response. In order to assess which mechanisms ANXA1 and CALD1 might been involved in, we performed Ingenuity pathway analysis (IPA) on ANXA1 and CALD1 correlated proteins in our MS cohort. ANXA1 (Hazard ratio [HR] = 1.83; 95% confidence interval [CI]: 1.22-2.75; P = 0.003) and CALD1 (HR = 1.57; 95% CI: 1.04-2.36; P = 0.039) based patient stratification showed significant association to TTP, while IHC staining on TMA showed that both ANXA1 (HR = 1.82; 95% CI: 1.12-3.00; P = 0.016) and CALD1 (HR = 2.29; 95% CI: 1.40-3.75; P = 0.001) expression was associated with shorter TTP independently of traditional predictive fa

Ferritin heavy chain in triple negative breast cancer: A favorable prognostic marker that relates to a cluster of differentiation 8 positive (CD8+) effector t-cell response

Ferritin heavy chain (FTH1) is a 21-kDa subunit of the ferritin complex, known for its role in iron metabolism, and which has recently been identified as a favorable prognostic protein for triple negative breast cancer (TNBC) patients. Currently, it is not well understood how FTH1 contributes to an anti-tumor response. Here, we explored whether expression and cellular compartmentalization of FTH1 correlates to an effective immune response in TNBC patients. Analysis of the tumor tissue transcriptome, complemented with in silico pathway analysis, revealed that FTH1 was an integral part of an immunomodulatory network of cytokine signaling, adaptive immunity, and cell death. These findings were confirmed using mass spectrometry (MS)-derived proteomic data, and immunohistochemical staining of tissue microarrays. We observed that FTH1 is localized in both the cytoplasm and/or nucleus of cancer cells. However, high cytoplasmic (c) FTH1 was associated with favorable prognosis (Log-rank p = 0.001), whereas nuclear (n) FTH1 staining was associated with adverse prognosis (Log-rank p = 0.019). cFTH1 staining significantly correlated with total FTH1 expression in TNBC tissue samples, as measure

Carbon nanotube-mediated wireless cell permeabilization:drug and gene uptake

AIM: This work aims to exploit the 'antenna' properties of multiwalled carbon nanotubes (MWCNTs). They can be used to induce cell permeabilization in order to transfer drugs (normally impermeable to cell membranes) both in in vitro and in vivo models. MATERIAL & METHODS: The performance of the MWCNTs as receiver antenna was modeled by finite element modeling. Once the appropriate field has been identified, the antenna properties of MWCNTs were investigated in sequential experiments involving immortalized fibroblast cell line (drug model: doxorubicin chemotherapeutic agent) and living mice (drug model: bcl-2 antiapoptotic gene) following stereotactic injection in the cerebral motor cortex. RESULTS: Finite element modeling analysis predicts that our MWCNTs irradiated in the radiofrequency field resemble thin-wire dipole antennas. In vitro experiments confirmed that combination of MWCNTs and electromagnetic field treatment dramatically favors intracellular drug uptake and, most importantly, drug nuclear localization. Finally, the brain of each irradiated animal exhibits a significantly higher number of transfected cells compared with the appropriate controls. CONCLUSION: This wireless application has the potential for MWCNT-based intracellular drug delivery and electro-stimulation therapies