Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Specific delivery of therapeutic agents against cancers

Conventional treatments for solid tumors involve removing the malignant tissues by surgery followed by chemotherapy and/or radiotherapy. Despite the effectiveness of these therapies, the side effects of chemotherapy and radiotherapy are very high because of lack of specificity to target malignant cells. Another problem is the development of resistance to these treatments by cancer cells Targeted drug delivery strategies have been developed to improve therapeutic agents efficacy and to reduce non-specific side effects by delivering the therapeutic agents specifically to target cells. Several approaches have been investigated to achieve this aim. The differences in cell surface receptors expression have widely been used to deliver therapeutic agents by conjugating them with specific ligands such as hormones, vitamins, folic acid, peptide, transferrin, aptamers, monoclonal antibodies and their fragments. In order to deliver therapeutic agents specifically to cancer cells we have conjugated therapeutic agents to a single chain antibody or to an aptamer that bind specifically to cancer cell receptors. Two types of therapeutic agents were conjugated to the scFv-425 single chain recognizing EGFR, the photosensitizer chlorin e6 and the chemotherapy payload polyglycerol-doxorubicin dendrimer. Standard methods for protein conjugation yield heterogeneous products containing a mixture of conjugated antibodies with the effector molecules conjugated at different sites and a variable number of effector molecules conjugated to each antibody resulting in a range of molar ratios and different pharmacokinetic, efficacy and safety profiles. To avoid the protein conjugation limitations, we investigated the use of SNAP-tag technology to provide a unique conjugation site on the antibody allowing the production of homogeneous conjugate preparations. We engineered a construct in which the coding sequence of a scFv that binds specifically to EGFR (scFv-425) was genetically fused to the SNAP tag cassette, which endows the antibody with a SNAP-tag and therefore allows site-specific conjugation to BG-modified substrates. This conjugation method can be applied to any antibody–therapeutic agent combination as long as the antibody carries the SNAP-tag and the substrate is modified with a BG group. We found that scFv-425-CAT-AD-SNAP conjugated to BG-modified Ce6 can selectively kill EGFR+ cells in four human tumor-derived cell lines representing epidermal, breast and cervical carcinomas (A431, MDA-MB-231, MDA-MB468 and SiHa) after exposure to light. Also scFv-425-CAT-AD-SNAP conjugated to BG-modified dendritic polymer that loaded with doxorubicin molecules kill three human tumor-derived cell lines representing epidermal, breast and pancreatic carcinomas (A431, MDA-MB-468 and Panc-1). In conclusion, we have demonstrated for the first time the conjugation of therapeutic agents to a scFv using SNAP-tag technology. This rapid and efficient method produces a homogeneous conjugate with defined pharmacokinetic and therapeutic profiles, and provides proof of principle that SNAP-tag technology can be used to conjugate therapeutic agents for cancer therapy, avoiding the off-target effects that have thus far limited the development of targeted drug delivery. In another approach RNA aptamer that binds specifically to cancer cell receptor has been used to deliver siRNA against cancer cells. siRNAs silence gene expression by triggering the sequence-specific degradation of mRNAs, but the targeted delivery of such reagents remains challenging and a significant obstacle to therapeutic applications. One promising approach is the use of RNA aptamers that bind to tumor-associated antigens as targeting moieties to achieve the delivery of siRNAs to tumor cells bearing specific antigens. RNA-based constructs are advantageous because they are inexpensive to synthesize and they have a low immunogenicity. We therefore joined an aptamer recognizing avB3 integrin to a siRNA that targets eukaryotic elongation factor 2 gene, and achieved for the first time the targeted delivery of a siRNA to tumor cells expressing avB3 integrin causing inhibition of cell proliferation and induction of apoptosis in tumor cells. In conclusion, we have demonstrated for the first time that an aptamer specifically binding to avB3 integrin can be used to deliver a cytotoxic siRNA to tumor cells expressing avB3 integrin. This allows the development of therapeutic modalities composed solely of RNA, reducing production costs and potential immunogenicity compared to protein therapeutics. The low cost, low immunogenicity and minimal off-target effects achieved by the specific targeting of tumor cells suggest that aptamer-siRNA chimeras could represent an exciting and efficacious new paradigm for cancer therapy with a significant potential impact in the clinic

