Pancreatic ductal adenocarcinoma (PDAC) is an intractable cancer and a leading cause of cancer deaths worldwide. Over 90% of patients die within 1 year of diagnosis. Deaths from PDAC are increasing and it remains a cancer of substantial unmet need. A number of factors contribute to its poor prognosis: namely, late presentation, early metastases and limited systemic therapy options because of chemoresistance. A variety of research approaches underway are aimed at improving patient survival. Here, we review high-risk groups and efforts for early detection. We examine recent developments in the understanding of complex molecular and metabolic alterations which accompany PDAC. We explore artificial intelligence and biological targets for therapy and examine the role of tumour stroma and the immune microenvironment. We also review recent developments with respect to the PDAC microbiome. It is hoped that current research efforts will translate into earlier diagnosis, improvements in treatment and better outcomes for patients. Despite advances in uncovering the biology underpinning this disease, and improvements in diagnostic and cancer registration practices in some countries, deaths from PDAC are projected to increase in the coming years2. The only possibility of cure is appropriate surgery with adjuvant chemotherapy3. Patients classically present with obstructive jaundice (when the tumour arises in the pancreatic head); however, many present with vague symptoms if tumours arise in the body or tail of the pancreas4. Clinical vigilance is key. Thus, at presentation, almost 80 to 85% already have locally advanced or distant metastatic disease, and treatment options offering curative intent are limited6. Although patients can be brought to surgery using neo-adjuvant regimens7, those with metastatic disease are currently unsuitable for surgery. The success of systemic therapies has also been limited because of the intense resistance of the disease to current treatment regimens. Progress in treatment, including personalised approaches, in combination with earlier detection could significantly contribute to better patient outcome  has a 5-year survival of around 7 to 10%, which remains almost unchanged in 40 years outcome .8, hereditary syndromes predisposing to PDAC9, intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms10, chronic pancreatitis11, , obesity12, new-onset diabetes (NOD)13 and smoking14. The European Prospective Investigation into Cancer and Nutrition (EPIC) highlighted that individuals with high healthy lifestyle index scores  have a decreased likelihood of developing PDAC15.Risk factors for PDAC include a history of familial pancreatic cancer (FPC)18. Similarly, some animal and small case-control studies in humans have related fatty infiltration of the pancreas to PDAC development19. It has been suggested that obesity may drive tumorigenesis through the activation of KRAS signalling pathways, although our understanding of this process is in its infancy20. Understanding the molecular mechanisms behind obesity-driven tumorigenesis would facilitate the development of risk reduction strategies and targeted screening for obese individuals. Furthermore, understanding how to effectively implement public health interventions will be key in primary prevention at a population level.The rise in obesity and diseases associated with it, including type 2 diabetes, remains a major significant public health challenge in higher socio-economic index countries and increasingly in the developing world. Evidence from case-control and cohort studies suggests an association between obesity and PDAC2). The World Health Organization advocates screening in all cancers, and anticipated increases in curative treatment are up to 30% from earlier detection (www.who.int/cancer/en/index.html). Five per cent to 10% of cases of PDAC are due to inherited risk factors: either a family history of the disease or germline mutations which give rise to PDAC syndromes9. The most studied of those syndromes include Peutz-Jeghers syndrome (STK11)21, hereditary breast-ovarian syndrome (BRCA2)22, familial atypical multiple mole melanoma (CDKN2A)23 and Lynch syndrome (MLH1/2/6)24. The International Cancer of the Pancreas Screening (CAPS) Consortium recommends screening for high-risk individuals, notably carriers of high-risk germline mutations, such as those mentioned above, or individuals with defined FPC. This screening should be offered in a research setting until the benefits, risks and costs of pancreatic cancer surveillance are elucidated25. Established research screening programmes include the following: the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer (EUROPAC), Liverpool, UK26; the North American National Familial Pancreatic Tumour Registry27 and the German National Case Collection for FPC28. There are specific guidelines for screening patients with chronic pancreatitis for the risk of pancreatic cancer. The current literature supports screening only for those patients with an autosomal dominant history of hereditary pancreatitis with or without a mutation in cationic trypsinogen (PRSS1 gene)29.Worldwide, there are currently no recommended population-wide screening programmes for PDAC in asymptomatic adults and this is due in part to the relatively low incidence of the disease in the general population  have a 5.4-fold relative risk of developing PDAC32. For about 1% of individuals who are at least 50 years old and whose diabetes has been diagnosed, PDAC is the underlying cause of diabetes. PDAC-associated diabetes is a form of type 3c diabetes33. Hyperglycaemia in PDAC occurs up to 3 years before diagnosis and begins before pancreatic tumours are visible on imaging, suggesting that the pathophysiology of PDAC-associated glucose dysregulation is more than the destruction of the gland34. Further support for this is shown by the improvement in glucose dysregulation after surgical resection of the tumour35. Despite recent advances, our understanding of the biology underpinning the development of diabetes in PDAC is in its infancy. However, with about 80% of individuals presenting with glucose dysregulation at the time of diagnosis of PDAC, including a large proportion presenting with NOD, screening strategies for this high-risk group are urgently needed30. Cohorts of patients in both the USA  and the UK (UK Early Detection Initiative for Pancreatic Cancer37) have been established in response to this need to investigate how the presence of NOD may be used to expedite the detection of PDAC in this high-risk group.Individuals with NOD are the largest high-risk group who may benefit from screening38. Cross-sectional imaging is used for screening patients with hereditary pancreatitis, as the calcium load in these individuals will prevent adequate EUS assessment. Future developments with positron emission tomography (PET) and the use of molecular imaging techniques that target proteins overexpressed by the tumour, signalling pathways, or the stroma may improve detection of early lesions40. Further development of hyperpolarised magnetic resonance imaging, which can identify metabolic aberrations in the pancreas indicative of pre-neoplasia, may also prove to be a useful adjunct to early detection and screening41.The capacity for early detection is limited by current imaging modalities, which lack the sensitivity to identify small lesions. Endoscopic ultrasound (EUS), which has a sensitivity of 72% for identifying T1 and T2 cancers, is advocated in FPC but suffers from inter-user variability, inconsistency in patient access, and invasiveness44. As our understanding of the molecular changes that occur during the transition from pre-neoplastic lesion to early PDAC and advanced disease improves, rational strategies have been applied to identify novel biomarkers across genomic, transcriptomic, metabolomic and proteomic platforms utilising a variety of specimens, including microvesicles (exosomes), ctDNA and methylated DNA47. New pre-diagnostic cohorts and large collaborative studies will be crucial to the development of biomarkers and their rapid translation to the clinic. It is widely accepted that biomarkers will be essential in the refining of inclusion criteria for future PDAC screening programmes. Much research has been published in this area; however, no biomarkers have been carried through to clinical use. The failure to translate biomarker development into the clinic lies in part with the widespread validation of biomarkers using a diagnostic sample, which are compromised by late changes during tumorigenesis that are not seen in early-stage disease. Second, where existing pre-diagnostic cohorts exist, they often lack adequate control groups and do not contain data relevant to PDAC, such as diabetes status or presence of chronic pancreatitis, limiting the interpretation of findings. Finally, and perhaps most importantly, PDAC within the population is relatively rare, which demands high levels of specificity from biomarkers to minimise false-positive rates. To comprehensively test biomarker accuracy, large numbers of samples are required from target populations and these cohorts are hard to come by. Of those published biomarkers, carbohydrate antigen (CA) 19-9 remains the only validated biomarker clinically used in the management of PDAC. Although CA19-9 may hold future utility as a component in a panel of markers for the identification of PDAC, it lacks the sensitivity and specificity to act as a stand-alone biomarker for asymptomatic diseaseTo meet with the demand for rapid evaluation of an increasing number of early-stage tumours, it is likely that diagnostic, prognostic and predictive algorithms, based on a combination of imaging, molecular and health data, will be needed. Here, artificial intelligence (AI), and more specifically machine learning (ML) and deep learning (DL) (a subset of ML applied to large datasets), have emerged as valuable tools for disease risk-stratification and identification in general populations. ML and DL automate analytical model building. They use a variety of operations to examine and compare small to large datasets to find common patterns and explore nuances. Both ML and DL can be performed in a supervised or unsupervised manner, with unsupervised learning training models and unlabelled data.48. More recently, AI methods (ML and DL) have been explored to provide a better quantitative description of biological processes in PDAC49, to identify novel biomarkers50, and to undertake large-scale analysis of clinical records to develop algorithms for automatic identification of at-risk individuals51.Early detection and management of pancreatic cancer rely on an understanding of the basic biology of the disease as well as an appreciation of the disease course at a population level . AI methods are making contributions here across the spectrum of basic research areas, and progress requires interdisciplinary collaboration between experts in AI, disease physiology and molecular cell biology. Perhaps most widely explored is the use of ML and DL approaches for the extraction and interpretation of features in medical imaging, although we are still only just scratching the surface of what is possible for image-based modelling of pancreatic cancer riskIncreasingly extensive datasets are being generated as a result of advances in high-throughput proteomic, transcriptomic and genomic