Total hip arthroplasty (THA) is one of the most successful and widely accepted orthopedic procedures. Instability after THA is one of the most significant postoperative complications. Dual-mobility THA components were introduced in 1974 to overcome the risk of instability by increasing the jump distance. Dual-mobility bearings couple two articulations, namely, one between a 22-28 mm prosthetic head and polyethylene liner and another larger articulation between the polyethylene liner and the metal cup. Dislocation of the polyethylene liner and the consequent direct articulation between the prosthetic head and metal cup is recognized as intraprosthetic dislocation (IPD). This mode of THA failure is specific to dual-mobility implants. Despite the reduced incidence of IPD in modern dual-mobility implants compared to the early designs, iatrogenic IPD can occur during closed reduction of dislocated polyethylene liner-metal cup articulation. IPD requires timely diagnosis and early surgical intervention to minimize the necessity of major revision surgeries. This study presents a comprehensive review for dual-mobility-bearing THA, including the history and biomechanics, and focuses on the pathomechanics, diagnosis, and management of IPD. Total hip arthroplasty (THA) is one of the most successful orthopedic procedures widely used to treat advanced hip joint arthritis and femoral neck fractures ,2. More History of dual-mobility componentsThe concept of THA dual-mobility cups was first introduced in France by Bousquet and Rambert in 1974 to overcome the risk of dislocation after THA ,17. The The NOVAE acetabular cup was uncemented plasma-sprayed alumina coated with an inner stainless steel-polished surface ,17. The Biomechanics and design of dual-mobility implantsDual-mobility-bearing THA incorporates a small prosthetic head (22 or 28 mm in diameter) which is freely mobile but constrained within a larger polyethylene liner ,17,18. TIncidence of intraprosthetic dislocationIPD is a specific complication of THA incorporating dual-mobility-bearing components -17. The Types and causes of intraprosthetic dislocationIPD may not be proceeded by a traumatic event and can occur at any point after dual-mobility THA surgery. The leading cause of IPD is polyethylene liner wear, especially before introducing ultra-high-molecular-weight polyethylene liners . In 2013Iatrogenic IPD may occur without polyethylene liner wear during closed reduction of the polyethylene liner-metal cup dislocation Figures , 2. The Presentation and diagnosis of intraprosthetic dislocationDue to the direct articulation between the prosthetic head and the metal cup, limbing, leg shortening, and grinding sensations in the affected limb are common findings in IPD. Rarely, IPD may only present with hip discomfort and leg weakness .Plain radiographs can help identify an eccentric position of the prosthetic head within the cup due to the direct contact between the head and the metal cup Figure . This siIf the “bubble sign” is not clear or the plain radiographs are not conclusive, a computed tomography (CT) scan should be obtained . CT scanManagement of intraprosthetic dislocation of dual-mobility implantsMetal cup in dual-mobility THA implants is not designed for direct articulation with the prosthetic head. The delay in IPD diagnosis may cause damage and wear of the prosthetic head and/or the acetabular metal cup. The friction between the prosthetic head and metal cup wear may result in significant soft tissue metallosis and raised cobalt and chromium levels ,31. EarlIPD requires an operative intervention to revise the damaged polyethylene liner and restore dual-mobility articulations . It is recommended to obtain a CT scan to assess the alignment of the acetabular cup and identify any prosthetic damage prior to the surgery. During the revision surgery, the acetabular metal cup should be checked for stability and signs of wear. Revision of the cup should be performed at the same stage if signs of loosening and wear are noticed. The prosthetic head and the prosthesis neck should also be examined carefully to identify any signs of wear or damage that require revision. IPD without prosthetic head damage and stable, intact acetabular shell can be managed by only revising the polyethylene liner.IPD is a specific complication noted in dual-mobility-bearing THA. This mode of failure is mainly caused by the polyethylene liner wear resulting in failure of the captive mechanism and dissociation of the prosthetic head-polyethylene liner articulation. Iatrogenic IPD may result from closed reduction attempts involving dual-mobility-bearing THA. Post-manipulation radiographs showing eccentric head position within the metal cup and the “bubble sign” of the migrated polyethylene liner are diagnostic for IPD. If radiographs are not conclusive and IPD is suspected, a CT scan should be obtained. IPD requires operative intervention to revise the polyethylene liner and any other damaged or loose implant components.


Randomised controlled trials (RCTs) for surgical interventions have often proven difficult with calls for innovative approaches. The Imperial Prostate (IP4) Comparative Health Research Outcomes of Novel Surgery in prostate cancer (IP4-CHRONOS) study aims to deliver level 1 evidence on outcomes following focal therapy which involves treating just the tumour rather than whole-gland surgery or radiotherapy. Our aim is to test the feasibility of two parallel RCTs within an overarching strategy that fits with existing patient and physician equipoise and maximises the chances of success and potential benefit to patients and healthcare services.IP4-CHRONOS is a randomised, unblinded multi-centre study, including two parallel randomised controlled trials targeting the same patient population: IP4-CHRONOS-A and IP4-CHRONOS-B.IP4-CHRONOS-A is a 1:1 RCT and the other is a multi-arm, multi-stage (MAMS) RCT starting with three arms and a 1:1:1 randomisation. The two linked RCTs are discussed with patients at the time of consent and the choice of A or B is dependent on physician and patient equipoise. The primary outcome is the feasibility of recruitment, acceptance of randomisation and compliance to allocated arm.This paper describes the statistical analysis plan (SAP) for the feasibility study within IP4-CHRONOS given its innovative approach. Version 1.0 of the SAP has been reviewed by the Trial Steering Committee (TSC), Chief Investigator (CI), Senior Statistician and Trial Statistician and signed off. The study is ongoing and recruiting. Recruitment is scheduled to finish later in 2021. The SAP documents approved methods and analyses that will be conducted. Since this is written in advance of the analysis, we avoid bias arising from prior knowledge of the study data and findings.Our feasibility analysis will demonstrate if IP4-CHRONOS is feasible in terms of recruitment, randomisation and compliance, and whether to continue both A and B or just one to the main stage.ISRCTN17796995. Registered on 08 October 2019ISRCTN  Randomised controlled trials (RCTs) for surgical interventions have often proven difficult with calls for innovative approaches. The Imperial Prostate (IP4) Comparative Health Research Outcomes of Novel Surgery in prostate cancer (IP4-CHRONOS) study aims to deliver level 1 evidence on outcomes following focal therapy which involves treating just the tumour rather than whole-gland surgery or radiotherapy. Our aim was to test the feasibility of two parallel RCTs within an overarching strategy that fits with existing patient and physician equipoise. The study protocol has been published .The target sample size for IP4-CHRONOS-A was adjusted because of centre opening problems and allocated resources to the study due to the Coronavirus Disease 2019 (COVID-19) pandemic. The minimum number of participants required to assess the IP4-CHRONOS-A feasibility of recruitment is 36 in 8 months (recruitment rate of 33% ± 15%). The maximum number of participants remains at 60 as per the study sample size calculation.Tumour grade Local stage  T3)Previous or current 5-alpha reductase inhibitor use (for A only)Randomisation is blocked and stratified by the following stratification factors:The feasibility objective for IP4-CHRONOS-A is to determine if patients agree to participate in an RCT that randomly assigns them to focal therapy alone or radical therapy (radiotherapy or prostatectomy). The feasibility objective for IP4-CHRONOS-B is to determine if patients expressing a preference for focal therapy agree to participate in a multi-arm, multi-stage (MAMS) RCT that randomly assigns them to focal therapy alone or focal therapy in combination with neoadjuvant and/or adjuvant agents.The primary outcome measures for both A and B are the feasibility of recruitment, acceptance of randomisation and compliance to allocated arm. The feasibility of recruitment will be determined by recruitment rates to each study. Recruitment rate is defined as the total number of patients recruited (consented) out of the total number of patients approached. Randomisation rate is defined as the total number of patients randomised out of the total number of patients recruited (consented). Recruitment and randomisation rates will be calculated for A and B separately. Recruitment to the study is defined as the patient giving informed consent, and so the date of recruitment is equal to the date of informed consent.Compliance comprises treatment compliance and drug compliance. Treatment compliance is measured in both A and B. Treatment compliance is defined as the proportion of patients who underwent treatment as randomly allocated. Drug compliance is only measured in IP4-CHRONOS-B for those patients who are randomly allocated to receive focal therapy plus a neoadjuvant drug treatment. Drug compliance is defined using two definitions: the proportion of patients who return their empty blister packs and the proportion of patients who are given the neoadjuvant drug and who do not have a registered protocol deviation (stating that the drug was taken for less than 8 weeks). Both estimates for drug compliance will be presented, and the Trial Management Group, independent Trial Steering Committee (TSC) and future funders will determine what constitutes feasibility of compliance. Participants who drop out of the study before compliance data has been collected will be recorded as missing and will