Specific delivery of therapeutic agents against cancers

Conventional treatments for solid tumors involve removing the malignant tissues by surgery followed by chemotherapy and/or radiotherapy. Despite the effectiveness of these therapies, the side effects of chemotherapy and radiotherapy are very high because of lack of specificity to target malignant cells. Another problem is the development of resistance to these treatments by cancer cells Targeted drug delivery strategies have been developed to improve therapeutic agents efficacy and to reduce non-specific side effects by delivering the therapeutic agents specifically to target cells. Several approaches have been investigated to achieve this aim. The differences in cell surface receptors expression have widely been used to deliver therapeutic agents by conjugating them with specific ligands such as hormones, vitamins, folic acid, peptide, transferrin, aptamers, monoclonal antibodies and their fragments. In order to deliver therapeutic agents specifically to cancer cells we have conjugated therapeutic agents to a single chain antibody or to an aptamer that bind specifically to cancer cell receptors. Two types of therapeutic agents were conjugated to the scFv-425 single chain recognizing EGFR, the photosensitizer chlorin e6 and the chemotherapy payload polyglycerol-doxorubicin dendrimer. Standard methods for protein conjugation yield heterogeneous products containing a mixture of conjugated antibodies with the effector molecules conjugated at different sites and a variable number of effector molecules conjugated to each antibody resulting in a range of molar ratios and different pharmacokinetic, efficacy and safety profiles. To avoid the protein conjugation limitations, we investigated the use of SNAP-tag technology to provide a unique conjugation site on the antibody allowing the production of homogeneous conjugate preparations. We engineered a construct in which the coding sequence of a scFv that binds specifically to EGFR (scFv-425) was genetically fused to the SNAP tag cassette, which endows the antibody with a SNAP-tag and therefore allows site-specific conjugation to BG-modified substrates. This conjugation method can be applied to any antibody–therapeutic agent combination as long as the antibody carries the SNAP-tag and the substrate is modified with a BG group. We found that scFv-425-CAT-AD-SNAP conjugated to BG-modified Ce6 can selectively kill EGFR+ cells in four human tumor-derived cell lines representing epidermal, breast and cervical carcinomas (A431, MDA-MB-231, MDA-MB468 and SiHa) after exposure to light. Also scFv-425-CAT-AD-SNAP conjugated to BG-modified dendritic polymer that loaded with doxorubicin molecules kill three human tumor-derived cell lines representing epidermal, breast and pancreatic carcinomas (A431, MDA-MB-468 and Panc-1). In conclusion, we have demonstrated for the first time the conjugation of therapeutic agents to a scFv using SNAP-tag technology. This rapid and efficient method produces a homogeneous conjugate with defined pharmacokinetic and therapeutic profiles, and provides proof of principle that SNAP-tag technology can be used to conjugate therapeutic agents for cancer therapy, avoiding the off-target effects that have thus far limited the development of targeted drug delivery. In another approach RNA aptamer that binds specifically to cancer cell receptor has been used to deliver siRNA against cancer cells. siRNAs silence gene expression by triggering the sequence-specific degradation of mRNAs, but the targeted delivery of such reagents remains challenging and a significant obstacle to therapeutic applications. One promising approach is the use of RNA aptamers that bind to tumor-associated antigens as targeting moieties to achieve the delivery of siRNAs to tumor cells bearing specific antigens. RNA-based constructs are advantageous because they are inexpensive to synthesize and they have a low immunogenicity. We therefore joined an aptamer recognizing avB3 integrin to a siRNA that targets eukaryotic elongation factor 2 gene, and achieved for the first time the targeted delivery of a siRNA to tumor cells expressing avB3 integrin causing inhibition of cell proliferation and induction of apoptosis in tumor cells. In conclusion, we have demonstrated for the first time that an aptamer specifically binding to avB3 integrin can be used to deliver a cytotoxic siRNA to tumor cells expressing avB3 integrin. This allows the development of therapeutic modalities composed solely of RNA, reducing production costs and potential immunogenicity compared to protein therapeutics. The low cost, low immunogenicity and minimal off-target effects achieved by the specific targeting of tumor cells suggest that aptamer-siRNA chimeras could represent an exciting and efficacious new paradigm for cancer therapy with a significant potential impact in the clinic

Inhibitors of PD-1/PD-L1 and ERK1/2 impede the proliferation of receptor positive and triple-negative breast cancer cell lines