technologies. As ML researchers and practitioners gain more experience, the capacity for ML to interrogate and integrate this information to aid in our understanding of PDAC biology, and to inform how to manage and detect the disease at the earliest time point, will be immense. This will be an exciting area to follow in the coming years.52. The major genetic mutations responsible for PanIN progression are well established. Grade 1 and 2 PanIN are characterised by a point mutation in the KRAS oncogene (90% of tumours)53, and telomerase shortening is also characteristic of this stage54. Grade 2 is associated with increased activation of CDKN2A and CDKN1A55. Grade 3 and 4 are associated with mutations in TP53 (50–70%) and SMAD4 (60–90%)57. There are many more somatic mutations described in PDAC specimens, and over 100 to 150 have been identified using next-generation sequencing58. Most somatic mutations occur in common pathways: RAS signalling, transforming growth factor beta (TGFβ) pathways, cell-cycle control, WNT signalling, NOTCH signalling and DNA damage repair59. As in other gastrointestinal tumours, the majority of somatic mutations are not currently targetable; however, there is some evidence that our understanding of underlying mutations is translating into clinical practice, including small molecule inhibitors of KRASG12C and PD-1 blockade in tumours deficient in the mismatch repair (MMR) genes MLH1 and MLH261.PDAC is preceded by the progression of precursor lesions, such as pancreatic intraepithelial neoplasia (PanIN), IPMN and mucinous cystic neoplasm. PanIN, which arises from a stepwise accumulation of somatic genetic mutations, is the most common of these premalignant lesions and represents a key target for early detection and treatment62. A more recent analysis of 309 resected PDAC tumours confirmed the progenitor, squamous and quasi-mesenchymal subsets but also further characterised the TME, identifying three additional subsets: desmoplastic, immune classical and stroma-activated63. It has been suggested that tumours of the squamous subset are associated with an adverse prognosis and are more resistant to current chemotherapy regimens compared with other subtypes; however, fully translating this work into clinical applications is still in the early stages64.Recent global transcriptomic analysis has allowed PDAC to be subclassified based on distinct molecular signatures. Early work classified these molecular subtypes as classical (progenitor), quasi-mesenchymal (squamous) and exocrine-like (aberrantly differentiated endocrine exocrine), although this classification was based on analysis of the epithelial aspects of PDAC tumours only and did not take into account the wider tumour microenvironment (TME)66. Much of this programme is focussed on patients with metastatic disease, and the most common actionable alterations are seen in DNA repair genes (BRCA1/2 or ATM mutations) or cell-cycle genes . It should be noted that, despite genetic testing, the majority of patients (68%) enrolled in this trial nonetheless underwent standard-of-care chemotherapy with either FOLFIRINOX or gemcitabine with nab-paclitaxel rather than targeted therapy66. Other classes of drugs utilised based on molecular signatures of PDAC include inhibitors of MEK67 and PARP68. Data published from the “Know Your Tumour Programme” suggest that patients who underwent therapy that were matched to molecular profiling had better overall survival than those who did not , suggesting that it is feasible to tailor therapy on the basis of individual molecular characteristics. However, of the 1,856 patients who were referred to the programme, only 46 (2%) underwent matched therapy and this was due to the aggressiveness of the disease and limited treatments available at the time of the trial for some of the identified molecular signatures, such as NTRK alterations69. Additional limitations relating to targeted therapies should be noted. These include the difficulty in obtaining samples of adequate yield and quality for molecular subtyping in non-resected patients following fine-needle aspiration as well as intra-tumour heterogeneity of PDAC70. Targeted treatments based on molecular profiling, therefore, can currently be viewed as an encouraging proof-of-concept idea that requires ongoing global efforts before the majority of patients will benefit from such methods.There are a number of research programmes aiming to apply the expansion of molecular subtypes of PDAC into clinical trials, and this is a major area of collaboration across industry and academia. The “Know Your Tumour Programme”, one such example, aims to determine whether targeting actionable molecular signatures can improve outcomes71. Tissue stroma plays a role in the response to injury utilising the immune, vascular and connective tissue components within it. Understanding and trying to develop treatments to target aspects of the TME have therefore been the focus of research over the last decade. The characteristic dense stroma induced by PDAC includes an array of cell types, including cancer-associated fibroblasts (CAFs), inflammatory cells, blood vessels and nerve cells, as well as the extracellular matrix (ECM) produced by the CAFs, including collagen, fibronectin, laminin and hyaluronic acid73.PDAC exhibits a strong desmoplastic reaction characterised by a hypoxic and hypovascular TME. This desmoplasia is argued to be a principal contributor of resistance to standard chemotherapy in PDAC74. This resultant desmoplasia creates a mechanical barrier around tumour cells and prevents vascularisation, which limits the delivery of chemotherapy and also immune cell infiltration75. Thus, a key area of research is to understand how to target the development of this dense stroma, either by targeting the extracellular components of the fibrosis itself or by targeting stromal cells (PSCs) and attempting to revert them to their quiescent form. Matrix metalloproteinases (MMPs) are a group of proteins that remodel the ECM. MMP276, MMP778 and MMP9 and tissue inhibitors of MMPs79 are shown in pre-clinical models to be differentially expressed between normal pancreas and PDAC, and higher levels are associated with worse prognosis and metastatic disease80. Marimastat81 and tanomastat82, two inhibitors of MMP, showed promising results in pre-clinical xenograft models of melanoma83, gastric cancer84 and colon cancer85. When these were applied to metastatic PDAC in phase 3 trials, however, there was no survival benefit82.Pancreatic stellate cells (PSCs) are myofibroblast-like cells that are activated by PDAC cells and become CAFs, producing ECM resulting in fibrosis in the tumour86. PEGPH20 is a human recombinant PH20 hyaluronidase, which in a mouse model led to depletion of hyaluronan, improved vascular permeability and increased drug delivery of gemcitabine and chemotherapeutic efficacy86. Clinical trials, however, have shown differing results; phase II trials of PEGPH20 with gemcitabine plus nab-paclitaxel have improved progression-free survival but reduced overall survival when combined with FOLFIRINOX88. Also, a subsequent phase III trial combining PEGPH20 with gemcitabine plus nab-paclitaxel did not improve overall survival compared with chemotherapy alone 89. Much of this was due to the side effects of PEGPH20 plus the worsening of chemotoxic symptoms with FOLFIRINOX leading to reduced treatment regimens and dosages.Interest in targeting hyaluronan within the ECM, stemming from the observation that high deposition of hyaluronan in PDAC is associated with poor prognosis, has developed over a number of yearsIn utero, repression of endodermal Sonic hedgehog (SHH) by inhibin-βB and FGF2 allows the expression of Pdx1 and insulin, initiating pancreatic differentiation90. Dysregulated hedgehog signalling is implicated in pancreatic carcinogenesis91, and mouse models showed that hedgehog signalling promotes desmoplasia and antibody-mediated inhibition reduced this desmoplastic reaction92. There is also some evidence of paracrine stimulation of PSCs through this pathway93. Translation of this in phase II studies, however, has been disappointing. A trial combining the SHH inhibitor saridegib and gemcitabine in metastatic PDAC was stopped early in 2012 because it led to higher rates of progressive disease94. Similar trials combining gemcitabine with vismodegib also showed no benefit to overall or disease-free survival compared with chemotherapy alone95.Subsequently, attempts to target signalling pathways responsible for the development of stroma rather than particular components of the ECM have focussed on the hedgehog signalling pathway. 96. Similar to results in colorectal cancer, the success of such treatments in PDAC has been limited96. The use of a small molecule inhibitor UAMC-1110 was not effective in a recent mouse model97. Indeed, studies of genetic deletion of fibroblasts in mouse models of PanIN and PDAC led to disease with more aggressive phenotypes, indicating that fibroblasts play a complex role in tumour development98. CAFs are also shown to be important for shaping the antitumour immune response99. Of particular importance is the role of CXCL12-CXCR4 signalling in stromal-immune crosstalk100. The COMBAT trial combined motixafortide, a CXCR4 inhibitor, with PD-1 inhibition in a phase IIa open-label study in metastatic PDAC and showed modest but insignificant changes to overall survival101. Thus, it is likely that future efforts will be focussed on exploiting reprogramming of the fibroblasts and the role they play in altering the TME rather than the cruder method of attempting to eliminate them. The results of ongoing studies focussing on CXCR4 inhibition  are eagerly awaited.Efforts to target CAFs and PSCs in an attempt to reduce the desmoplastic reaction have focussed mainly on inhibition of fibroblast activation protein (FAP)102. However, PDAC is a relatively immunologically “cold” tumour, and molecular profiling indicates that only a subset of the immune cells present within the tumour are immunologically active103. With respect to immune cells, the myeloid compartment dominates the TME and includes tumour-associated macrophages (TAMs), granulocytes and inflammatory monocytes. These are actively recruited to the TME during carcinogenesis orchestrated by KRAS mutations in the epithelial compartment104. TAMs can be categorised as either M1 or M2 macrophages; M1 generally shows tumoricidal activity acting via tumour necrosis factor (TNF), interleukin-12 (IL-12), IL-1α and interferon-gamma (INF-γ), and M2 produces anti-inflammatory tumour-promoting cytokines such as TGFβ and IL-10105. Myeloid-derived suppressor cells (MDSCs) are also produced from myeloid cells and these recruit regulatory T cells to the TME107. There is also evidence to suggest that this myeloid cell infiltration is critical for PDAC initiation (induction of immune checkpoint ligands) and it promotes the formation and maintenance of pre-neoplastic lesions108.The TME in PDAC is rich with immune cells, and it has long been established that chronic inflammation is an important characteristic of PDAC109, TAM M1 macrophages110 and MDSCs111, have been targeted. Further targets include CD40 agonists113, chemokine modulation114 and immune checkpoint inhibitors115. Despite showing promise in other solid organ tumours, the most studied of these—CTLA-4 (ipilimumab), and PD-1 (nivolumab)—have