not be included in the compliance analysis.Patients’ experience of each treatment arm including systemic issues, erectile dysfunction, urinary symptoms and rectal symptoms will be summarised using their responses to Patient Reported Outcome Measures (PROMS): EQ-5D-5L, IIEF-15, EPIC-26 and EPIC-Urinary Domain. These will be investigated using frequency tables and graphical visualisations at the relevant time points.For each of IP4-CHRONOS-A and IP4-CHRONOS-B, progression to the main stage will be deemed appropriate if the recruitment rate is above 21.1% .If the overall recruitment rate, estimated at the end of the feasibility study, is below 21.1%, this will suggest that the trial would have low recruitment feasibility and, after completion of patient follow-up for a minimum of 3 months, should not proceed to the main stage. The results will be presented to the sponsor, Trial Management Group, the TSC and future funders. If the recruitment rate is between 21.1 and 33%, then identifiable remedial work to improve the recruitment rate will be implemented in the main phase of the trial. If the recruitment rate is greater than or equal to 33%, the trial will be deemed feasible.If the trial does not proceed to the main stage following the analysis of the feasibility study, patients will still be followed up for a minimum of 3 months and will then revert to standard of care in which the clinical care provided to patients will not differ from the clinical follow-up stipulated in the protocol. At the end of the study, patients will continue to be followed up locally within their recruitment centres with the ICE  or the HEAT international HIFU registry as per NICE guidelines IPG432/IPG42.For each of IP4-CHRONOS-A and IP4-CHRONOS-B, if the lower end of the confidence interval for the proportion of patients who underwent treatment is ≥ 80%, then we have viable compliance to progress to the main phase. If the lower end of the confidence interval for the proportion of patients who underwent treatment is between 70 and 80%, then this indicates that identifiable remedial work might be needed to improve this for the main stage. If the lower end of the confidence interval for the proportion of patients who underwent treatment is < 70%, then this could affect the primary outcome analysis and threaten the validity of the study.For IP4-CHRONOS-B only, if the lower end of the confidence interval for the proportions of patients who complied to taking the neoadjuvant drug is between 80 and 90%, then this indicates that identifiable remedial work may be needed to improve this for the main stage. If the lower end of the confidence interval for the proportions is < 80%, then this could affect the primary outcome analysis and threaten the validity of the study and may require sample size re-estimation.The integrated qualitative component is designed to inform the primary and secondary trial objectives in the trial recruitment and testing stages. Participant interview data highlighting trial processes in need of improvement may be used in real time to allow timely protocol amendments to improve recruitment and retention of participants. Healthcare professionals  responsible for recruiting patients will also be interviewed.A data analysis plan of the qualitative component has been written, and the analysis will be conducted by the team at the School of Medicine at Cardiff University. Anonymised transcripts will be analysed using deductive thematic analysis techniques, with a coding framework developed to reflect the trial outcomes. Analysis will begin with two qualitative researchers individually coding the first three interview transcripts. The data sets will be coded in full and organised into themes and subthemes. This data will be presented in a narrative format and interpreted within the context of patient experience to inform outcomes and reflect the aims of the trial.All feasibility outcomes will include all patients recruited (consented) to the trials (IP4-CHRONOS-A and B) during the feasibility phase, by arm.Summaries of continuous variables will be presented as means and standard deviations if approximately normally distributed, and as medians and inter-quartile ranges for skewed data; categorical variables will be presented as frequencies and percentages. Normality of continuous variables will be checked visually by plotting the data and inspecting the distribution. Proportions calculated as part of the feasibility outcomes analyses will be presented along with corresponding 95% confidence intervals. A 5% significance level will be used.Baseline characteristics will be summarised by trial and by arm. These include demographics  decile), Digital Rectal Examination results, details of current medications, International Prostate Symptom Score (IPSS) score, and Maximum Cancer Core Length (MCCL) and Gleason grade at pre-enrolment biopsy. Baseline characteristics will also be summarised for those who withdrew and those who completed each trial.The total number of patients recruited to IP4-CHRONOS will be calculated and reported. Also, the number of patients recruited to A and B will be reported to estimate the feasibility for each RCT.The primary analysis of the feasibility study will calculate the mean number of patients recruited and randomised per month, per centre (including only the months in which the centre is open and recruiting). Overall recruitment and randomisation rates will also be calculated, along with their corresponding 95% confidence intervals. Graphs displaying the recruitment and randomisation rates over time will be presented. A graph displaying the actual versus target recruitment rates will also be presented.The proportions of patients in each treatment arm who underwent treatment will be presented, along with their corresponding 95% confidence intervals. Treatment compliance will be evaluated by arm and by each RCT (A or B). Viable treatment compliance is defined as the lower end of the confidence interval for the proportion of patients who underwent treatment being ≥ 80%.Neoadjuvant drug treatment in IP4-CHRONOS-B is prescribed at randomisation. The proportions who returned their empty blister packs, and the proportions who took the drug and did not have a registered protocol deviation, stating that they took the drug for less than 8 weeks, in each treatment arm will be reported, along with their corresponding 95% confidence intervals. Drug compliance will be evaluated by arm for each of the drug compliance definitions. Viable drug compliance is defined as the lower end of the confidence interval for the proportions being ≥ 90%.https://www.ctu.mrc.ac.uk/our-research/methodology/conduct/kmunicate/) which includes displaying the uncertainty around the survival curves and risk tables for the number of patients at risk at each time point. Safety data will be reported in line with the Imperial Clinical Trials Unit (ICTU) standard operating procedures.Other analyses for the primary outcomes include Kaplan-Meier curves presenting the time from randomisation to withdrawal, summary statistics of time between key events throughout the trial  and safety (adverse and serious adverse events). Kaplan-Meier analysis will be reported using the KMunicate format (Reported adverse events (AEs) and serious adverse events (SAEs) will be listed and then summarised, by treatment arm, in terms of severity grade (using v4.0 Common Terminology Criteria for Adverse Events (CTCAE) grading) and causal relationship to treatment, for IP4-CHRONOS-A and B separately.Patient-reported outcome measures (PROMs) are collected at baseline and 3 months. These include EQ-5D-5L, IIEF-15, EPIC-26 and EPIC-Urinary Domain. These analyses will be conducted for IP4-CHRONOS-A and IP4-CHRONOS-B.EQ-5D-5L has five dimensions: mobility, self-care, usual activities, pain/discomfort and anxiety/depression, each measured using five levels of response . SummaryEQ visual analogue scale (VAS) is an additional question on the EQ-5D-5L . This wiIIEF-15 consists of five domains: erectile function, orgasmic function, sexual desire, intercourse satisfaction and overall satisfaction . EPIC-26On 05/03/2020, COVID-19 was added to Public Health England’s list of notifiable diseases in England and Wales . This daPrimary outcome analysis concerning recruitment and randomisation will be calculated, separately, for those patients who were randomised before and during/after the COVID-19 pandemic. Primary outcome analysis concerning treatment compliance will be calculated, separately, for those patients who started treatment before and during/after the COVID-19 pandemic. Drug compliance analysis will be calculated, separately, for those patients who were randomly allocated to focal plus neoadjuvant drug treatment  in IP4-CHRONOS-B and started their neoadjuvant drug treatment before and during/after the COVID-19 pandemic.Summary statistics of time between key events throughout the trial  will be calculated for patients whose most recent event is before and during/after the COVID-19 pandemic. Reasons for withdrawal will be reported for patients who withdrew from the study before and during/after the COVID-19 pandemic.These analyses will be presented if there exist patients in both the before and during/after COVID-19 analysis populations. Analyses for populations which do not contain any patients will be omitted as they are already included in the main study analysis.COVID-19-related protocol deviations will be summarised alongside other protocol deviations for the study.A specific missing data mechanism is not required for the feasibility study. No formal method will be used for handling outliers.STATA v17 (or above) will be used for all analyses.IP4-CHRONOS provides an innovative trial design in what is recognised as a difficult-to-recruit disease space, localised prostate cancer, especially given the surgical interventions involved. The study attempts to match patient and physician equipoise and might provide valuable insights into whether such an overarching strategy might provide some solutions to the ongoing problems we have had in delivering randomised comparative trials in these areas. Our planned analysis strategy for the feasibility phase has been set out here to reduce the risk of reporting bias and data-driven analysis. Any deviations from the methods described in this paper will be detailed and justified fully in the final statistical report.