Purpose!#!Triple-negative breast cancer (TNBC) is characterized by an unfavorable prognosis and missing systemic therapeutic approaches beside chemotherapy. Targeting the immune checkpoint PD-1/PD-L1 showed promising results in breast cancer and especially in TNBC. The extracellular signal-regulated kinase 1/2 (ERK1/2) is an important driver of carcinogenesis. Here, the effect of combined PD-1/PD-L1 and ERK1/2 inhibitor treatment is investigated of cell growth and intracellular impact of breast cancer cell lines.!##!Methods!#!The IC!##!Results!#!Both inhibitors PD-1/PD-L1 and ERK1/2 impeded the proliferation of TNBC to a higher extent than of non-TNBC. By combined treatment, cell lines were inhibited either synergistically or additively. ERK1/2 and S6 phosphorylation were reduced and expressions of c-Fos and FosL were diminished after ERK1/2 inhibitor as single and combined treatment. Between genes involved in immune modulation, IL-8 was upregulated in TNBC cells after combined treatment.!##!Conclusion!#!In conclusion, combination of PD-1/PD-L1 and ERK1/2 inhibitors showed favorable effects for a new therapy strategy, with better results in TNBC cell lines than in non-TNBC cells. The effects have to be validated in models that can reflect the interaction between immune and tumor cells like the situation in the tumor micro-environment

Near Infrared Photoimmunotherapy: A Review of Recent Progress and Their Target Molecules for Cancer Therapy

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed molecular targeted cancer treatment, which selectively kills cancer cells or immune-regulatory cells and induces therapeutic host immune responses by administrating a cancer targeting moiety conjugated with IRdye700. The local exposure to near-infrared (NIR) light causes a photo-induced ligand release reaction, which causes damage to the target cell, resulting in immunogenic cell death (ICD) with little or no side effect to the surrounding normal cells. Moreover, NIR-PIT can generate an immune response in distant metastases and inhibit further cancer attack by combing cancer cells targeting NIR-PIT and immune regulatory cells targeting NIR-PIT or other cancer treatment modalities. Several recent improvements in NIR-PIT have been explored such as catheter-driven NIR light delivery, real-time monitoring of cancer, and the development of new target molecule, leading to NIR-PIT being considered as a promising cancer therapy. In this review, we discuss the progress of NIR-PIT, their mechanism and design strategies for cancer treatment. Furthermore, the overall possible targeting molecules for NIR-PIT with their application for cancer treatment are briefly summarised

A specific photoimmunotheranostics agent to detect and eliminate skin cancer cells expressing EGFR

Purpose: The term “theranostics” represents a new paradigm in medicine especially for cancer treatment. This term was coined by Funkhouser in 2002 and defines a reagent that combines therapeutic and diagnostic properties. It is widely believed that theranostics agents will have considerable impact on healthcare before, during, and after disease by improving cancer prognosis and management simultaneously. Current theranostics approaches still rely on passive tumor targeting strategies, which have scattergun effects and tend to damage both neoplastic and non-neoplastic cells. Methods: Here we describe a simple, controlled, and efficient method to generate homogeneous photoimmunotheranostics reagents. This method combines molecular optical imaging, photodynamic therapy, and immunotherapy using SNAP-tag technology. SNAP-tag is a derivative of the O(6)-alkylguanine-DNA alkyltransferase (AGT) which has the ability to efficiently conjugate to O(6)-benzylguanine (BG) molecules under physiological conditions depending on its folding pattern. Results: The theranostics agent was able to specifically recognize various epidermal growth factor receptor (EGFR)-expressing skin cancer cell lines using flow cytometry analysis and confocal microscopy and eliminate them at EC50’s of 32–55 nM. Conclusions: These experiments provide a framework for using SNAP-tag technology to generate homogeneous photoimmunotheranostics reagents with unified pharmacokinetic and therapeutic profiles. Furthermore, the reagent generated in this work could be used to simultaneously monitor and suppress the growth of skin squamous carcinoma and melanoma cells expressing EGFR.National Research Foundation (South Africa

EpCAM- and EGFR-Specific Antibody Drug Conjugates for Triple-Negative Breast Cancer Treatment

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC

Targeted Delivery of Dendritic Polyglycerol–Doxorubicin Conjugates by scFv-SNAP Fusion Protein Suppresses EGFR+ Cancer Cell Growth

Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-Tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with single-chain antibodies to specifically target and destroy cancer cells