been disappointing in PDAC116. Another approach is to enhance antigen presentation and drive the expansion of tumour-specific T-cell clones through “vaccination”. Whilst multiple studies have shown that it is possible to yield antigen-specific immunological responses in patients with PDAC, vaccination strategies alone might not be enough to generate clinically meaningful antitumour effects117.Immunotherapy is an active area of interest, most commonly aimed at augmenting the antitumour adaptive immune response. Most of the above-mentioned immunosuppressive cells, including TAM M2 macrophages119. It has been suggested that there is a symbiotic relationship between PDAC cells and the microenvironment, including CAFs and TAMs. CAFs release non-essential amino acids through enhanced autophagy to support tumour cell needs through the TCA cycle120. Moreover, there is some evidence that PDAC cells reprogramme the stroma into a tumour-promoting metabolic environment that hinders T cells121. Blocking glutamine metabolism augmented with anti-PD-1 led to cytotoxic T-cell activation and a reduction in hyaluronan synthesis in a mouse model of PDAC123. There is much to learn in this area, and limited data are available from the small number of trials conducted. However, these studies remain of great interest as they suggest that focussing on the metabolic remodelling of the TME may influence desmoplasia, cancer metabolism and the immune response in a more orchestrated way.The limitations of direct stroma or immune-based therapeutic targeting perhaps allude to the logic that a better strategy would be to exploit integrated aspects of the TME, such as specific points of biological convergence. An example of this is targeting cancer cell metabolism. Cancer cells maintain high glycolytic activity in order to grow as well as needing glutamine to fuel the tricarboxylic acid cycle (TCA) cycleet al. profiled the bacteria present in seven different human tumours, demonstrating that bacteria were located intracellularly in both cancer and immune cells124, raising the possibility that they influence the immune state of the tumour environment and have potential implications for responses to immunotherapy. Interestingly, the bacterial composition of tumours varied between tumour types.Characterising the pancreatic tumour microbiome is providing insight into carcinogenesis. It is also uncovering the potential of the tumour microbiome as a therapeutic biomarker. Whilst the presence of bacteria in tumours is well recognised, their exact purpose or the consequences of their presence remain unclear. In an attempt to answer this question, Nejman 126. Significant variation in methodology and results, however, currently prevents consensus opinion. Elevated levels of intracystic bacterial DNA were found in patients with IPMN (both with high-grade dysplasia and with cancer)127, raising pertinent questions regarding the potential of iatrogenic bacterial translocation via endoscopy. Of course, it must be appreciated that other routes of translocation exist. Furthermore, exploration of pancreatic cystic fluid (PCF) has revealed the existence of a unique bacterial ecosystem, which may play a role in oncogenesis128.The notion that certain microbes play a causative role in oncogenesis is gaining momentum129. LTSs were found to have a more diverse tumour microbiome, and an intra-tumoral microbiome signature was found to be predictive of long-term survival. Human-to-mice faecal microbial transplants demonstrated attenuated tumour growth and immune cell infiltration with LTS faeces when compared with STS faeces. This pivotal study demonstrated that crosstalk occurs between the gut and tumour microbiome, which can directly influence and predict the outcome of disease129.The pancreatic cancer microbiome may also act as a clinical prognostic biomarker. Via 16S rRNA gene sequencing, the tumour microbiome of long-term survivors (LTSs) was compared with that of short-term survivors (STSs)+ T helper cells and CD8+ T-cell activation130. Reducing the bacterial content of tumours upregulates PD-1 expression, increasing the efficacy of checkpoint-targeted immunotherapy130. A number of studies in tumours such as lung and melanoma have addressed the proposal that gut dysbiosis may affect response to treatments133. The idea that we may be able to use microbiota composition to define groups most likely to respond to treatment is gaining traction and may help inform future clinical trials.The immunosuppressive environment of PDAC to date has hindered the effective use of immunotherapy. Targeting the intra-tumoural microbiome may be a strategy to increase efficacy. Bacterial ablation of tumours results in immunogenic reprogramming of the PDAC microenvironment, reducing MDSCs, increasing macrophage differentiation, and promoting CD4134. The results from emerging clinical trials in this area are thus eagerly awaited.It can no longer be assumed that the bacterial populations found inside and around PDAC are merely environmental bystanders. What remains unclear is whether bacteria in these regions contribute to causing the cancer or whether they populate as a result of oncogenesis. What is recognised, however, is that crosstalk between organs and their microbiomes exists and is far greater than anticipated. A barrier to translating scientific findings into large-scale clinical trials and clinically meaningful results is the issue of bias in the studies published, which can occur at any stage of the analysis. Consequently, there are relatively few clinical trials aimed at evaluating therapies manipulating the pancreatic microbiomePDAC remains a major global health problem. Improvements in survival have been made over the last 20 years because of advances in perioperative care, meaning that patients who undergo surgery can also benefit from adjuvant therapy. Many patients continue to present late with a disease that is too advanced for surgical intervention with curative intent, and this remains a significant challenge. Our understanding of high-risk groups and how to detect disease early in these groups continues to be central to improving outcomes, and ongoing research using high-risk cohorts is vitally important. Developments in AI and ML will hopefully improve early detection initiatives taking into account large datasets. There has been an increase in our understanding of the biology of PDAC, its microenvironment and the microbiome across the genetic, epigenetic, transcriptomic, proteomic and metabolomic spectrums. The translation of these findings into clinical trials remains an aim for the future.


The immune system is essential in the process of nerve repair after injury. Successful modulation of the immune response is regarded as an effective approach to improving treatment outcomes. T cells play an important role in the immune response of the nervous system, and their beneficial roles in promoting regeneration have been increasingly recognized. However, the diversity of T-cell subsets also delivers both neuroprotective and neurodegenerative functions. Therefore, this review mainly discusses the beneficial impact of T-cell subsets in the repair of both peripheral nervous system and central nervous system injuries and introduces studies on various therapies based on T-cell regulation. Further discoveries in T-cell mechanisms and multifunctional biomaterials will provide novel strategies for nerve regeneration. Human nerves are divided into two main types: the peripheral nervous system (PNS) and the central nervous system (CNS), including the brain and spinal cord –3. PNS aIn the early clinical treatment of nerve injury, allografting was considered one of the most effective ways to bridge the injury gap, as it elicits a foreign body immune response to avoid graft failure , 10. TheTherefore, in the clinical management of treating neurotrauma, it is of paramount importance to be able to expand the immune response that promotes tissue repair while inhibiting excessive inflammation that leads to further damage . At pres+ and CD8+ , they can be divided into two main subgroups, CD4and CD8+ . Additioand CD8+ . The infe repair , 25.T helper (Th) cells have been widely recognized as one of the essential members of both innate and adaptive immune responses, which also contribute to neutrophilic inflammation, remyelination, and neuropathic pain after nerve injury –28. Th cin vitro studies have shown that the supernatants of Th1 or Th17 cells are cytotoxic to human fetal oligodendrocyte progenitor cells (OPCs) and reduce the differentiation of OPCs into via proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) or astrocyte-derived CXCL10  (Regulatory T cells (Treg cells) are also a subset of CD4 (FOXP3) , 47. By via IL-10 after intracerebral hemorrhage-induced injury  mouse model . MeanwhiThermal, mechanical, chemical, or ischemic damage to the CNS or PNS results in neuronal loss and neuronal dysfunction, usually accompanied by sensorimotor impairment, which alters the quality of life of patients –64. For As the effect of T cells on nerve regeneration has been gradually explored, strategies for T-cell modulation have started to draw the attention of researchers , 80. ForFor central nerve injury, Laura’s team found that transplantation of human neural stem cells (hNSCs) into a mouse model of demyelination experimental autoimmune encephalomyelitis during the chronic phase achieved successful remyelination and neuroinflammation suppression by increasing central Treg cells, demonstrating that Treg cells can directly interact with stem and progenitor cells within tissues to promote central nerve regeneration . In Sun’Clinically, biomedical nerve grafts are usually required for repairing traumatic nerve defects. Recent studies have found that the behavior of T cells plays an important role in nerve defect repair using acellular nerve grafts (ANAs). Deng’s study found that insufficient accumulation of T cells within long-scale ANAs resulted in reduced expression of both IFN-γ and IL-4, which may limit functional recovery and nerve regeneration across the ANAs , 92. To Desirable nerve repair and functional recovery of traumatic nerve injuries remain great challenges in the clinic, and the immune response is essential in the pathological process of neural regeneration. However, there is a certain need for a deeper understanding of cellular immunity in the nervous system and how T cells contribute to the immune response after injury, especially in exploring the roles of different T-cell subsets at various stages of the repair process. Significantly, several achievements have been made for effectively modulating T cells in the immune response using biomaterials, stem cells, antibodies, or immunosuppressive drugs. The combination of biomedical scaffolds with cell therapy or spatiotemporal delivery of immunomodulators/cytokines/chemokines can provide great opportunities, which can be facilitated by further research into immunology.XT and JL drafted the main body of this manuscript. YY modified the manuscript. QL, TH, HZ, and XC helped with the manuscript editing and discussions. All authors contributed to the article and approved the submitted version.This work was financially supported by the National Science Foundation of China (Project No: 31830028) and Jiangsu Provincial Key Medical Center.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.