Polyoxalate (POx) and copolyoxalate (CPOx) smart polymers are topics of interest the field of inflammation. This is due to their drug delivery ability and their potential to target reactive oxygen species (ROS) and to accommodate small molecules such as curcumin, vanilline, and p-Hydroxybenzyl alcohol. Their biocompatibility, ultra-size tunable characteristics and bioimaging features are remarkable. In this review we discuss the genesis and concept of oxylate smart polymer-based particles and a few innovative systemic delivery methods that is designed to counteract the inflammation and other aging-associated diseases (AADs). First, we introduce the ROS and its role in human physiology. Second, we discuss the polymers and methods of incorporating small molecule in oxalate backbone and its drug delivery application. Finally, we revealed some novel proof of concepts which were proven effective in disease models and discussed the challenges of oxylate polymers. Aging-associated disease (AAD) is an umbrella term that describes simple and complex pathological conditions which are only observed in people of advanced age in both male and female affecting one or more organs . Oxygen-Understanding the mechanisms involved in aging is an arduous task. A shift in balance between free radicals and oxidants within the cell depends upon its genetic makeup. Genes play a decisive role in aging and determining the redox balance. While genetic association with AAD is beyond the scope of this work, the production of free radicals and ROS are AAD-linked. It is difficult to differentiate normal aging and AAD, but free radicals and ROS are a key culprit in majority of the cases .2O2), superoxide (O2•–), singlet oxygen (1O2), peroxyl (RO•2), alloxyl (RO•), the hydroxyl radical (•OH), reactive nitrogen intermediates—nitric oxide (NO•), and nitrogen dioxide (NO•2)) .,28.26,28Based on the biomedical application, oxalate-based polymers can be classified into first, second, and third generations of CPOx formulation . The genThese are polymers designed with redox responsiveness polyoxalate group. The molecular weight of the polymers was approximately in the range of 11,000 Da. These particles were early proof of concept and were designed to target macrophages in the cell.These are polymeric particles designed with a redox responsive-polyoxalate group. In addition, a small molecule was covalently linked to the polymer backbone. The precursor is a co-polyoxalate polymer, hence this is considered the first generation of co-polyoxalate nano formulation. These particles were sophisticated in comparison to their polyoxalate predecessors. Since the formulations were in their infancy no systemic delivery applications were carried out. Second generation of copolyoxalate nanoformulation are polymeric particles derived from the previous generation’s redox responsiveness concept. However, the biomedical applications of these polymers were extended to in vivo models. The third generation of copolyoxalate nanoformulation are polymeric particles designed to be theragnostic in nature. These particles delivered therapeutic small molecules in an in vivo animal models and were simultaneously imaged after the drug was delivered.2O2 . CP. CP27]. In vivo animal experiments in mice were performed by injecting 3 mg/kg per day of CPOx and PLGA nano formulations. In the PLGA-treated group, there were inductions of NADPH oxidase 2 which signifies induction of intracellular ROS. There was no alteration of intracellular NADPH oxidase 2 activity  34]. CP. CP34]. The concept behind the CPOx polymers and their corresponding nano formulation were from the polyaspirin polymer, a study conducted by Uhrich and co-workers . POx polGastrodia elata and it is an aromatic diol compound incorporated in oxalate backbone. p-Hydroxybenzyl alcohol-incorporated copolyoxalate (HPOX) was the first CPOx synthesized. HPOX nanoformulation was evaluated in RAW 264.7 macrophage cells with lipopolysaccharide (LPS)-induced inflammation. Protein expression studies confirmed that p-Hydroxybenzyl alcohol from the HPOX nanoparticles exhibited anti-inflammatory and antioxidant activity ..Curcuma Skeletal muscle is one of the largest organs in the body, irrespective of sex. It is subjected to wear and tear with age. Often physiological changes depend upon the physical activity of the individual. Skeletal muscle is one of the largest consumers of oxygen in the body: approximately 20% per minute, and hence superoxide anions are produce at several sites and released to the muscle cells’ cytosol ,44. WithPoly  PVAX-based nano formulation was evaluated in contusion injury of triceps surae muscles and Achilles tendons. The study showed that PVAX nano formulation exhibit anti-inflammatory activity by reducing macrophage accumulation at the site of injury. Protein expression of caspase-3, Bcl-2, and Bax confirms the anti-apoptotic potential for the PVAX particle formulation. The integration of diagnosis and therapeutic nature of PVAX, using ultrasound imaging of musculoskeletal injuries was tested and proved to be an added advantage to age-related skeletal muscle dysfunction .Many people live in an air polluted environment where people with high sensitivity are susceptible to foreign bodies. The inflammatory cells defense is sensitized by the foreign bodies which leads to mucoid secretion and shortness of breath. Delivering small molecules that could exhibit antioxidant and anti-inflammatory could mitigate the ill effects of ROS in the lung. Biocompatible CPOx-containing PVAX polymeric particles could help in easing the inflammation. An in vivo study performed with porous PVAX microparticles loaded with allergy medicine dexamethasone (DEX), followed by intratracheal injection of particles, showed a rapid drug release in porous PVAX microparticles loaded with DEX. DEX combined with vanillyl alcohol was shown to enhance therapeutic outcome . Since tIschemic tissue after blood perfusion is subjected to ROS insults that can overwhelm redox balance, damage the surrounding tissues, and induce apoptosis. Biodegradable hydroxybenzyl alcohol containing polyoxalate copolymer (HPOX) with multifunctional capability was used by Lee et al. in their study to evaluate HPOX’s therapeutic efficacy. HPOX’s particles showed localized antioxidant and anti-apoptotic activity at the site of inflammation. In vivo hind-limb reperfusion injury treated with HPOX’s loaded with 4-amino-1,8-napthalimide showed reduced poly ADP ribose polymerase-1 and caspase-3 protein expression which corresponds to the antioxidant and anti-inflammatory properties of HPOX . In anotLee et al. and Bae et al. investigated the vanillyl alcohol incorporated PVAX in myocardial I/R (ischemia/reperfusion) injury. The first objective of the study was to provide an insight on the biocompatibility of the potential for PVAX formulation . The latAccording to the World Health Organization’s Global Health Observatory, in 2018 alone, 9.6 million people are estimated to have died from cancer, and 30–50% of cancer incidences could have been prevented ,53. As p2O2 as a trigger. The naturally occurring small molecules were pre-treated to sensitize the cancer cells, followed by photosensitizer molecule encapsulated CDNP. H2O2 present in the cancer cells permeates and enters the CDNP core containing HPOX and protoporphyrin IX (PPIX). This rapidly gives rise to high energy 1,2-dioxethanedione intermediate. Energy released from the decomposition of these unstable 1,2-dioxethanedione results in chemi-dynamic therapy (CDT), i.e., PPIX activation, 1O2 generation, and results in programmed cell death of the cancer cell ” showed that there is a light-free photodynamic therapeutic system for cancer treatment . The meccer cell . The stu2O2 concentration in the breast-tumor and then the CPPO containing particle was administered systemically. Enhanced permeability and retention of the nano formulation increase the possibility of the nanoparticles to be activated at the site of the cancer cells. Particle accumulation served as an image-guided tumor therapy aided in fluorescence and chemiluminescence bioimaging of the tumor  oxalate (CPPO) encapsulated with a specially designed photosenzitiser TPE-BT-DC . In vivohe tumor . In compFDA approved PLGA has been used in the field of biomaterial since the early 1960s. POx and CPOx are relatively new polymeric biomaterials. Despite its advantages POx and CPOx material synthesis involves a complicated process and various hazardous solvents. To be commercially viable, methodology of the synthesis must be modified and simplified. Translation of these smart polymer are reduced due to limited investment in research and development to improve the methods of POx and CPOx synthesis. It is hoped that this review would shed a positive light over POx and CPOx and gain traction in biomedical sciences.Polymeric prodrug has gained a considerable amount of interest as a naturally occurring phenolic compound incorporated in CPOx, an emerging avenue of prodrug mediated drug delivery. Within the past decade, POx and CPOx based novel micro and nano formulations were intensively investigated in inflammatory diseases ranging from wound to light free tumor therapy. In the future we anticipate (a) CDT’s application in animal models and human trials; (b) POx and CPOx nano formulation that could cross the blood–brain barrier and treat ailments that are as a result of redox imbalance in brain; (c) multi targeted nano formulation with more narrow targeted action; and (d) POx and CPOx particle formulations from bench to bedside.


Salmo salar) is a major species produced in world aquaculture and an important vertebrate model organism for studying the process of rediploidization following whole genome duplication events . The current Salmo salar transcriptome is largely generated from genome sequence based in silico predictions supported by ESTs and short-read sequencing data. However, recent progress in long-read sequencing technologies now allows for full-length transcript sequencing from single RNA-molecules. This study provides a de novo full-length mRNA transcriptome from liver, head-kidney and gill materials. A pipeline was developed based on Iso-seq sequencing of long-reads on the PacBio platform (HQ reads) followed by error-correction of the HQ reads by short-reads from the Illumina platform. The pipeline successfully processed more than 1.5 million long-reads and more than 900 million short-reads into error-corrected HQ reads. A surprisingly high percentage (32%) represented expressed interspersed repeats, while the remaining were processed into 71 461 full-length mRNAs from 23 071 loci. Each transcript was supported by several single-molecule long-read sequences and at least three short-reads, assuring a high sequence accuracy. On average, each gene was represented by three isoforms. Comparisons to the current Atlantic salmon transcripts in the RefSeq database showed that the long-read transcriptome validated 25% of all known transcripts, while the remaining full-length transcripts were novel isoforms, but few were transcripts from novel genes. A comparison to the current genome assembly indicates that the long-read transcriptome may aid in improving transcript annotation as well as provide long-read linkage information useful for improving the genome assembly. More than 80% of transcripts were assigned GO terms and thousands of transcripts were from genes or splice-variants expressed in an organ-specific manner demonstrating that hybrid error-corrected long-read transcriptomes may be applied to study genes and splice-variants expressed in certain organs or conditions . In conclusion, this is the single largest contribution of full-length mRNAs in Atlantic salmon. The results will be of great value to salmon genomics research, and the pipeline outlined may be applied to generate additional de novo transcriptomes in Atlantic Salmon or applied for similar projects in other species.Atlantic salmon ( Salmo Salar) is a species with significant value both economically and scientifically. It is an important aquaculture species, and there is also substantial commercial harvesting of wild salmon  event,  approximately 80 million years ago . They arSalmo Salar genome has been publicly available since 2015 thanks to the efforts of the International Cooperation to Sequence the Atlantic Salmon Genome (ICSASG)  of transcriptomes on platforms producing short-read data  (A chromosome level assembly of the (ICSASG) , but themo Salar . The vast-reads) . While tt-reads) . Thus, tt-reads) . A full-t-reads) .Long-read sequencing based on the PacBio Iso-Seq SMRT sequencing technology produce full-length transcript sequences  by sequencing single molecules . This mede novo full-length transcriptome with a quality comparable to any reference resource without the use of the current genome assembly as an error-correcting source . RNA extraction was performed using the Qiagen RNeasy Mini Kit , with On-column DNase Digestion using the RNAse-Free DNase Set, according to the manufacturer’s protocol. The total RNA extracts were used for both PacBio long-read sequencing and Illumina short paired-end sequencing. The PacBio non-size selected Iso-seq library preparation was used for the for long-read sequencing. The Express Template Prep 2 protocol requiring RIN-values > 8 on the RNA-samples was applied, and each of the nine samples were individually processed. The resulting cDNA-adapter complexes from the nine samples were sequenced on one PacBio Sequel II 8M SMRT Cell each. For short-read sequencing on the Illumina platform, the automated NEBNext Ultra II Directional RNA-Seq library kit with Poly-A selection  was used for library preparation, and the paired-end sequencing (150 bp) was performed using one Illumina NovaSeq 6000 SP flow cell for all nine samples multiplexed together.The RNA extraction, library preparation and sequencing of the two SAV samples was carried out by Genewiz Germany GmbH . RNA was extracted with the RNeasy Plus Mini Kit  according to the manufacturer’s protocol and the samples used for sequencing had a RIN > 8. For long-read PacBio Iso-seq sequencing, the cDNA synthesis was performed using the SMRTer PCR cDNA synthesis kit  without size-selection, and the cDNA-adapter complex was generated using the SMRTbell template prep kit V1.0. . Each sample was sequenced using the PacBio Sequel I. Each sample was sequenced on two 1M v2 SMRT cells to compensate for the lower read count on the Sequel I compared to the Sequel II. The short-read sequencing applied the NEBnext Ultra RNA library preparation kit  in accordance with manufacturers protocol. The paired-end (150 bp) sequencing was carried out on the Illumina HiSeq 4000 platform.The Iso-Seq raw long-reads from the PacBio Iso-seq sequencing were processed through the IsoSeq3 pipeline  as illusCutadapt 1.18  was applThe error-corrected HQ reads (EC-HQ reads) were then filtered using an inhouse python script which removed any EC-HQ read that was less than 99% covered by the De Brujin graph generated from the Illumina reads  were removed using an inhouse python script  with default parameters, apart from allowing up to 10 000 bp overhangs in the 5′ end and 1000 bp overhangs in the 3′ end. This allowed any EC-HQ reads to be grouped by genome co-ordinates and assigned into groups of transcripts originating from same locus. Furthermore, reads with the same splice-pattern, representing the same isoform, were clustered together keeping the longest one as the representative isoform sequence of the cluster. The groupings of clusters and genome mapping co-ordinates were retained in the fasta headers as information that was utilized by downstream applications (SQANTI3). The relaxed 5′ and 3′ overhang cutoffs were used to allow shorter EC-HQ reads that represented fragments of other full-length EC-HQ read transcripts to be clustered together with the full-length transcripts representing this isoform, rather than being erroneously identified as separate isoform variants.A grouping of the EC-HQ reads likely to originate from same genome locus was carried out applying cdna_Cupcake version 12.1.0  with minSalmo trutta genome (RefSeq accession no GCF_901001165.1) applying cdna_Cupcake and same parameters. The remaining sequences that did not map to either the Salmo salar or the Salmo trutta genomes were mapped against the SAV genome (GenBank accession no. KC122923) as one of the individual samples was SAV infected, and the matching reads were discarded.The EC-HQ reads which did not map well to the Atlantic Salmon genome were mapped against the RefSeq version of the fSalTru1.1 assembly of the The remaining EC-HQ reads that did not map to either of the Salmonid genomes were clustered using Cogent 6.1.0 using default parameters  Cogent,. Cogent All the EC-HQ reads, both the ones that were mapped to either of the genome assembly sequences as well as those grouped by Cogent were finally clustered with CD-Hit version 4.8.1  CD-Hit,. This fi1. Sequence identity threshold 0.99.2. Local sequence alignment.3. Cluster reads to most similar longer EC-HQ read if there were more than one fitting the alignment criteria.4. The short EC-HQ read must align with 99% of its length to the longer (1% “uncovered” bases). If the shorter EC-HQ read were larger than 3000 bp the 1% limit was replaced by 30 bp or less uncovered bases.5. The one long EC-HQ read that other shorter reads were aligned to was allowed to have any amount of overhang.This final clustering assured that identical structural variants were aligned into a single representative EC-HQ read. In most cases the ones aligned to a longer EC-HQ reads would be 5′ incomplete EC-HQ reads or EC-HQ reads with an incomplete 3′UTR due to mispriming in the cDNA synthesis. This assured that every different structural isoform was represented by a single full-length EC-HQ read. An inhouse python script was used to identify FLNC support and contributing samples for each non-redundant EC-HQ read following the final clustering. This non-redundant EC-HQ read transcriptome was further analyzed by classification of structural variants  and functional annotation .Salmo salar or the Salmo trutta genome were classified and compared against the existing annotation for the respective genomes. This analysis was carried out with SQANTI3 (v 1.0.0) with default parameters (The EC-HQ reads that had been clustered by cDNA_cupcake using the rameters  (SQANTI3rameters . SQANTI3Full Splice Match (FSM) which is an identical match to a transcript isoform present in the genome annotation with all the same splice junctions and exons.Incomplete Splice Match (ISM) representing an incomplete but otherwise identical match to a known isoform. All the present splice junctions match, but there are exons missing in either or both ends of the EC-HQ transcript.Novel In Catalog (NIC), a novel isoform with another combination of exons than those isoforms annotated in the genome, but with a combination of known splice-junctions from previously annotated isoforms.Novel Not in Catalog (NNC), novel isoform containing at least one splice junction not present in the annotation. Consequently, these have at least one novel exon.Intergenic, meaning a novel transcript that maps to a locus with no previously annotated genes in the current version of the Atlantic salmon genome.Genic intron, meaning the transcript maps entirely within the intron of an annotated gene in the Atlantic salmon genome.Genic genomic, which means the transcript overlaps annotated introns and exons.Antisense, which means there is no annotated gene at the locus on the strand where the transcript matches, but there is one on the reverse strand.Fusion, meaning the read spans across two different annotated loci in the current genome annotation.Salmo trutta genome if they were successfully mapped against the Salmo salar genome. A structural classification indicating the EC-HQ was of any other category than FSM in Salmo salar does therefore not exclude that it would have mapped different  in Salmo trutta.Sequences were not mapped against the SQANTI also provides additional useful information like number of exons, splice junction signals , CDS length, polyA-signals upstream on 3′end and genomic percentage of A’s downstream of a 3′ termination site (used to judge whether there was cDNA synthesis mispriming).All EC-HQ reads in the final non-redundant transcriptome dataset  were subAll complete CDS sequences were fed into the functional annotation workflow of OmicsBox   with theSalmo salar mitochondrial sequence by SQANTI were also fed to TransDecoder using the Vertebrate Mitochodrial genetic code to search for ORFs. Any complete CDS from these sequences were also functionally annotated as described above.Sequences that were mapped onto the Additionally, for the transcripts in the final full-length mRNA transcriptome , we identified subgroups of full-length mRNAs expressed in at least three samples from a specific organ type, but not in any of the other organ types in our materials. For these, we generated multi-level GO charts, showing the most specific GO-terms appearing in the dataset over the default abundance cutoffs suggested by OmicsBox, in a non-redundant way. These cutoffs were respectively 144 transcripts for gills, 98 in head-kidney, and 116 in liver.An inhouse python script Filter,  was usedGIYK00000000. The version described in this paper is the first version, GIYK01000000.The PacBio and Illumina raw sequencing data was submitted to NCBI’s SRA database, and the final transcript sequences in our full-length mRNA transcriptome were submitted to the Transcriptome Shotgun Assembly under the accession de novo mRNA transcriptome and the Salmo salar mRNAs in the RefSeq database.Blast analysis was applied to find the degree of sequence similarity between the final Salmo salar mRNA transcripts was identified and downloaded as a full record fasta files using a filtered nucleotide search through the NCBI RefSeq database. These were searched against our full-length mRNA transcriptome using blast 2.9.0 + with e-value cutoff 1e-15, and outputfmt “6 std qcovhsp slen.” The same search settings were used in the reverse comparison with our transcriptome as query against the RefSeq sequences. An inhouse python script was used to filter the blast results. The filter classified transcripts into three categories. Matches between query and subject meeting the following criteria were categorized as identical isoforms: E-value less than 10–50, percentage identity ≥ 99%, and either query coverage per high-scoring segment pair >99% or alignment length ∗ 100/subject length > 99. This ensured that any match meeting the e-value and identity thresholds had a greater than 99% coverage of the query by the subject sequence, or a greater than 99% coverage by the subject of the query sequence. These thresholds ensured that matches categorized as identical isoforms were consecutively matching sequences originating from the same isoforms, but allowed either of them to differ in UTR length compared to the other. Matches within this category were further grouped depending on whether the RefSeq RNAs had the longer UTR (query coverage per high-scoring segment pair < 99%) or whether the matching mRNA in our full-length mRNA transcriptome had the longer UTR . The second category, named significant hits, were all matches not meeting the identical isoform criteria, but with an E-value of less than 10–15. The remaining query sequences returning E-values more than 10–15 were categorized as non-matching RefSeq mRNAs (or non-matching full-length mRNAs in the reverse search).The complete set of RefSeq The overlap between mRNAs in our dataset and the genome-annotation based mRNAs (genome reference sequence GCF_000233375.1) would be the number of sequences classified as FSM by SQANTI3. These were retrieved from the final.tsv file . The numRefSeq mRNAs with sequences that are mismatches if compared with the correlated mRNAs (exons) in the Atlantic salmon genome assembly was identified by adding the term ‘AND “assembly gap:” [All Fields]’ to the search in the RefSeq database. These represented RefSeq RNAs that are not supported by the current genome sequence. Furthermore, the number of such RefSeq mRNAs that are supported by our full-length mRNAs was retrieved by searching their accession numbers among those categorized in the identical isoform category described above.The results from the Pacbio sequencing and Illumina sequencing are summarized in The coverage distribution by Illumina reads on the HQ reads is illustrated in The EC-HQ read dataset were analyzed by Repeatmasker to identify transcripts originating from interspersed repeats  could not be mapped to either of the two salmonid genomes. It is very unlikely that eight percent of the transcripts would map well in the Salmo trutta genome if the reason for mismatch against the Salmo salar genome was low quality or error in the EC-HQ reads. On the contrary, this indicated that there are missing or misassembled sequences in the current Atlantic salmon genome sequence that prohibited a successful mapping of a surprisingly large proportion (22%) of the of the unique Atlantic salmon EC-HQ reads. Following from this, errors in the genome sequence would be the likely explanation for why 14% of transcripts grouped by Cogent could not be mapped to the Salmo salar genome sequence . The fact that our final full-length transcriptome matches the current RefSeq mRNAs much better than the transcripts (annotated exons) in the genome sequence  also points to incorrect annotation of splice products. The de novo full-length transcriptome from this study may therefore aid to improve the current transcript annotation in the genome sequence. In light of this, the 22% of transcripts not currently mapped to the Atlantic salmon genome sequence represents a very useful source for long range linkage information that may be used to improve the genome sequence assembly.The mapping of the EC-HQ reads to the _cupcake  showed aSalmo salar genome and the 8721 that had been mapped to the Salmo trutta genome were structurally annotated using SQANTI3  in terms of splice pattern and sequence identity (SQANTI3 default 95% cutoff). The majority of such matching transcripts in the current Atlantic salmon annotation have been generated from predictions based on the genome sequence with variable support from short HTS reads or ESTs . The FSMs in our dataset, therefore, represents experimental validation of 17 787 transcript isoforms by single molecule sequenced full-length mRNAs. Surprisingly, there were 960 EC-HQ reads mapped as FSMs in the Salmo trutta genome. Obviously, these are also true Atlantic salmon full-length mRNAs, but inconsistencies in the current genome assembly of Salmo salar prevented these transcripts from mapping to the Salmo salar genome sequence.The EC-HQ reads in the final mRNA dataset categorized as Full Splice Match (FSM) final in  represenSalmo trutta genome.The transcripts mapped as either Novel in Catalog (NIC), or Novel Not in Catalog (NNC) with exons defined by canonical splice junctions, represents novel isoforms not currently annotated in the genome sequences. The NICs have combinations of known splice sites which makes them isoforms with new combinations of annotated exons. There were 17 039 such novel transcripts in the final dataset, approximately the same number as all FSMs. There were an even larger number of NNCs, a total of 25 581 transcripts, illustrating the capability of long-read based methods to identify new isoforms not possible to predict reliably using short-reads and the genome sequence alone. Again, a substantial proportion  of the transcripts in these categories could only map to, and be classified by SQANTI3, using the Salmo trutta. Again, this illustrates the ability of