The importance of the cytoskeleton not only in cell architecture but also as a pivotal element in the transduction of signals that mediate multiple biological processes has recently been highlighted. Broadly, the cytoskeleton consists of three types of structural proteins: (1) actin filaments, involved in establishing and maintaining cell shape and movement; (2) microtubules, necessary to support the different organelles and distribution of chromosomes during cell cycle; and (3) intermediate filaments, which have a mainly structural function showing specificity for the cell type where they are expressed. Interaction between these protein structures is essential for the cytoskeletal mesh to be functional. Furthermore, the cytoskeleton is subject to intense spatio-temporal regulation mediated by the assembly and disassembly of its components. Loss of cytoskeleton homeostasis and integrity of cell focal adhesion are hallmarks of several cancer types. Recently, many reports have pointed out that lncRNAs could be critical mediators in cellular homeostasis controlling dynamic structure and stability of the network formed by cytoskeletal structures, specifically in different types of carcinomas. In this review, we summarize current information available about the roles of lncRNAs as modulators of actin dependent cytoskeleton and their impact on cancer pathogenesis. Finally, we explore other examples of cytoskeletal lncRNAs currently unrelated to tumorigenesis, to illustrate knowledge about them. Recently, extensive advances in transcriptomic and genomics have led to elucidate that the genome is far more pervasively transcribed than was previously appreciated, highlighting the importance of the non-coding genome in several cellular processes ,2,3. BroClassically, the cytoskeleton has been defined as a complex and dynamic meshwork composed of different structural proteins and a multitude of accessory components, mostly of a protein nature that mediates formation and stabilization of cytoskeletal structures themselves and the interactions between them. Broadly, cytoskeletal architecture is formed by three pivotal protein structures: (1) microfilaments of actin, responsible for shaping the cell and movement in a multitude of cells and unicellular organisms; (2) microtubules consisting of tubulin dimers, which facilitate the anchoring of the different cellular organelles in the cytosol as well as in the movement of the genetic material during cell cycle progression; and (3) intermediate filaments formed by proteins of different natures with a purely structural function . The intCytoskeletal homeostasis is under intense and tight regulation at both transcriptional and post-transcriptional levels, triggering cytoskeleton remodeling and turnover of cytoskeletal structures. Transcriptional regulation involves activation and inactivation of conserved gene pathways such as RHO GTPases /Rho or Wnt/β-catenin signaling, while post-transcriptional regulation modulates the processing of several genes of these pathways ,22. In mAlteration of cytoskeletal architecture is involved in the genesis of several diseases such as cardiovascular diseases , neurodegeneration related diseases (Alzheimer’s disease), and chronic liver diseases  ,43,44,45Another example of cytoskeletal lncRNA was provided by Chen et al. (2017). They demonstrated a pivotal role of taurine upregulated gene 1 (TUG1) in cortex cytoskeleton formation of vascular smooth muscle cells (VSMCs). TUG-1 is required for the correct assembly of EZH2 and the α-actin complex, participating on it. Downregulation of TUG1 has revealed that impaired expression of TUG1 triggers the breakdown of EZH2-α-actin interaction, leading to accelerated depolymerization of F-actin in VSMCs. Curiously, EZH2 mediated methyltransferase action upon α-actin is necessary for correct lysine-methylation, and thus the formation of the cortex cytoskeleton . FunctioActin microfilaments are considered the most dynamic of the three cytoskeletal protein structures capable of strong structural changes within minutes, determining the shape of a cell . Under pAn example of the most direct interaction of lncRNA-actin filament in a cancer context has been provided by Pei et al. (2018) , which dLNC CRYBG3 is not the only example of lncRNA involvement in cell cycle progression by modulation of cytoskeletal actin dynamics. Grembergem et al. (2016) showed that breast cancer cell lines require upregulation of a long non-coding RNA cytoskeleton regulator RNA (CYTOR), previously annotated as LINC000152, to increase cellular proliferation and to promote the transition between the G2/M phase in malignant cells. Functional assays have demonstrated that downregulation of CYTOR leads to the repression of several proteins involved in cytoskeletal homeostasis such as Golph3, Rhobtb3, and plakophilin4, which in turn leads to the failure of actin filament remodeling. Furthermore, Golph3 is essential to cytoskeletal molecular pathway activation such as in mTOR while PKP4 is recognized as a structural protein in cell–cell and cell–matrix binding ,63. As aCytoskeletal dynamics require the participation of several modulating factors which regulate G-actin assembly and de-assembly to form filaments. Among the modulators reported, those in the actin depolymerizing factor (ADF)/cofilin protein family are essential for actin dynamics, cell division, chemotaxis, and tumor metastasis. CFL-1 is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filament turnover in vitro and in vivo . InteresRecently, two lncRNAs have been identified as modulators of Cofilin-1 expression/function in different types of gliomas–Growth Arrest-Specific 5 (Gas5) and Aldoa repressor specify transcript (ARST) ,76. CuriUnlike Gas5, ARST does not affect Cofilin-1 mRNA and/or protein expression levels but modulates functional binding of Cofilin-1 to F-actin C. Gain aCells are usually surrounded by an extracellular matrix (ECM), and adhesion of the cells to the ECM is a pivotal step in their migration process ,34,82,83Chang et al. (2021)  describeSeveral gene pathways have been related to focal adhesion establishment, and their deregulation is reported in several tumors. Extensive sequence transcriptome analysis performed in samples of hepatocellular carcinoma have identified DARS-AS1 as oncogene positively modulating the expression of Cytoskeleton associated protein 2 (CKAP2), which in turn promotes the expression of two pivotal pathways required for remodeling cytoskeleton derived focal adhesions, focal adhesion kinase (Fak) and extracellular signal-regulated kinases (ERK) ,91. FuncThe dynamic movement of malignant cells through different tissues is related to the emission of cytoplasmic projections resulting in the reorganization of actin filaments based on the cell surface. Interestingly, the ratio of cellular protrusions determinates the migratory and invasive abilities of malignant cells . Long noCytoskeletal remodeling is subject to the activity of several receptor proteins on the surface of the cell, which in turn trigger the activation or repression of different intracellular signal cascades involved in cytoskeleton dynamics and turnover of actin dependent structures. Broadly, the RHO GTPases family acts as nexus between signals that come from transmembrane protein receptors and different modulators of cytoskeletal architecture A. CuriouRegulation of Rho/ROCK signaling by metastasis-associated lung adenocarcinoma transcript 1  has been described in both osteosarcoma and breast cancer ,124. LosThe molecular function of Rho and Rac members is required for correct actin remodeling in several carcinomas ,125. TwoROCK proteins constitute pivotal effectors downstream of RHO GTPases signals and disruption of their expression leads to failure of RHO GTPases dependent cytoskeleton remodeling . RecentlThe analysis of the human transcriptome has made possible to elucidate the importance of the non-coding genome in the regulation of multiple diseases such as cancer. Interestingly, tumorigenesis requires remodeling of the cytoskeleton, and thereafter, malignant cells can migrate and disperse through the organism, colonizing other tissues in a process term metastasis, which promotes the aggressiveness of cancer and therefore is correlated with a poor diagnosis of disease. The regulatory potential of lncRNAs has been described in all cellular processes including the modulation of the cytoskeletal architecture by regulating both structural cytoskeletal proteins and the signaling pathways that trigger the formation of actin-dependent structures. Several reports have highlighted the importance of lncRNAs as oncogenes in multiple carcinomas suggesting their pivotal role in the necessary remodeling of the cytoskeleton that leads to metastasis. Furthermore, knockout of most lncRNAs involved in cytoskeletal homeostasis leads to loss of ability to spread out and lower proliferation and survival rate pinpointing that lncRNAs can be considered as potential therapeutic targets against several carcinomas. Curiously, just a few cases of lncRNAs have been reported as regulators of cell–cell interactions in other contexts such as atrial fibrillation or even in non-pathological processes, demonstrating the importance of lncRNAs in cytoskeletal homeostasis. Given the relevance of cytoskeletal lncRNAs as modulators of migration and cell cycle regulation described above, it would be interesting to analyze their role during embryonic development since this complex process requires a tight and precise migration of different cell subpopulations. Failure of proper cell migration during embryonic development would result in malformation of the embryo and thus in many cases in congenital diseases that ultimately might lead to embryonic death.


Fusion and fission are essential for maintaining mitochondrial homeostasis through control of morphology, content exchange, inheritance of mitochondria, maintenance of mitochondrial DNA, and removal of damaged mitochondria by autophagy. Mitochondrial motility occurs through mechanical and molecular mechanisms which translocate mitochondria to sites of high energy demand. Motility also plays an important role in intracellular signaling. Here, we review key features that mediate mitochondrial dynamics and explore methods to advance the study of mitochondrial motility as well as mitochondrial dynamics-related diseases and mitochondrial-targeted therapeutics.Mitochondria are cell organelles that play pivotal roles in maintaining cell survival, cellular metabolic homeostasis, and cell death. Mitochondria are highly dynamic entities which undergo fusion and fission, and have been shown to be very motile  The origins of mitochondria are widely accepted to be traced to an ancestral endosymbiotic bacterium known as alphaproteobacterium . PhylogeMitochondria are commonly known as the “powerhouse” of the cell, but this overly simplistic description ignores many important aspects of these intracellular organelles. Mitochondria are indeed the intracellular site for aerobic respiration and the subsequent synthesis of ATP. Extensive research over the past half a century has revealed numerous biochemical processes involving mitochondria such as protein synthesis, cell metabolism, cell death, calcium signaling, and gene expression in the nucleus that are critically important in the survival of eukaryotes .via motor proteins and adaptors  , is also required for mitochondrial fission  . The yea fission . The exadivision .Fzo1) and Ugo1 and the third is the inner-membrane protein, Mgm1. The mammalian orthologs of these proteins are mitofusins 1 (MFN1) and 2 (MFN2) in relation to Fzo1 and optic atrophy protein (OPA1) in relation to Mgm1. The loss of any of the genes for these proteins leads to a disruption in the balance of mitochondrial fission and fusion and results in severe pathophysiological consequences. Mouse models lacking MFN1, MFN2   . New insin (GED) . The larin (GED) . Recent in (GED) . The posin (GED) . Fzo1 alethering . While Munctions . MFN1 haMgm1 and OPA1 are DRPs which reside in the mitochondrial inner-membrane and which have exposure to the intermembrane space   . OPA1 hal fusion . Original fusion , at leas reviews .OPA1 has two distinct isoforms: short and long. The long isoform is generated after OPA1 is imported into the IMM. The precursor protein contains what is known as a mitochondrial targeting sequence. Cleavage of this sequence generates membrane-anchored long OPA1 isoforms (l-OPA) (s-OPA) is a result of proteolytic processing of OPA1 by OMA1 and YME1L, creating a balance between the two  . The gen the two . It was l fusion , but the fission . Howeverl fusion .Mgm1 also has short and long isoforms. After outer membrane fusion has occurred, the Mgm1 isoforms come together to form a functional heterodimeric unit that mediates mitochondrial fusion , Mitochondrial fission protein 1 (Fis1), and adaptor proteins Mdv1 and Caf4.There are four key proteins in yeast involved in mitochondrial fission: Dynamin-1  which is also referred to as the B-insert  and mitochondrial division protein 1 (Mdv1)  .Fis1 is a protein in the outer membrane of mitochondria that recruits Dnm1 to the surface via a molecular adaptor (either Mdv1 or Caf4). Mdv1 acts as a protein bridge between Fis1 and Dnm1 by binding its N-terminus to Fis1 and its C-terminus to Dnm1  (MiD49) and 51 (MiD51)  DRP1  , and Mit (MiD51) . The comongation . This imchondria . Studies of Fis1 . In addiof) DRP1 . Studiestogether . The datications .The endoplasmic reticulum (ER) interacts with mitochondrial membranes at ER contact sites see . These cThe highly motile nature of mitochondria in mammalian cells is chiefly motivated by molecular motors that actively translocate mitochondria along the microtubule cytoskeleton. In some cell types, approximately 80% of the cell volume is explored by mitochondria within 15 min . MicrotuDrosophila, dynein heavy chain 64C is the primary motor for both anterograde and retrograde transport of mitochondria  and interacts with Milton, a kinesin-binding protein crucial for the mediation of mitochondrial motility discovered through genetic screening for function . Milton osophila . This shTRAK1) and 2 (TRAK2), similarly engage with kinesin-1 and dynein/dynactin to induce mitochondrial motility. We have demonstrated that TRAK1 plays an important role in mitochondrial trafficking in a study that identified deleterious variants in TRAK1 from encephalopathic patients  membrane protein that targets kinesin to the ER membrane , and syn2+ and it acts as a brake by tethering mitochondria to the microtubule track (Syntaphilin (Snph) anchors mitochondria to microtubules, rendering them stationary. Snph behaves as an engine off-switch by detecting Miro-Cale track . Snph fale track .via a protein known as plectin (Links between intermediate filaments (IFs) and mitochondria appear in the colocalization of mitochondria with neuronal IFs . There i plectin . Mutatio plectin . In addi plectin . Vimenti plectin . The modA 1995 study was the first to show that mitochondria directly interact with the actin cytoskeleton, and that there is an implication in motility . The actMyosin is a motor protein known mostly for its role in muscle contraction and is ATP-dependent and responsible for actin-based motility. Myosin directs movement toward actin filament plus-ends, such as myosin V , or minuAdvances in imaging techniques and technology have enabled mitochondrial fusion and fission events to be quantitatively ascertained and visually observed. The best indirect methods for the assessment of mitochondrial fusion and fission in mammalian cells include cell-cell fusion, photoactivatable mitochondrially target Green Fluorescence Protein (mitoGFP), and fluorescence recovery after photobleaching (FRAP). Cell-cell fusion is a technique used to observe the mixing of mitochondrial matrices in mammalian cells. It was experimentally induced between two cell populations labeled with two differing mitochondrial markers, either mitoGFP or mitochondrially targeted Red Fluorescence Protein .C. elegans Bcl-2 homolog, bolsters mitochondrial fusion in HeLa cells . Mitochondrial connectivity is confirmed with rapid recovery of fluorescence while a failure of recovery is an indication of mitochondrial fragmentation or discontinuity . An examLa cells .BAX and BCL2 antagonist/killer (BAK) to have direct roles in mitochondrial fusion  , which chttps://github.com/vianamp/MitoGraph, is a program that is fully automated for analyzing the 3D morphology, volume and topology of mitochondria in living cells. This software has been optimized and validated for tubular mitochondrial networks in budding yeast  and statistical analysis for comparison between image stacks obtained from individual experimental conditions. In addition, the software has provision for subdivision of the measured parameters to be analyzed in two distinct intracellular regions, a thin perinuclear region and the remaining cell periphery, as depicted in Our own Mitochondrial Single Particle Tracking (MitoSPT) , is avaihttps://github.com/ThomasSchwarzLab/QuoVadoPro, utilizes ImageJ macros to quantify movement of a fluorescently tagged organelle. The extent of intracellular movement is inferred by quantifying the variance of pixel illumination over a series of time-lapse images. QVP gives only a motility score as a motility metric on an image-wide basis and not by frame-by-frame quantification. Mitochondrial morphometrics are not analyzed nor are fission and fusion events. QVP is implemented as an ImageJ plugin.QuoVadoPro (QVP)  is also https://github.com/MitoSegNet, uses a pre trained deep learning model that segments mitochondria and analyzes mitochondrial morphometrics. There is no need for user-input parameters with this software, which handles batch input and thus eliminates bias. MitoSegNet analyzes 2D images from fluorescence microscopy but does not perform analysis of temporal variation. It can be used only to visualize and quantify individual mitochondrial morphometrics. This program is based in Python and runs on a standalone software platform.Mitochondrial Segmentation Network (MitoSegNet) , found ahttps://github.com/fiji/MTrack2, utilizes ImageJ’s particle analyzer plugin to link particles between frames of 2D images. This is a fairly simple 2D tracking software package that gives centroid positions of objects in a binary image and tracks them over time. No morphology or fission and fusion events can be analyzed through this software. MTrack2 is an ImageJ plugin written in Java, and its GUI is relatively limited because it relies on ImageJ’s templates and results page to provide results output.MTrack2, found at Mitochondria form tubular networks that undergo changes in morphology to correspond with cellular needs through fusion and fission events. The shape of mitochondria can have an effect on their distribution to specific subcellular locations. This has been found to be especially important in neurons, in which small mitochondria are more efficiently transported to nerve terminals. It is however worth noting that smaller mitochondria do not always engage in efficient transport and that fragmented mitochondria resulting from a fusion impairment show signs of critical transport defects . MitochoMitochondria are known to be responsive to cues that change their morphology. Under conditions of starvation and stress, stress-induced mitochondrial hyperfusion (SIHM) is triggered. During SIHM episodes, mitochondria will hyperfuse into large complexes to offer cytoprotection and enhanced resistance to apoptosis, increasing opportunity for the cell to return to homeostasis . ApoptosA primary function of mitochondrial fission and fusion is the promotion of the membrane and contents mixing between mitochondria. Kiss-and-run fusion events, or transient fusion, progress content exchange without having an effect on mitochondrial morphology . LabeledThe benefits of content exchange include the promotion of homogenization of the mitochondrial population as well as amelioration of injurious effects of heteroplasmic mtDNA mutations. Since the mtDNA only encodes for 13 polypeptides, most of the mitochondria’s proteasome is imported from the cytosol. The inhibition of mitochondrial fusion causes individual mitochondria to deviate in their properties, indicating that fusion assists in the reduction of variability between organelles. As most mtDNA mutations are recessive in nature, the mutational load must attain high levels of around 60–90% heteroplasmy before any appreciable dysfunction in the respiratory chain is encountered. The capacity to withstand these excessive levels of mtDNA mutations is extremely reliant on mitochondrial fusion .de novo; thus, they must be inherited by daughter cells through mitosis. Elongation of mitochondria occurs during the G1-S phase in tissue culture cells but then fragmentation begins at the G2 and M phases  activity . The losWhen mitochondrial fission is absent, the mtDNA nucleoids begin to aggregate and form large structures that distort mitochondrial tubules . This agDRP1 and mitochondrial fission have previously been thought to be necessary for mitophagy to occur in mammalian cells, some recent research findings demonstrate that certain forms of mitophagy do in fact occur independent of DRP1  . As a result of MOMP, there is a release of pro-apoptotic factors including cytochrome c from the intermembrane space to enter into the cytosol, and this triggers downstream cell death pathways. is event . BAX is ne (OMM) . This poThe fusion and fission proteins discussed in the previous section are targets for a number of post-translational modifications (PTMs). PTMs facilitate quick responses to alterations in physiological demands and are common mechanisms to modify protein activity. The most studied modifications include acetylation, phosphorylation, ubiquitination, O-GlcNAcylation, and SUMOylation. Mitochondrial fusion is the result of adjacent mitochondria tethering to one another followed by the fusion of the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM), in that order . The regMFN2 expression in mice due to an increased demand for energy by the protein peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a), such as skeletal muscle due to increased mechanical load/exercise or brown adipose tissue reacting to cold environments   . Another is seen .MFN2, such as mi2R-761 in hepatoma cells  show that MTCH2 may serve as a novel regulator of mitochondrial fusion, because knockout of MTCH2 from ESCs results in failure of mitochondria to elongate  and YME-like protein 1 (YME1L) have been shown to cleave OPA1  in mice and Serine 637 (SER637) in human DRP1 are the most well studied sites of regulation. SER616 is phosphorylated by cdk1, PKC, ERK1-2, and CaMKII resulting in DRP1-induced mitochondrial fission. On the other hand, this process is inhibited by PKA phosphorylation of SER637  and Threonine 586 (THR586) are O-GlcNAcylated leading to a reduction in the phosphorylation of DRP1 at SER637. SER637 as discussed above, inhibits DRP1 activity, so a reduction in phosphorylation at SER637 is followed by an increase in mitochondrial fragmentation and a reduction in mitochondrial membrane potential   . This stThe mechanics behind mitochondrial motility have been best studied in the neuron and the molecular mechanisms that engage mitochondria in their motility are well established. As neurons are postmitotic cells that survive as long as the organism is alive, mitochondria need to be cycled when they are subject to dysfunction or age. Conditions of stress and impaired integrity also lead to shifts in mitochondrial motility. Regulation of mitochondrial motility is therefore incredibly important to meet the necessary metabolic requirements to adapt to stressors, as well as to remove dysfunctional and aged mitochondria while replenishing the cell’s supply of healthy ones. The implications of a defect in mitochondrial transport are seen in the pathogenesis of a multitude of major neurological disorders. The coordination between these mechanisms to distribute mitochondria in neurons and in other cells, however, is not very well understood.The bidirectional transport of mitochondria along microtubules is carried out by kinesin and dynein motors as discussed previously, and is frequently described as a “tug of war.” Our mechanistic understanding of how motor proteins, adapters, and corresponding regulatory proteins interact at the molecular level is lacking. With the amount of research that has been done, we cannot accurately predict how disturbances at a cellular level play a role in the determination of net retrograde or anterograde movement, but there are hypotheses as to how this occurs. Much of the research done regarding how kinesins and dyneins regulate movement of a cargo being transported along a microtubule is based on mechanics and the velocity or static behavior of mitochondria are dependent on the balance of forces. Retrograde and anterograde velocity is thought to be regulated by the amount of kinesin and dynein molecules acting on a mitochondria as well as the overall load force the motors need to act against, such as the dynein stall force . Other f2+ levels draw mitochondria to synapses by activation of the voltage-gated calcium channels at presynaptic terminals or NMDA receptors at postsynaptic sites  is a rare heterogenous inherited neuropathy. Those affected experience peripheral neuropathy, hypotonia and severe progressive muscle weakness in the distal limbs resulting in gait defects. The most common type, CMT Type 2A (CMT2A), is known to be caused by defects to the inner mitochondrial membrane fusion protein, ondition . Current neurons . MFN2 mucultures . This clhe axons .MFN2 in cells concluded that the defect in locomotion was not a result of the depletion of MFN2, but instead due to the incorrect mitochondrial shaping. They came to this conclusion after seeing that mouse embryonic fibroblasts that were isolated from MFN2 knockout embryos exhibited normal mitochondrial locomotion. Both short tubular and round mitochondria were present and even though the round mitochondria seemed to lose their directed movement, they retained the ability to fuse with the mitochondrial network to create mitochondria having undirected movement , Parkinson’s disease (PD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS). Abnormal mitochondrial dynamics lead to the progressive loss of structure and function of neurons within the central and peripheral nervous systems. With the discovery of fusion and fission proteins, namely DRP1 through the enhancement of its GTP-hydrolyzing activity  gene . The mutactivity , thus call death . Mutant ll death .PINK1 and Parkin have been linked to PD and mutations in either result in impaired mitophagy and early accumulation of DRP1 in neurons. The excessive amount of fission leads to excessive fragmentation accompanied by oxidative stress and reduced ATP production  have also been seen in patients with syndromic parkinsonism as well as dementia  injury  injury . Use of ) injury . Mdivi-1in PINK1 . The con2+  is an effective nutritional intervention that helps in the prevention of age-related metabolic disorders . The molOther pharmacological interventions for mitochondrial disorders include mitochondria-targeted antioxidants and a CR mimetic, resveratrol, which has been demonstrated to affect mitochondrial biogenesis in liver, muscle, and brain. Resveratrol has also been shown to promote the expression of proteins involved in mitochondrial fission and fusion resulting in a protective effect in mitochondria . OxidatiMitochondria are complex and dynamic intracellular organelles that undergo highly regulated motile interactions involving numerous complexes . They are also subject to fusion and fission events under the control of regulating proteins. Mitochondrial fission and fusion maintain the functionality of mitochondria and plays an incredibly important role in cell fate. Fission is responsible for proper distribution and number of mitochondria while fusion ensures the exchange of content for optimal mitochondrial activity. Disruption of mitochondrial motility or an imbalance in mitochondrial fusion/fission is associated with organ dysfunction and diseases known to involve the neurological, vascular, and skeletal systems. With an evolving understanding of mitochondrial dynamics, some therapeutic avenues are being researched to combat dysfunctional dynamics with many trying to reverse excess fission . There is a lack in knowledge of the molecular events involved in mitochondrial fusion and fission, requiring more mechanistic understanding. Further studies regarding how fusion and fission is related to mitophagy is promising as it relates to mitochondrial disorders and cancer. The field of mitochondrial dynamics is expanding and more research regarding mitochondrial motility and its role in the pathogenesis of disease states and dysfunction is necessary. An expanding pool of experimental techniques and imaging tools offer potential for novel discoveries in mitochondrial dynamics understanding as well as novel treatment approaches for mitochondrial diseases involving errors of dynamic functions.