long-read based transcriptome sequencing to identify transcripts not detected by short-read supported genome predictions.The remaining SQANTI3 categories  comprised the smaller part of the final mRNA transcriptome . Despite their structural classification by SQANTI3 bringing into question if these were true mRNAs, they were all supported as full-length mRNAs by OmicsBox. Also, as a threshold of at least 5 supporting FLNCs was used to filter out possible artifacts from these categories, they are likely to represent true Atlantic salmon protein-coding transcripts. Although the ISM reads were supported by the genome annotation as matching a known transcript but missing exonic sequence in the 5′ or 3′ end (or both), they all had a complete CDS. We did employ the more conservative FLNC-support criteria (at least 5 FLNCs) for ISMs, and given this threshold, it is less likely that they are incomplete, but rather represent full-length mRNAs. A small number of ISM sequences (3.7%) had 80% or higher A content in the 20 bases immediately downstream of where they mapped in the genome sequence. This could have allowed mispriming during cDNA synthesis leading to incorrect 3′UTR lengths in these transcripts. The fraction of transcripts grouped by Cogent with at least 5 supporting FLNCs is approximately as large (4803 transcripts) as those mapped to An overview of the number of FLNCs supporting each EC-HQ mRNA in the final full-length mRNA transcriptome is given in A python script was used to estimate the number of instances were transcripts annotated as encoding the same gene by SQANTI or OmicsBox had been assigned to different loci by cdna_Cupcake, or different gene families by Cogent. In all, 12% of genes had at least two EC-HQ mRNA transcripts that were mapped to different loci. This indicates that at least 12% of the expressed genes were likely represented by multiple paralogs in our dataset.GIYK00000000. The version described in this paper is the first version, GIYK01000000. The TSA Contig ID for each sequence is listed in The transcripts have been deposited at DDBJ/EMBL/GenBank as a Transcriptome Shotgun Assembly project under the accession Salmo salar reference genome sequence. The other source is the current collection of Salmo salar mRNA transcripts in the RefSeq database. Although these would be expected to correspond well, they differ in the sequence information given for thousands of transcripts. There are 4475 RefSeq mRNAs that are annotated as either having a gap or additional sequence that is not present in the current genome reference sequence. The long-read based de novo mRNA transcriptome from the present study could possibly aid to resolve what are the correct transcript sequences. The single-molecule based method applied here is also expected to be the superior one for isoform identification. This potential for increasing the quality of annotated transcript isoforms with our dataset was investigated by comparing our long-read transcriptome to each of the available sources (the RefSeq mRNA sequences and the annotation of transcripts in the RefSeq genome sequence).The current transcript information on Atlantic salmon mRNA sequences is provided from two NCBI sources. One is the transcript information (transcript and isoform variants defined by their exons) given in the annotation to the current Salmo salar mRNAs in RefSeq consist mostly of transcripts that are predicted by use of the genome sequence but supported and error-corrected based on EST and short-read sequences as part of the NCBI Eukaryotic Genome Annotation pipeline1. All 97 604 Salmo salar mRNA sequences in the RefSeq database were blasted against our dataset, classifying hits into three categories  to already annotated transcripts in the RefSeq version of the Salmo salar genome assembly. The majority of mRNA isoforms predicted in the Salmo salar genome annotation . Furthermore, 1268 of the transcripts that did not map to the Salmo salar genome were in the category identical isoforms in the blast comparisons against the RefSeq mRNAs. A similar comparison where the RefSeq transcripts not matching the genome reference sequence  were compared to our de novo transcriptome showed that 673 of these were in the category identical isoforms. All together, these comparisons indicate that the full-length de novo transcriptome may contribute considerably in improving the current transcript annotation quality and aid in improving the reference genome assembly.The fact that there were considerably fewer FSMs 19%,  than thealb1, constituted 3,8% of all FLNCs on its own, and the top five most abundant transcripts represented 6.5% of all FLNCs. Serum albumin  and the two other top expressed genes (fgg and itih3) were all encoding secretory proteins from liver tissues. Other highly expressed genes like two splice variants of actin (actb), selenoprotein P (SelP), and apolipoprotein Eb (apoeb) were expressed in all tissues. A surprising finding was that one of the highly expressed transcripts annotated as complement factor H-like (cfhr5) did not map to the Atlantic salmon genome sequence, but was a FSM in the Salmo trutta genome. Furthermore, two other highly expressed transcripts (prothrombin-like and transferrin A-like) were annotated as fusion-products by SQANTI. Again, this is likely due to incorrect genome-annotation rather than due to sequence artifacts, given the high number of FLNCs supporting these transcripts in several tissues.Ten percent of all FLNCs in the dataset were from only 17 mRNAs (listed in fgg and itih3). Again, the transcripts exclusively expressed in this organ were, as expected, among those coding for proteins associated with liver function. Taken together, the results here showed the potential of our transcriptome pipeline to identify genes and splice variants that have particular organ-specific functions.The final mRNA transcripts were from three organs: liver, gills, and head-kidney. Several of the highly expressed transcripts were only present in liver samples, indicating that the transcriptome pipeline could identify transcripts expressed in an organ specific manner. Applying a conservative approach to identify such organ specific transcripts, we searched the full-length mRNA transcriptome for transcripts that were expressed in at least three samples from one organ, while they were absent in samples from either of the other two organs. This revealed that there were 2717 transcripts expressed in gills only, either from genes expressed only in gills (1811) or splice-variants expressed only in gills (906). In liver there were 1784 transcripts, of which 1113 were from liver-specific genes and 671 were liver-specific splice-variants. In head-kidney there were 1757 transcripts, 700 from head-kidney specific genes and 1057 were head-kidney specific splice-variants. Our pipeline aimed to generate a full-length mRNA transcriptome with reference level sequence accuracy from a variety of organ samples by processing PacBio long-reads that were hybrid error-corrected with Illumina paired-end reads from the same samples. Similar approaches have been used to generate high quality transcriptomes in other species , but thiThe initial examination of the HQ reads in the materials indicated there were many cases of frame shift errors leading to incorrect CDS’s or premature stop codons (data not shown). While we could not conclude for certain that these were sequencing errors in all cases, such errors were not unexpected given a sequence accuracy of 99% and the fact that PacBio reads are prone to numerical errors in homopolymers . This acde novo transcriptome sequences had, in agreement with findings in similar studies  or the blast analysis against the Atlantic salmon RefSeq mRNAs, about 70% of the final mRNA transcriptome are novel isoforms. These novel isoforms are either splice variants, (each locus had on average three splice variants) or paralogs. This showed that our long-read transcriptome sequencing approach led to a substantial increase in number of Atlantic salmon transcript isoforms. Such high success rate in discovery of novel isoforms agrees well with findings from similar studies . A cutofA large fraction of the contributing FLNCs in this study belonged to a few transcripts with extremely high levels of expression (10% of FLNC reads were from the 17 most abundant transcripts). In future projects aiming to characterize all full-length mRNAs in a sample material, removal of such transcripts  would greatly increase the likelihood of identifying the more rarely expressed transcripts .The sequencing depth applying one Sequel II cell was approximately eight times higher than from one Sequel I cell. This is in agreement with other studies . CombiniFull-length transcriptomes have a range of useful applications . Among tIn conclusion, the hybrid corrected long-read pipeline employed here successfully generated high-quality full-length mRNA transcripts. The long-read approach led to the detection of novel splice variants and validated a quarter of all predicted Atlantic salmon mRNAs by transcripts originating from single molecule sequenced long-reads. Comprised solely of mRNAs with complete CDSs, more than 80% were assigned GO terms, and thousands of genes or splice variants from genes expressed in an organ specific manner were identifies. This full-length transcriptome will be an important resource for functional genomics in salmon aquaculture research.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by the National Research Authority in Norway (NARA).SR, RA, BH, and T-KØ contributed to conceptualization and final editing of the manuscript. SR and RA contributed to analysis pipeline design, formal analysis, and wrote, reviewed and edited the manuscript. SR contributed to pipeline implementation, additional programming, and writing the original draft. RA, BH, and T-KØ contributed to resources. RA supervised and acquired funding. RA and BH contributed to project administration. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bone and soft tissue sarcoma (STS) are heterogeneous diseases comprised of various molecular and histologic subtypes . During Osteosarcoma is not a cancer clearly characterized by genetic mutations or oncogenic expressions. Therefore, it is remarkably interesting to investigate new prognostic signatures and therapeutic targets. Pandya et al. found that the identification of the over-amplification of the MYC-RAD21 genes associated with patients that have lower overall survival (OS), and the therapeutic approach to use a BET inhibitor and CHK1 inhibitor appears to have significant negative consequences on the osteosarcoma cells.In addition to osteosarcoma, Ewing’s sarcoma (ES) is also the most frequently observed primary malignant bone tumor in the second decade of life . MullardThere is no single clinical examination to facilitate the accurate diagnosis of STS, which requires comprehensive consideration of several examinations. Fujibuchi et al. demonstrated the relationship between pretreatment clinical parameter and accurate diagnosis of STS. Multivariable analysis revealed that tumor size, white blood cell count, hemoglobin count, C-reactive protein level, and lactate dehydrogenase level were significant predictive factors for sarcoma.In the patients with STS, surgical resection is the standard treatment for STS. Götzl et al. reported that the retrospective analysis of 290 patient showed significantly clearer margin resections in cases with free-flap reconstructions. The surgical margin is a critical variable for achieving local tumor control.Adjuvant radiotherapy is also important for achieving local tumor control . SenolytUsing a resected tumor sample, Nakamura et al. described that a high expression of both interleukin-6 (IL-6) and IL-6 receptors was a prognostic factor for OS and metastasis-free survival (MFS). They found that this high expression indicated that the patient had a poor prognosis for OS and MFS. IL-6 is an essential cytokine in the regulation of inflammation . The infThe median survival time of patients with advanced STS is typically <12 months . TherefoDadone-Montaudié et al. evaluated the therapeutic potential of the pan-FGFR inhibitor erdafitinib to treat dedifferentiated liposarcoma (DDLPS). They detected overexpression of FGFR1 and/or FGFR4 in a well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS) and demonstrated correlation of this expression with poor prognosis. Erdafitinib treatment reduced cell viability, inducing apoptosis and strong inhibition of the ERK1/2 pathway.Miolo et al. tried to identify new serum prognostic biomarkers in STS patients treated with the trabectedin regimen. They showed that citrulline, an amino acid belonging to the arginine metabolism, represents an important metabolic signature that may related with survival in patients with STS.Recent studies on metastatic colonization have confirmed the hypothesis that certain tissue microenvironments may be preordained to be intrinsically hospitable to disseminated cancer cells . PouliquIn addition to original articles, this Special Issue includes two reviews. Fujiwara et al. reviewed role of tumor-associated macrophage in sarcomas. Tumor-associated macrophages (TAMs) are a major component of tumor-infiltrating immune cells in the tumor microenvironment and have a dominant role as orchestrators of tumor-related inflammation. TAM-targeted therapy as a novel treatment approach for sarcomas should be expected. Napolitano et al. reviewed the treatment of desmoid including low-dose chemotherapy and treatment with tyrosine kinase inhibitors.We expect that these possible biomarkers could be useful for detecting sarcomas earlier and making aggressive treatment strategies to improve survival in patients with sarcomas when those markers are raised.We also hope that researchers enjoy reading this Special Issue on present and future biomarkers and therapeutic strategies in sarcomas.


Socioeconomic differences in receipt of adjuvant treatment contribute to persistent disparities in breast cancer (BCA) outcomes, including survival. Adjuvant endocrine therapy (AET) substantially reduces recurrence risk and is recommended by clinical guidelines for nearly all women with hormone receptor-positive non-metastatic BCA. However, AET use among uninsured or underinsured populations has been understudied. The health reform implemented by the US state of Massachusetts in 2006 expanded health insurance coverage and increased the scope of benefits for many with coverage. This study examines changes in the initiation of AET among BCA patients in Massachusetts after the health reform.We used Massachusetts Cancer Registry data from 2004 to 2013 for a sample of estrogen receptor (ER)-positive BCA surgical patients aged 20–64 years. We estimated multivariable regression models to assess differential changes in the likelihood initiating AET after Massachusetts health reform by area-level income, comparing women from lower- and higher-income ZIP codes in Massachusetts.p-value< 0.001) relative increase in the likelihood of initiating AET among BCA patients aged 20–64 years in low-income areas, compared to higher-income areas, after the reform. The increase was more pronounced among younger patients aged 20–49 years (7.1-percentage point increase).There was a 5-percentage point (The expansion of health insurance in Massachusetts was associated with a significant relative increase in the likelihood of AET initiation among women in low-income areas compared with those in high-income areas. Our results suggest that expansions of health insurance coverage and improved access to care can increase the number of eligible patients initiating AET and may ameliorate socioeconomic disparities in BCA outcomes.The online version contains supplementary material available at 10.1186/s12885-021-08149-0. Breast cancer (BCA) is the second most common cancer and the second leading cause of cancer death among women in the US . This isAdjuvant endocrine therapy (AET) is recommended for BCA patients with estrogen receptor (ER) or progesterone receptor (PR) positive BCA. About 60–75% of invasive BCA cases are ER-positive , 6, and Previous research has shown that personal, social, and structural factors are associated with guideline-concordant use of AET among BCA patients , 22. OldTo better understand the impact of insurance coverage on initiation of AET and associated socioeconomic disparities among BCA patients, we examine the impacts of the Massachusetts Health Care Insurance Reform Law passed in 2006 , which increased the rate of insurance coverage in the state as well as the scope of covered benefits for many who were already previously insured . The refThis study examines changes in initiation of AET among non-elderly women in low-income areas diagnosed with BCA following health reform in Massachusetts. We hypothesize that changes in the proportion of BCA patients who initiate AET will be more substantial among women from lower-income areas, who are most likely to be impacted by the reform, than those from higher-income areas in Massachusetts.Our primary data source is the Massachusetts Cancer Registry (MCR), which is administered by the Massachusetts Department of Public Health and follows standards set by the North American Association of Central Cancer Registries (NAACCR), the Commission on Cancer (CoC), the National Cancer Institute (NCI), and the Centers for Disease Control and Prevention (CDC) . The MCRn = 20,713).Out of BCA cases diagnosed during the study period of 2004–2013 in Massachusetts, we eliminated cases among non-female patients; cases in patients under 20 or over 64 years of age, given they were not directly impacted by the insurance reforms; cases for which the patient had another BCA diagnosis within the study period on or before the date of diagnosis of the current case; cases for which diagnosis was established by autopsy or death certificate or where diagnosis date was the same as death date; cases for which the patient had another BCA diagnosed within 365 days; cases that did not receive surgery; cases with ER-negative status; cases with incomplete PR status (data not collected or not documented); cases with stage IV diagnosis; cases with no information on initiation of AET; and cases whose date of endocrine therapy preceded the date of surgery. Figure We estimated a linear probability regression model to examine differential changes in the likelihood of initiating AET among BCA patients residing in lower-income areas compared to those in higher-income areas in Massachusetts. Our model includes a binary variable identifying lower-income ZIP code areas, another binary variable indicating the post-reform period, and an interaction term between these two binary variables to assess population-level differential impacts of the state health reform on initiation of AET among BCA patients living in lower- versus higher-income areas of Massachusetts. The outcome variable is a binary variable indicating whether the patient initiated AET after surgery. ZIP code areas with median household income below the state median value in 2006 , were categorized as low-income areas; this designation was used to capture a population most likely impacted by the reform. The comparison group was BCA patients who lived in ZIP code areas with median household income above the state median income. The pre-reform period includes 2003–2006; the post-reform period includes 2007–2013. We adjusted for patient age, marital status, race/ethnicity, and stage at diagnosis. We controlled for secular trends in BCA treatments by adjusting for each calendar year in the model. An individual-specific error term was estimated using Huber-White robust standard errors.We conducted two additional sets of models to test whether the changes in the initiation of AET were sensitive to definition of low-income areas and study period. The first model defined low-income areas as the lowest tertile ZIP code areas and high-income areas as the highest tertile ZIP code areas in Massachusetts and compared subgroups of patients most likely and least likely to be impacted by the health reform. The second model truncated the post-reform period in 2010 to account for the introduction of generic AIs in 2010 . In addiTo supplement our main results comparing population-level average changes in proportions of BCA patients residing in low-income areas who initiate AET after surgery to those in high-income areas, we estimated a linear model to assess overall changes in temporal trends in AET initiation. This model includes a time variable that measures the study period by quarter and ranges from 0 (first quarter of 2003) to 39 (fourth quarter of 2013), a binary variable that indicates the pre-reform period as 0 and the post-reform period as 1 which indicates the level change following the state health reform, and an interaction variable between these two variables which indicates the slope change following the state health reform. We adjusted for patient age, marital status, race/ethnicity, stage at diagnosis, and county-level characteristics described above.Table More BCA patients were diagnosed at stage 0 or I in ZIP code areas above state median household income in the pre-reform period; however, this difference was attenuated in the post-reform period. The number of BCA patients who had a mastectomy with reconstruction increased statewide in the post-reform while the number of BCA patients who had breast-conserving surgery (BCS) decreased more in higher-income ZIP code areas. Women residing in high income areas were more likely to be married and less likely to be non-Hispanic White compared to women residing in low-income areas.p-value< 0.001) in the likelihood of initiating AET following the Massachusetts reform in low-income ZIP codes, relative to the increase in the higher income ZIP codes. This effect was more pronounced among younger BCA patients ages 20–49 years, for whom we observed a 7.1-percentage point  relative increase in the likelihood of initiating AET relative to the comparison group.Table When we shortened the post-reform period to isolate changes after health insurance expansions prior to the introduction of generic AIs .Massachusetts health reform increased access to medical care, improved financial support for safety-net hospitals, and provided more expansive prescription drug coverage. In particular, younger patients were more likely to gain coverage under state health reform. A 2004 Massachusetts Health Insurance Survey found that over 90% of newly enrolled Medicaid enrollees after Massachusetts health reform were previously unenrolled . PreviouSocioeconomic disparities in mortality among BCA patients have persisted despite increases in overall survival rates over recent decades. According to a recent report by the American Cancer Society, the mortality rate among BCA patients in poor counties was about 1.16 times higher than that in affluent counties. The observed relative increases in likelihood of initiating AET among younger women in lower-income areas who were more likely to have been without health insurance prior to the reform implies that Massachusetts health reform reduced disparities in receipt of adjuvant therapy and has important implications for health outcomes among BCA patients. This study examines initiation of AET, though AET adherence is also critical for reducing breast cancer recurrence rates, thus future studies estimating the effects of state and federal health reforms on adherence to AET would provide additional insight regarding the impact of health insurance policy changes on health outcomes.This study has limitations. First, the limited pre-reform period might not have captured other secular trends that contributed to the increased likelihood of initiation of AET among BCA patients. Second, we did not have comparable data available from other states, limiting the geographic generalizability of our findings and the ability to include a control group that was not impacted by the reform in any way. However, this study compared ZIP codes where the median household income was below the state median household income to those where median household income was above the state median household income in an effort to compare a population most likely to be impacted by the reform to a comparable group. Given that those in higher-income areas also stood to benefit from certain provisions of the health reform, our estimates of the differential impact on patients from lower- versus higher-income areas may represent an underestimate of the full impact of reform. Fourth, due to the nature of cancer registry data, there was no relevant clinical information, including menopausal status and comorbidities, and other potential socioeconomic information including patient’s education and income that can impact initiation of AET. However, regarding patient’s menopausal status, we adjusted for BCA patients’ age as a proxy. Fifth, local area (such as ZIP code-level) health care capacity characteristics, such as the number of providers, were not adjusted for in the model. Estimates from models that adjusted for county-level health care capacity characteristics were strikingly similar to estimates from our main model.Disparities in BCA outcomes by socioeconomic factors, such as poor insurance coverage and lack of financial resources, persist. Given that about two-thirds of early-stage BCA cases are hormone-responsive, our findings indicate that expansion of health insurance coverage increases the number of eligible patients initiating AET and insurance coverage expansion may be an important policy tool for reducing income disparities in BCA outcomes, including survival. Timely initiation of and adherence to AET will result in better prognosis, which will prevent recurrence rates and improve survival of patients. This evidence from Massachusetts health reform underscores the significance of continued efforts to expand coverage across the US and emphasizes the importance of evaluating the effect of other relevant health insurance policies, such as the Affordable Care Act (ACA), on the uptake of adjuvant treatment among cancer patients.Additional file 1: Table S1. Event study estimates to test parallel trends in the pre-reform period for all models. Table S2. Comparing various definitions of low-income areas to estimate changes in likelihood of initiating AET after state health reform in low-income areas in Massachusetts.