In metastatic stages of gastric cancer, surgical intervention with curative or life-prolonging intention is still a highly individualized as well as experimental approach, which is unfortunately often performed in daily routines without the sufficient data of well-powered randomized trials. In particular, a small subgroup of metastatic patients showing a limited number of distant metastases in a restricted number of organs, termed limited or oligometastatic disease, needs further evaluation and is discussed in the current manuscript.Gastric adenocarcinoma and esophageal cancer are the fifth and seventh most common cancer types worldwide. At the time of initial diagnosis, up to 50% of esophagogastric cancers present with distant metastatic lesions and are candidates for chemotherapy. Curative surgery in this stage is still an experimental approach. Only a small number of these metastatic patients show an oligometastatic disease with no uniform definition of what oligometastatic means in gastric cancer. Nevertheless, the question remains unanswered as to whether these patients are still candidates for curative concepts. Some studies have attempted to answer this question but have not been adequately designed to address the role of a curative-intended multimodal therapy in this setting. The current FLOT-5 is designed to potentially provide a definitive answer to the question of whether curatively intended surgery plays a role or is a disadvantage in this setting. Gastric adenocarcinoma and esophageal cancer, with an estimated 1,033,701 and 572,034 new cases annually, are the fifth and seventh most common cancer types, respectively, worldwide .In metastatic stages of gastric cancer, surgical intervention with curative or life-prolonging intention is still a highly individualized as well as experimental approach. It has been evaluated in several trials with a limited number of patients only and subgroup analyses of studies as well as retrospective patient cohorts. Only a small number of these metastatic patients show distant metastases with a limited number of lesions in a limited count of organs: so-called limited or oligometastatic disease .Obtained data indicate that surgery could possibly provide a benefit for a highly selected subgroup such as, e.g., patients aged 70 years or less with one metastatic site only (lymph nodes or liver)  and an eA research group from Germany identified D3 lymph nodes as an independent predictor for overall survival (OS) based on the data of 48 gastric cancers with gastric primary resection . In addiThe concept of limited/oligometastatic disease (OMD) was introduced in 1995 and described a clinical disease state of only limited metastatic burden . In 2020A pilot of the German Gastric Group (FLOT study group)  establisn = 60/238) showed limited metastatic disease and more than half of them (n = 36) underwent surgery after induction with four cycles of FLOT regimen. These patients with surgical resection showed a survival benefit with a median OS of 31 months compared with only 16 months in the same population but without curatively intended surgery .,23.n = 6The FLOT-5 trial was a prospective, multicenter, randomized, investigator-initiated phase III study to evaluate the effect of induction FLOT chemotherapy in chemo-naïve oligometastatic gastric or GEJ adenocarcinoma patients without prior resection of the primary or of the metastases. For HER2-positive and PD-L1-positive (combined positivity score ≥ 5) trastuzumab and nivolumab, respectively, were added to the FLOT regimen. The systemic treatment was combined with curatively intended gastrectomy/esophagectomy and resection of all metastatic lesions or local ablation/radiation procedures with the goal to achieve a R0- situation at all sites. Due to the randomized nature of the study, after 4 cycles of FLOT without signs of progressive disease, patients were randomized in a 1:1 fashion into Arm A with surgery following adjuvant systemic treatment or Arm B without surgery, the standard arm with continuation of systemic therapy only . All patAnother highly published trial in the OMD arena was the randomized Asian phase III REGATTA trial  that enrPeritoneal carcinomatosis in gastric cancer is a special issue and should also be addressed in OMD trials, e.g., RENAISSANCE.In individual cases, patients could possibly benefit from an interdisciplinary, coordinated, multimodal therapy if the following conditions are present: synchronous peritoneal metastases, isolated peritoneal involvement with a Peritoneal Cancer Index (PCI) ≤ 6 at laparoscopy, induction with neoadjuvant chemotherapy and a high probability of complete macroscopic cytoreduction.Numerous cohort studies are available on this. In a well-selected group of patients with peritoneal metastases, a French study demonstrated a long-term remission rate of 11% after resection and hyperthermic intraperitoneal chemotherapy (HIPEC). In multivariate analysis, a PCI < 7 synchronous metastasis and achievement of complete cytoreduction were shown to be independent prognostic factors . Resultsp = 0.046). A multivariate analysis performed on the 68 patients of this study suggested that ≥6 cycles of systemic chemotherapy, synchronous metastasis, complete cytoreduction and the absence of postoperative complications were indicative of a more favorable outcome [A prospective phase III trial from China showed a significant increase in median survival from 6.5 months for CSR alone to 11 months for CSR plus HIPEC  .In addition, a meta-analysis of prospective randomized trials  from 1985 to 2016 showed survival benefits for patients with isolated peritoneal metastases after resection and HIPEC. Patients with positive peritoneal cytology or even those with lymph node metastases appeared to benefit from therapy . HoweverIn gastric carcinoma, there has been a long debate as to whether diagnostic techniques can reliably detect a positive endoperitoneal cytologic finding, and, even more important, what this means for prognosis and how it influences the therapeutic strategy .The urgent need to increase the survival of patients with an advanced disease status led to the suggestion of intraperitoneal chemotherapy even in patients with positive lavage cytology without macroscopic peritoneal metastasis (Cy+/PC−). The impact of a combination of peritoneal lavage to wash out tumor cells and intraperitoneal chemotherapy in those patients was described in a meta-analysis. The results showed that the OS of patients was increased by intraperitoneal chemotherapy and that peritoneal lavage further increased these survival rates accompanied with a decrease in the peritoneal recurrence rate . NeverthAs mentioned by the CYTO-CHIP investigators, positive peritoneal cytology after gastrectomy corresponds to an R1 resection, with an equivalently bleak prognosis .Based on these data, the results obtained in patients with tumor-positive peritoneal cytology without macroscopic signs of peritoneal seeding within the ongoing PERISCOPE II will be of special interest.n = 172) was able to show that conversion of CY+ to CY− after neoadjuvant FLOT–chemotherapy was prognostic regarding overall survival [Interestingly a retrospective single-center cohort analysis from the University Hospital of Zurich . In particular, the group with only one metastatic site showed a clinically significant survival improvement due to surgical resection  compared with the group with two or even more metastatic sites . The maximum benefit from surgical resection was detected in the subgroup of patients younger than 70 years combined with only one metastatic site, indicating that further factors besides the tumor burden will influence the outcomes of OMD gastric cancer patients [Non-curatively treated patients with incurable tumors from the Dutch Gastric Cancer Trial  were stuA multicenter study in patients with esophagogastric cancer and oligometastatic disease, performed in Dutch as well Swiss cancer centers, was able to show that the combination of local and systemic treatments in the limited metastatic situation was associated with an improvement in OS compared with chemotherapy alone. However, there was a mix of histology and localization with 73% esophageal cancer including 79% adenocarcinomas and metachronous seeding in half of the population indicating the need of a clearly defined characterization of this special subgroup . SimilarAcross multidisciplinary tumor boards, oligometastatic esophagogastric cancer was discussed within the OMEC-2 study to investigate the agreement in the definition and treatment of oligometastatic esophagogastric cancer in European-experienced centers. There was consensus that the definition of oligometastatic applies to cancers with only 1–2 metastatic lesions in either the liver, lung, retroperitoneal lymph nodes, adrenal gland, soft tissue or bone. Furthermore, there was consensus that oligometastatic disease can only be considered when the disease is at least stable after a median of 18 weeks of systemic therapy. However, there was no unity regarding the optimal treatment in the case of oligometastatic disease. Additionally, the cases discussed varied in terms of location and number of metastases, histology and time of detection, synchronous vs. metachronous. The results of OMEC-2 show even more that there is a big need for data of a randomized controlled trial in a clearly defined population of patients with only synchronous limited metastatic burden in a pure adenocarcinoma histology group without a mix of histology .p < 0.001), especially in cases of single liver lesions compared with multiple hepatic metastases  [A systematic review and pooled analysis by Markar et al. , includi= 0.011) . This onThere is a Chinese consensus on the diagnosis and treatment of gastric cancer with liver metastases, dividing into Type I resectable, Type II potentially resectable and Type III unresectable.Type I shows 1–3 liver metastases; maximal diameter ≤ 4 