We are presenting to you an magnetic resonance imaging (MRI) finding of a 27-year-old male who presented to us with complains of gradually progressing pain over the neck along with radiation to the upper extremity with more involvement of the right side than the left. He also complained of insidious onset of weakness in the upper extremity. Magnetic resonance imaging findings revealed T2 hyperintense mass (red arrow) in the intradural extramedullary mass involving the C3-C4 cervical vertebrae and the intervertebral disc in between, highly suggestive of neurofibroma. Spinal neurofibromas are frequently observed over the cervical cord and are usually asymptomatic. Our patient showed mild symptoms, probable reasons might be the slow growth rate of neurofibroma. He was referred for physiotherapy for further opinion. On physical examination, he had weakness in upper limb movement (MRC grade 2+) and weak grip strength. Surgery was the treatment of choice for him and he underwent surgical excision of it along with pre-operative physiotherapy management for 5 days. Post-surgery, there was an enormous improvement in his symptoms i.e. residual weakness persisting without any pain. He was prescribed strengthening and gripping exercises. Before discharge, he had a grade of 4+ on the MRC grading scale. Early medical and surgical management along with pre- and post-operative rehabilitation is the road to recovery in such patients.


Ixekizumab is monoclonal antibody targeted against interleukin-17 (IL-17) and has been approved for use in chronic plaque psoriasis. Despite its efficacy in treating psoriasis, concerns have been raised regarding Ixekizumab’s potential to induce and exacerbate inflammatory bowel disease (IBD).Here we report the new onset of severe drug-associated colitis with surgical complications in a 45-year-old male patient who was receiving Ixekizumab therapy for chronic plaque psoriasis. Review of the patient’s colonic pathology demonstrated acute inflammatory changes with features of Crohn’s disease. The patient remained disease-free 9-months following his hospitalization and cessation of Ixekizumab.This case raises suspicion for an association between Ixekizumab and IBD and calls on clinicians to have heightened awareness of potential risks before prescribing anti-IL-17 agents. Interleukin-17 (IL-17) is a proinflammatory cytokine that plays a complex role in T-cell-mediated immunity and has emerged as a key target in the biologic therapy of various autoimmune diseases . In psorA 45-year-old male patient presented to our institution with a 3-week history of diffuse abdominal pain, tenesmus, and over 10 episodes of non-bloody diarrhea per day. The patient did not have nausea or vomiting nor did he report fever, chills, or unintentional weight loss. In the week prior to his presentation, he was assessed by his family physician who had ordered a panel of stool nucleic acid amplification tests which were negative for gastrointestinal infections. He was then trialed on a brief course of proton pump inhibitor as an outpatient with no improvement in his symptoms before presenting to the emergency department.The only significant past medical history for our patient was a long-standing history of plaque psoriasis for which he was being treated with Ixekizumab. He had previously undergone multiple courses of biologic treatments with adjunctive phototherapy but did not achieve reasonable control of his disease until he was switched to Ixekizumab approximately 9 months ago. The patient did not have a family history of IBD or early onset colorectal cancer. He is a life-long non-smoker and he does not report excessive alcohol use. There was no history of recent travel, blood transfusions, intravenous drug use, or new sexual contacts.On initial assessment, the patient was hemodynamically stable with diffuse non-peritonitic abdominal pain on physical examination. His laboratory investigations demonstrated marked elevation in his inflammatory marker (C-reactive protein of 388 mg/L) as well as a hepatocellular pattern of liver enzyme elevation . A computed tomography (CT) of the abdomen and pelvis was performed revealing continuous circumferential bowel wall thickening of the left colon from the rectum to the splenic flexure on a background of reactive retroperitoneal lymphadenopathy  colitis, the patient was started empirically on oral Mesalamine and IV Ganciclovir while awaiting the results from his endoscopic biopsy. Interestingly, the biopsy showed no viral cytopathic changes and CMV immunohistochemical staining was negative. The specimen did however have non-specific changes of cryptitis, epithelioid granulomas and mixed inflammatory infiltrates with areas of fibrinoid necrosis. Following discussions with our gastroenterology and pathology experts, Ganciclovir was discontinued and high dose corticosteroid therapy was initiated for probable drug-associated colitis in context of his Ixekizumab exposure.Despite initial clinical response to steroid therapy, the patient developed toxic megacolon 2 days later and was complicated by perforated viscus as evident from the presence of intraperitoneal free air on abdominal X-ray. Following an emergent total colectomy, the patient was transferred to the intensive care unit (ICU) for continued support. During his week-long ICU admission, the patient suffered from persistent intra-abdominal sepsis requiring high dose vasopressors which prompted repeated exploratory laparotomies revealing ongoing small bowel ischemia. The patient had further resection of his distal ileum and was successfully weaned from vasopressors before undergoing an end ileostomy. He continued rehabilitation for an additional 2 weeks on the surgical ward before being discharged home with follow-ups to our gastroenterology and general surgery specialists. At 9-months post-discharge, the patient had been progressing well with no recurrence of clinical disease in the absence of any immunosuppressive therapy. Further review of the patient’s surgical and endoscopic specimens revealed the presence of epithelioid histocytes and widespread fissuring ulcerations with intermittent regions of transmural inflammation favouring a Crohn’s-like pattern  regulates the secretion of IL-17 by T helper type 17 cells (Th17) which promote the release of pro-inflammatory mediators leading to subsequent intestinal mucosal damage . HoweverPsoriatic diseases have been associated with IBD in addition to many other gastrointestinal pathologies including celiac disease, acid reflux, and irritable bowel syndrome (IBS) . The undIxekizumab is 1 of 3 available anti-IL-17 biologics that have been approved for treatment of psoriasis by the United States Food and Drugs Administration. In the initial efficacy trials examining the use of Ixekizumab for plaque psoriasis, new presentations of IBD were not found to be a frequent adverse effect in the experimental groups . SimilarAnti-IL-17 agents—such as Ixekizumab—are becoming increasingly prevalent in their use against psoriatic diseases. Although their precise actions in the gut mucosa remain unclear, our case raises concern that IL-17 inhibitors may be complicit in the induction of inflammatory bowel pathologies. Physicians should be vigilant in their use of anti-IL-17 therapy and patients on active treatment should be monitored closely for the development of gastrointestinal symptoms.


The ongoing COVID-19 pandemic has highlighted a central principle of the host immune response to RNA virus infections: while most infected patients remain asymptomatic or mild symptomatic, a portion of patients experience severe clinical symptoms, suggesting the existence of host immune factors that determine susceptibility to symptomatic disease. One well-known RNA virus infection that is characterized by differential susceptibility to symptomatic disease due to host immune determinants is dengue. A well-balanced, early immune response is protective for the host and leads to asymptomatic infection or self-limiting disease. Exacerbated and skewed immune responses, mostly observed during secondary heterotypic infection, can result in dengue hemorrhagic fever or dengue shock syndrome. The aim of this Research Topic was to group research papers that provide insights into the mechanisms leading to protection from disease or immunopathology upon dengue infection.Rathore et al. of the old paradigm that dengue virus does not cause infection in immunocompetent mice. In this paper, the authors aimed to systematically test the ability of clinical isolates of DENV1-4 to induce replicating and systemic infection in wild type C57BL/6 mice and characterize the clinical outcome of infection. The authors found consistent and efficient viral replication in target organs and observed clinical symptoms that seemed to mimic human disease. Even though this proposed murine model for DENV can contribute to our understanding of immune-mediated pathology, limitations remain such as the reproducibility of utilizing clinical DENV isolates and the non-natural mode of infection via intraperitoneal injection. Future studies should be directed to understand the observed discrepancy between laboratory strains and clinical isolates, to assess the production of infectious virus in these models, and a reproducible method to induce either inapparent or more severe disease in order to identify correlates of protection.One of the largest obstacles to understand in depth the sequence of the immune response to dengue virus and the mechanisms behind an unbalanced immune response remain the absence of animal models representative for human disease . One steUpasani et al. assessed IFN-I response and frequencies of circulating pDCs in a cohort of hospitalized Cambodian children undergoing acute DENV infection. They found that patients with higher viral loads had elevated plasma level of IFN-I and increased percentages of circulating pDCs. Analyzing IFN-I concentration in plasma with ultrasensitive digital ELISA and expression of IFN-I and IFN-related genes in PBMCs revealed that IFN-I response were reduced in critically-ill patients, as compared to patients with mild disease. Thus, a robust and rapid IFN-I response, likely generated by pDCs, seems to be beneficial for DENV patients. It would be of interest to investigate further the link between pDC-mediated IFN-I responses and anti-DENV antibody production, and the predictive value of IFN-I as biomarker of severe disease.To explore putative biomarkers of severe dengue disease and their potential sources in humans the next study in this issue focused on the type I interferon response (IFN-I), which is induced by a broad array of viruses. Plasmacytoid dendritic cells (pDCs) are rare immune cells that circulate in the blood. In response to viral infection, they secrete large amounts of IFN-I. These cytokines are responsible for the induction of a rapid and efficient antiviral state. They also stimulate B cell responses. Pradeep et al. aimed to identify novel biomarkers of severe disease at hospital entry. The authors hypothesized that early secretion of cytokines by innate immune cells such as NKT cells, monocytes and granulocytes could be associated with an unbalanced immune response as observed in severe dengue cases. At admission, slightly lower, but significant percentages of circulating NKT cells, monocytes and granulocytes produced tumor necrosis factor (TNF)-α in dengue patients with warning signs compared to dengue patients without warning signs. A composite biomarker – IFN-γ+ NKT cells combined with interleukin (IL)-6+ granulocytes– could distinguish patients who progressed to worsened disease during hospitalization with a sensitivity of 90% and a specificity of 76%. However, the frequencies of these cells in blood are very low and hence interpretation should be with caution and how these parameters can translate into clinical practice remains to be determined. Further development of predictive biomarkers involves large scale multi-center translational studies to take these biomarkers from the bench to the clinic.Indeed, from the public health perspective, the ability to identify dengue patients which will progress to severe dengue will improve patient management and intervention allocation in resource constrained countries. The research presented here by Falconi-Agapito et al. use a library of 9,072 linear peptides covering the entire proteome of dengue, as well as of the 2 related flaviviruses Zika and yellow fever viruses. Sera from symptomatic individuals from Peru, which is an endemic area, or from primary-infected travelers returning to Belgium, were analyzed across four or two time points, respectively. The strongest IgG responses were detected in the early convalescent phase and were targeting peptides located mainly in 4 viral proteins . This IgG response was higher in secondary infections compared to primary ones. The magnitude of the IgM response was higher in primary infected travelers than in Peruvian patients, but the breadth and depth of the response was lower in this group compared with the endemic subjects. IgM and IgG exhibiting dengue-specific epitopes were identified. Validating the findings on larger cohorts of patients would be necessary. Upon validation, such peptide-based serological tests could be used to differentiate primary from secondary infection and would be easier to manufacture than conventional full-length recombinant protein assays.During secondary dengue infections, suboptimal levels of cross-reactive antibodies, mainly directed against the structural protein E, can drive antibody-dependent-enhancement of infection, resulting in more severe clinical signs than during primary infection . MoreoveEchegaray et al. evaluated the performance of two new lateral flow rapid diagnostic tests (RDTs) determining DENV seropositivity in samples from healthy volunteers non-endemic, endemic, and pediatric cohorts. The Dengvaxia dengue vaccine is approved for DENV-seropositive subjects 9 years of age and up whereas younger and dengue-naïve vaccinees are at increased risk of post-vaccination dengue disease; thus, rapid and accurate evaluation of serostatus would aid dengue vaccination efforts. One of the RDTs was based on standard DENV1-4 envelope (E) antigen, while the second contained as antigen recombinant DENV1-4 envelope (E) proteins with engineered glycosylation sites in the fusion loop to reduce false positives due to fusion loop binding as well as pre-adsorption with glycosylated ZIKV E to further reduce false positives in DENV/ZIKV-endemic areas. Performance of the first standard RDT was insufficient even with quantitative assessment, while the glycosylated E RDT exhibited acceptable sensitivity and specificity when evaluated quantitatively through pixel intensity analysis of the indicator strip on the lateral flow device. Performance of this new RDT was comparable to ELISA-based or gold standard neutralization tests currently available. This paper highlights that continual improvement of RDTs which are currently most often deployed to assess symptomatic patients could be field-adapted to be read on a device and eliminate the need and time required for laboratory analysis. Such advances will facilitate the use of RDTs to identify healthy, eligible candidates that can be safely vaccinated.Detection of prior dengue infection with high sensitivity and specificity remains challenging. Sanchez-Vargas et al. compared two T cell reactivation protocols in a pediatric dengue cohort using samples at timepoints before and after secondary dengue infection to ask if the dengue-specific T cell response pre-secondary infection would be changed upon secondary infection and if the response would correlate with clinical severity of secondary dengue. Brief ex vivo reactivation with peptides derived from structural or nonstructural DENV genes yielded low interferon (IFN)-γ responses and did not demarcate recent secondary infection or clinical severity. Instead culturing cells with non-structural peptides for up to two weeks yielded improved overall sensitivity with IFN-γ responses increased after infection in those subjects not requiring hospitalization. In this small study, NS-specific IFN-γ responses pre-secondary infection were higher in subjects who went on to require hospitalization after DENV2 secondary infection. The balance of serotype-specific or cross-reactive antibody responses and neutralization capacity thereof may be also important in clinical outcome  and is supported by NIH grant U01AI151758 – 01 and Institut Pasteur.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In a pooled analysis of 2148 TNBC-patients in the adjuvant setting, TILs are found to strongly up and downstage traditional pathological-staging in the Pathological and Clinical Prognostic Stage Groups from the AJJC 8th edition Cancer Staging System. This suggest that clinical and research studies on TNBC should take TILs into account in addition to stage, as for example patients with stage II TNBC and high TILs have a better outcome than patients with stage I and low TILs.The importance of integrating biomarkers into the TNM staging has been emphasized in the 8 Prognostic biomarkers are becoming increasingly important given that many low early-stage breast cancer patients have excellent outcomes with standard of care treatments. The clinical guidelines for systemic treatment in early-stage TNBC are not consistent. The evidence for the integration of biomarkers into the TNM staging system is mostly retrospective and with limited prospective data available1. In the case of multigene panels, their clinical validity for defining prognosis of particular subgroups of patients had been shown in several retrospective analyses and these were routinely used and incorporated into national (NCCN and ASCO) and international  treatment guidelines far before prospective data became available. In the 8th edition of the AJCC, histologic grading, molecular subtype  and multigene panels/genomic signatures were included into staging assessments. Whilst pathological and clinical stage remain a valuable aspect of the staging process and are strongly prognostic, we highlight here the evidence that supports Tumor Infiltrating Lymphocytes (TILs) as a biologic biomarker which improves discrimination over prognostic pathological and clinical staging for early stage TNBC.The importance of integrating biomarkers into the TNM staging has been emphasized in the 82. These data were from 2148 patients enrolled onto eight prospective clinical trials as well as one large institutional cohort. All patients had received anthracyclines with or without taxanes based regimens in the adjuvant setting. The median follow-up was 6.6 years. In the current study, using the Pathological and Clinical Prognostic Stage Groups from the AJJC 8th edition Cancer Staging Manual , three endpoints were calculated for each Stage group: invasive disease-free survival (iDFS), distant disease-free survival (D-DFS) as well as overall survival (OS). At a cut-point of 30% or more, TIL scores are found to strongly up and downstage traditional pathological-staging in this large data set 92. Theseth Edition were Adjuvant Online and PREDICT-Plus. We have developed a prognostic model for early-stage disease that includes age, number of positive lymph nodes, tumor size, grade, and stromal TIL percentage. This prognostic model is available on the website of the International Immuno-Oncology Biomarker Working Group (www.tilsinbreastcancer.org). This prognostic model could be used in routine patient care as well as future trial designs.Risk assessment models meeting the AJCC criteria and included in the 85, pembrolizumab, a PD-1 inhibitor, was found to significantly but moderately increase the rates of pathological complete response (pCR) and significantly improve event-free survival (EFS) when added to a neoadjuvant regimen of anthracycline, taxanes and carboplatin in patients with stage II and III TNBC. Similar magnitude of EFS improvements have been reported in smaller studies7. PD-L1 protein is known to be highly expressed on the breast cancer TIL and correlations between both markers have been observed8. In the KEYNOTE-522 study, patients designated as PD-L1 positive by the PD-L1 IHC 22C3 pharmdDx assay  were reported to have higher rates of pCR in both pembrolizumab and control arms across all TNM stages. An important treatment by PD-L1 interaction was not observed, in contrast to late-stage disease10. In any case, the increasing incorporation of PD-1 inhibition into the treatment of both early- and late stage TNBC further reinforces the need for routine evaluation of TIL in all TNBC patients, particularly given discordances in PD-L1 IHC assays. One could also speculate that patients with high expression of both immune biomarkers, i.e. those “immune enriched” could be strong candidates for future treatment optimization 11.The inclusion of immune biomarkers is timely given reporting of phase III registration trials of PD-1/PD-L1 targeting agents in early-stage TNBC. In the recently reported KEYNOTE-522 study (NCT03036488)2. The study protocol of this project was approved by the local institutional review committee at Gustave Roussy in November 2014 and authorized by the French National Committee on Computing and Liberty. Identified studies were prospective randomized clinical trials or large retrospective hospital series that evaluated the prognostic associations of TILs in patients who were diagnosed with early-stage TNBC treated with anthracycline-based chemotherapy with or without taxane in the adjuvant setting using the same prespecified method for reading TILS . TNBC was defined by estrogen receptor (ER), progesterone receptor (PR), and HER2 negativity as outlined in Loi et al. (ref).This is a follow-up analysis of the Loi et al 2019 studyThe prespecified primary end point was invasive disease-free survival (iDFS), defined as the date of first invasive recurrence, or second primary or death from any cause. Patients still alive without an event of interest were censored at the date of the last visit. Distant disease-free survival (D-DFS) is defined as the date of first distant recurrence or death from any cause. Patients still alive without an event of interest were censored at the date of the last visit. Overall survival (OS) was defined as the date of death from any cause. Patients still alive were censored at the date of the last visit. We used the Kaplan-Meier method to establish survival curves according to AJCC staging categories. The Kaplan–Meier estimates are provided together with bootstrap confidence intervals (1000 samples).Further information on research design is available in the Supplementary InformationReporting Summary