cm or limited to one liver lobe without involving important vessels or bile ducts with technological resectability judged by a hepatobiliary surgeon. Type II liver metastases according to the classification are out of the range of Type I, with potential technological resectability and Type III is further divided into Type IIIa and b. Type IIIa shows multiple diffusely distributed metastatic lesions in both lobes without extrahepatic metastases and IIIb shows extrahepatic metastases in one or more organs with or without peritoneal carcinomatosis. Regarding the primary, Type I tumors are T ≤ T4a, lymph node metastases are within the D2 lymph node dissection area (not including Bulky N2) whereas Type II primaries include T4b or Bulky N2 or Bulky No. 16a2, b1. Type III include primary lesion directly and considerably invade adjacent tissues or organs; regional lymph nodes such as mesenteric lymph nodes or paraaortic lymph nodes fixed, fused, or not resectable, as confirmed by imaging examinations or biopsy.The Chinese consensus recommends preoperative systemic treatments in Type I patients. In Type II patients, surgical resection is only recommended when R0 resection is intended. In Type III, cytoreductive surgeries are not encouraged .The observational registry study of the Spanish AGAMENON national registry  showed dp = 0.021) [AGAMENON analyzed data from 1792 subjects in 32 centers, of whom 5% received surgical resection of metastases for esophagogastric cancer. The most frequent sites of metastatic resections were carcinomatosis of the peritoneum (29%), liver lesions (24%) and metastases of extra-regional lymph nodes (11%). Patients who were selected for resection of metastases showed improved survival rates, HR 0.34  . Neverth= 0.021) ,49, Tablp = 0.009). The AGAMENON investigators concluded that subjects with limited metastatic disease, selected on a clinical basis, can benefit from early surgeries. However, data of highly selected patients should be interpreted with caution, because in terms on pure survival numbers they are numerically no better than data from pure systemic therapy studies without surgery, but have an additional risk of surgical morbidity and possible mortality.The 1- and 3-year relapse rates following R0 resection were high, with 58% and 65%, respectively, but interestingly the median time to recurrence since complete resection of metastases was up to 8.4 months  and the duration of systemic therapy before surgery increased mortality  of the French FREGAT group aims to evaluate overall survival after palliative gastric resection plus chemotherapy versus chemotherapy only in stage IV gastric cancer with only one solid organ metastatic site or with more than one metastasis in only one organ. Patients randomized to surgery can receive additional subtotal or total gastrectomy, depending on the location of the primary tumor. According to the original protocol, standardized D2-lymphadenectomy and resection of metastasis was not required, reflecting the situation of the REGATTA trial. With regard to the German FLOT-5/RENAISSANCE trial, SURGIGAST amended their protocol to the RENAISSANCE trial inclusion criteria of definition of oligometastatic disease and allowed surgery of the primary and treatment of the metastatic site.Currently, a Dutch group is also preparing the COSTA trial, evaluating the potential benefits of surgery in limited metastatic disease as with SURGIGAST or RENAISSANCE, but COSTA has several limitations. COSTA is a multicenter, single-arm phase II study in pts undergoing palliative chemotherapy plus palliative resection to evaluate OS/PFS/HRQL synchronous metastatic gastric cancer treated with resection of only primary in addition to chemotherapy.n = 115) and curative resection (n = 2087) in the Netherlands between 2011 and 2016. Although postoperative mortality was higher after non-curative surgery , after propensity score matching there was no difference between groups . Palliative surgery does not result in an additional risk of postoperative morbidity compared to curative gastrectomy. The oncological role as well as the safety aspects of a pure palliative gastric resection are therefore well defined and need no further evaluation [The COSTA idea is to evaluate the role of palliative resection, an issue already addressed by the REGATTA trial , so the aluation .The factors and variables discussed above should be addressed in modern OMD trials. In particular, the potential benefit from complete cytoreductive curatively intended surgery in cases with tumor regression after initial cycles of systemic induction chemotherapy needs further investigation.A palliative systemic therapy in a metastatic or even oligometastatic gastroesophageal disease is standard and substantiated by the good data of the combination of chemotherapy with new antibodies. Furthermore, the role of perioperative systemic therapy is also a standard of care in the current entity. There is therefore a strong rationale for discussing whether (pseudo-) curation is the goal to combine the standardized most effective systemic therapy components with radical local procedures.The ideal regimen should be based on 5-FU and platinum substances. Fluoropyrimidine plus platinum-based systemic therapy is a standard first-line regimen for unresectable advanced or metastatic human epidermal growth factor receptor 2 (HER2)- negative gastric and GEJ adenocarcinoma and should be part of the standard backbone in the limited metastatic disease ,63,64,65The cytotoxic drug docetaxel has shown efficacy in metastatic settings, in first-line  and second-line situation (docetaxel monotherapy) therapy .Several phase II studies demonstrated that the addition of docetaxel to the combination of fluorouracil, leucovorin and oxaliplatin (FLOT) was more tolerable than the parent DCF regimen for metastatic and locally advanced gastric and GEJ adenocarcinoma with FLOT. In addition, the FLOT regimen elicits a stronger tumor response than other combinations, including the anthracycline-based triplets in locally advanced, resectable tumors, which were substantial in a preoperative situation ,67,68. IThe TOGA trial established the addition of the HER-directed antibody trastuzumab to chemotherapy with fluoropyrimidine plus cisplatin-based systemic therapy in patients with HER2-positive status in the 1st line palliative treatment of esophagogastric adenocarcinoma .The non-interventional HERMES trial  followedThe HER-FLOT regimen was also found to be safe, with a promising pCR rate of >20% achieving pathological complete response (pCR) ,71. FurtIn the clear palliative 1st line treatment situation of esophagogastric adenocarcinoma, nivolumab was the first programmed cell death (PD-1) inhibitor to demonstrate superior OS, along with a clinically meaningful progression-free survival (PFS) benefit and improved and durable objective responses in combination with fluoropyrimidine plus oxaliplatin-based chemotherapy compared to chemotherapy alone in previously untreated patients in a phase III situation, thus establishing a new standard first-line treatment mainly in PD-L1-positive patients according to their combined positivity score (CPS) . In addiThus, the shown high response rates of fluoropyrimidine plus platinum-based chemotherapy plus docetaxel and/or PD-1 or HER-2-directed therapy ,49,70,72An interim analysis of the KEYNOTE-811, a randomized, global phase III study evaluating trastuzumab plus platinum and fluoropyrimidine doublet chemotherapy +/- pembrolizumab for unresectable or metastatic HER2-positive esophagogastric adenocarcinomas revealed that addition of the checkpoint inhibitor significantly induces tumor shrinkage and improves objective response rate . This coIn contrast to other trials in the limited metastatic situation, the German RENAISSANCE/FLOT-5 trial  is basedEven if the FLOT-5 trial does not show a benefit from curative intended surgery, this trial will still set a landmark by answering the question of pure systemic palliative intended vs. curative treatment by combining systemic and local therapies in a limited or oligometastatic gastric cancer disease.For the future, we will need a strong selection of patients as, in the definition of oligometastatic disease in the FLOT-3 and FLOT-5 trials and in this well-defined subgroup, randomized comparison between surgery and palliative 1st line systemic therapy is needed.Only a small number of esophagogastric cancers patients in formal stage IV of the disease show an oligometastatic or limited disease still with no uniform definition of what oligometastatic means in gastric cancer. Curatively intended treatment approaches in OMD esophagogastric cancers are still an experimental approach and the question remains unanswered as to whether these patients are still candidates for curative concepts because, until now, we have not had sufficient data from randomized phase III trials. The increasingly effective systemic therapies, such as triplet FLOT therapy or the addition of targeted therapies such as HER2-targeted therapy, anti-angiogenic therapy and/or the addition of checkpoint inhibitors, confront us with new questions. Do we still need local therapies such as surgery or radiotherapy when we have an increasingly effective system therapy, or should these methods be integrated all the more in order to combine all possible effective weapons for a maximum of tumor reduction?However, one question must be asked at the start of therapy in the treatment of OMD patients in real life situations as well in the design of studies for OMD esophagogastric cancer patients. If the treatment is intended to be formally curative and life-prolonging, then the patient must be given a maximum of therapy with the aim of maximum tumor-/cytoreduction, or if the orientation of the treatment is clearly palliative, then compromises can be made, since the aim of local therapies in this context is only sparing of systemic therapy and symptom control.


This study aims to assess the reliability of using a core-needle biopsy (CNB) for preoperative tumor characterization using gene expression analysis and conventional immunohistochemistry (IHC) analysis for clinical biomarkers in early breast cancer. Obtaining a preoperative CNB is a standard procedure in the evaluation of a primary breast cancer, with previous studies suggesting that it can be considered trustworthy to perform immunohistochemical analysis on CNB samples. However, little research has been carried out to evaluate whether gene expression-based biomarker assessment can be carried out reliably on the preoperative CNB. This information is important as genomic profiling is gaining ever-increasing importance in the treatment of early breast cancer.p = 0.011). Similarly, when using GEX, a significant shift from negative to positive ER (47%) and from low to high Ki67 (16%) was observed between the CNB and surgical specimen (McNemar’s p = 0.027 and p = 0.002 respectively). When comparing biomarker status between different techniques (IHC vs. GEX) performed on either CNBs or surgical specimens, the agreement in ER, PgR, and HER2 status was generally over 80% in both CNBs and surgical specimens (kappa range 0.395–0.708), but Ki67 and tumor molecular subtype showed lower concordance levels between IHC and GEX . These results suggest that both the techniques used for collecting tissue samples and analyzing biomarker status have the potential to affect the results of biomarker assessment, potentially also impacting treatment decisions and patient survival outcomes.In early breast cancer, a preoperative core-needle biopsy (CNB) is vital to confirm the malignancy of suspected lesions and for assessing the expression of treatment predictive and prognostic biomarkers in the tumor to choose the optimal treatments, emphasizing the importance of obtaining reliable results when biomarker status is assessed on a CNB specimen. This study aims to determine the concordance between biomarker status assessed as part of clinical workup on a CNB compared to a medically untreated surgical specimen. Paired CNB and surgical specimens from 259 patients that were part of the SCAN-B cohort were studied. The concordance between immunohistochemical (IHC) and gene expression (GEX) based biomarker status was investigated. Biomarkers of interest included estrogen receptor , progesterone receptor (PgR), Ki67, HER2, and tumor molecular subtype. In general, moderate to very good correlation in biomarker status between the paired CNB and surgical specimens was observed for both IHC assessment  and GEX assessment , respectively. However, using IHC, 52% of cases with low Ki67 status in the CNB shifted to high Ki67 status in the surgical specimen (McNemar’s  Obtaining a preoperative core-needle biopsy (CNB) on suspected breast lesions is a standard procedure in the primary evaluation of breast cancer and can be used to accurately differentiate invasive disease from cificity ,4,5,6,7.cificity ,9. In adcificity ,11,12,13The choice of systemic therapy in the preoperative setting is dependent on tumor biomarker classification performed on the preoperative CNB together with clinical and radiological tumor staging. Furthermore, the type of medical treatment offered to patients unfit for surgical excision of the tumor is also based on the biomarker status classification determined on a CNB. Although many local and international guidelines permit biomarker assessment on either CNB or surgical specimens, variability in biomarker status between paired CNB and surgical specimens, with the potential to impact choice of therapy have been previously reported ,15.in situ hybridization (ISH) for equivocal human epidermal growth receptor-2 (HER2) cases, is the method of choice for determining the status of the conventional histopathological biomarkers in breast cancer including estrogen receptor (ER), progesterone receptor (PgR), HER2, and the proliferation marker Ki67 [Immunohistochemistry (IHC), including ker Ki67 ,17,18. Tker Ki67 ,21,22,23ker Ki67 ,27,28,29This study aimed to determine the concordance of biomarker status assessed using IHC and mRNA sequencing (RNAseq) in paired, untreated preoperative CNB and surgical specimens from a consecutive population-based cohort of 259 patients with early breast cancer.The study is based on the Sweden Cancerome Analysis Network-Breast (SCAN-B) cohort which is an ongoing multicenter population-based observational study that was initiated in September 2010 with the purpose of analyzing breast tumors using next generation genomic technologies to facilitate translational research . As of 30 June 2022, over 18,600 patients were enrolled in SCAN-B, representing about 85% of eligible patients in the catchment area. Sample collection and processing within SCAN-B has been described previously . BrieflySelection of patients and tumors for this sub-study is presented in IHC analyses of biomarkers were performed as per routine clinical practice at five different local pathology departments  and reported in the medical records of the patients. Study-specific re-assessment of IHC biomarkers was not performed, to reflect the concordance levels in routine clinical practice. Other patient and tumor characteristics were obtained from the Swedish National Quality Register for Breast Cancer (NKBC)  and missin situ hybridization (FISH) or silver-enhanced in situ hybridization (SISH), and if HER2 gene amplification was detected, the tumor was classified as HER2-positive. Ki67 index was determined by counting at least 200 cells within areas with the highest observed Ki67 expression, i.e., “hotspots”. Both standard microscopic examination and digital image analysis were acceptable methods. Surrogate molecular subtypes were determined following the 2013 St. Gallen consensus with the slight modification of using a Ki67 low expression cutoff of <20% instead of <14% as specified in the consensus [Dichotomized variables for the conventional IHC biomarkers were used in statistical analyses as follows: positive vs. negative for ER, PgR, and HER2; high vs. low for Ki67. The cutoff used for ER and PgR expression was ≥10% for positive tumors, consistent with the national Swedish guidelines . For HERonsensus . At the onsensus , Swedishonsensus ,33.The RNAseq workflow for all tumors included in the SCAN-B cohort has been described in detail previously ,31. BrieThe multi-gene RNAseq based classifiers for predicting the status of the standard breast cancer biomarkers  developed by the SCAN-B work group were implemented in this study . These gp < 0.05 was considered to be statistically significant. Statistical analyses were performed using SPSS statistics version 27.The statistical significance for the concordance of biomarker expression between the paired CNB and surgical specimen and between different analytical methods was evaluated using both Cohen’s kappa and McNemar’s tests, wherever applicable. Two-sided statistical analyses were performed and The distribution of patient and tumor characteristics among the 259 patients is presented in p = 0.011). This shift in Ki67 corresponded to 60% (19/34) Luminal A-like to Luminal B-like subtype conversions between CNB vs. surgical specimens (p = 0.067). Similarly, using GEX, 16 out of the 21 (76%) ER discordant cases shifted from negative ER status in the CNB to positive ER status in the surgical specimen (McNemar’s p = 0.027). Ki67 status was discordant in 24 cases, 20 (83%) of which shifted from low expression in the CNB to high expression in the surgical specimen (McNemar’s p = 0.002). Interestingly, 9/10 (90%) tumors which were unclassifiable into any molecular subtype using GEX in the CNB were successfully classified into one of the five intrinsic molecular subtypes in the surgical specimen and Luminal A to Luminal B shifts were more prevalent (p = 0.168).First, the biomarker status of the paired CNB and surgical specimen were compared for each analytical method as reported in pecimens A; howeveNext, we investigated the agreement between IHC and GEX biomarker status separately for CNBs and surgical specimens . The resThree hundred and eleven (311) genes were significantly differentially expressed between paired CNB and surgical specimens; 172 with higher and 139 with lower relative expression in CNBs compared with the paired surgical specimens FDR = 0, . SignifiSeveral factors can impact the accuracy of analyses performed on preoperative CNBs, such as the size of the core-needle used, the number of samples taken, tumor size, and the experience and expertise of the radiologist performing the procedure. In this real-life study, these factors may have varied for the different patients, and the analyses presented herein focus instead on investigating how reliable a preoperative CNB is in routine clinical practice when determining the status of conventional breast cancer prognostic and treatment predictive biomarkers using IHC and GEX respectively.in situ cancer, and the spatial expression of biomarkers in the tumor. The area of the tumor that is represented in a tissue section is limited but is nevertheless larger compared to the area represented by a CNB, where there is a greater risk that the material taken does not fully represent the entire nature of the tumor, e.g., in terms of tumor heterogeneity.The surgical specimen is usually considered as the gold standard to obtain information about the status of different biomarkers for a breast cancer. The surgical specimen allows for an overview of the whole tumor area of that tissue section, which is essential for the assessment of the surgical margin. It also allows for the estimation of invasive areas, presence of GEX status are not necessarily classified as HER2-enriched by the conventional PAM50 molecular subtype classifier.In this study, we observed moderate to very good concordance of biomarkers between paired preoperative CNB and surgical specimen when the same technique (IHC or GEX) was used for biomarker assessment. The concordance for IHC biomarkers observed in this study is similar to results reported in previous studies; ER (68–100%), PgR (71–89%), HER2 (60–100%), Ki67 (80–82%) ,43,44,45Our study results also show that for most of the biomarkers, the concordance of GEX-based classifiers between the CNBs and surgical specimen were inferior to the concordance observed for the standard IHC. Tumor heterogeneity and sampling error are factors which can be accounted for when a biomarker is assessed using IHC by a trained pathologist. On the other hand, results of transcriptional profiling of tumors using techniques like bulk RNAseq represent the average gene expression levels in the sample, including non-malignant cells found in the sample, which may impact the classification of samples. The development of methods which ensure that the CNB collected for biomarker analyses is indeed rich in tumor cells can potentially improve the analytical validity of GEX profiling in CNBs. An example of a method under experimentation at our local pathology department is obtaining a punch biopsy from the surgical specimen and studying the tumor cellularity in the borders of the punch by H&E staining to ensure that the punch biopsy was taken from a section of the tumor with sufficient malignant cells, prior to extracting RNA for gene expression analysis. Tumor cellularity in the borders of CNBs will also be investigated, although this will be a more complex process.IHC positive and ERGEX negative by this multigene classifier showed an inferior survival compared to tumors concordantly classified as ER positive [The multigene classifiers have been shown to provide valuable added clinical information, particularly for IHC ER-positive tumors which were predicted to display ER-negative properties according to the GEX classifier . Specifipositive . In thisIHC status when a similar technique was used for biomarker analysis (IHC or gene expression) reported in this study is in line with previous research on this biomarker [Despite GEX-based biomarkers being incorporated into Swedish and international guidelines to help predict the benefit from adjuvant chemotherapy, current guidelines on neoadjuvant chemotherapy suggest that there is insufficient evidence for using genomic profiles in a neoadjuvant setting ,51. Remaiomarker . Howeveriomarker . Our resiomarker . Recent iomarker . HoweverA further consideration regarding Ki67 is the use of this biomarker to assess treatment response, where a relative reduction of Ki67 expression levels of the tumors after brief exposure to treatment may be interpreted as a good treatment response and is associated with a favorable long-term outcome. The results of the clinical trial POETIC aiming to assess the benefit of pre- and perioperative aromatase inhibitor (AI) treatment on the clinical outcome in operable breast cancer are strongly indicative of a prognostic value of the relative Ki67 reduction . In the When using a preoperative CNB to determine the biomarker status of a tumor using IHC or GEX, the limitations of both methods should be acknowledged to correctly reflect the nature of the tumor, and if possible, investigated for new/uncharacterized biomarkers. The proliferative status of the tumor should be interpreted with caution when determined immunohistochemically using Ki67 expression, especially in the ER+/HER2− group. Moreover, the endocrine responsiveness of a tumor as determined by its immunohistochemical ER status is an aspect where complementing with a GEX-based assay may add relevant clinical value. Future studies should determine ways to improve the quality of sample collection and the reproducibility of the results of biomarkers assessed in CNBs in the clinic to ensure that patients are always provided the best treatments and spared the toxicities of ineffective treatments.