Remote consultations (RCs) enable clinicians to continue to support patients when face-to-face appointments are not possible. Restrictions to face-to-face care during the COVID-19 pandemic has accelerated a pre-existing trend for their adoption. This is true for many health professionals including some chiropractors. Whilst most chiropractors in the UK have used RCs in some form during the pandemic, others have not. This study seeks to understand the views of chiropractors not using RCs and to explore perceived potential barriers.A national online survey was completed by 534 registered practicing UK chiropractors on the use of RCs. Respondents had the opportunity of providing open-ended responses concerning lack of engagement in RCs during the COVID-19 pandemic. Textual responses obtained from 137 respondents were coded and analysed using thematic analysis.The use of RCs provided an opportunity for chiropractors to deliver ongoing care during the COVID-19 pandemic. However, many chiropractors expressed concern that RCs misaligned with their strong professional identity of providing ‘hands-on’ care. Some chiropractors also perceived that patients expected physical interventions during chiropractic care and thus considered a lack of demand when direct contact is not possible. In the absence of a physical examination, some chiropractors had concerns about potential misdiagnosis, and perceived lack of diagnostic information with which to guide treatment. Clinic closures and change in working environment led to practical difficulties of providing remote care for a few chiropractors.The COVID-19 pandemic may have accelerated changes in the way healthcare is provided with RCs becoming more commonplace in primary healthcare provision. This paper highlights perceived barriers which may lead to reduced utilisation of RCs by chiropractors, some of which appear fundamental to their perceived identity, whilst others are likely amenable to change with training and experience. Telehealth is the use of electronic and telecommunication technologies for providing patient care. One form of telehealth, virtual or remote consultations (RCs), allows healthcare providers to communicate with patients remotely, via voice and/or video. These digital approaches in healthcare have experienced a slow but steady growth over the past two decades –4. They The start of the global COVID-19 pandemic in 2020 posed a challenge to provision of healthcare due to the way in which the SARS-CoV-2 virus is transmitted, especially for clinicians who provide manual therapy which is close-contact by nature. SARS-CoV-2 virus particles are spread more easily in indoor settings due to poorer ventilation, close range distances of less than 1 m, and through direct contact  all of wLike those from other countries, some UK chiropractors turned to RCs to continue to support their patients when face-to-face care was no longer an option . The useThe aim of this nested qualitative work was to explore the perceptions, views and perspectives of chiropractors who had not adopted RCs as a method of consulting during the pandemic, and to understand potential perceived barriers to their wider implementation in UK chiropractic practice following the COVID-19 pandemic.An online national cross-sectional survey was sent to all registered practicing chiropractors in the UK in May 2020 (n = 3131). Requests to complete the survey were sent via email by the UK national registration body, the General Chiropractic Council (GCC) as well as national member associations (British Chiropractic Association (BCA), McTimoney Chiropractic Association (MCA), United Chiropractic Association (UCA), and Scottish Chiropractic Association (SCA)). Potential participants were provided with information about the study prior to recruitment and were informed that submitting the completed survey would constitute consent to participate. This report is one of two reports derived from this cross-sectional survey, the other report  addresseSurvey respondents who reported not providing RCs were asked to provide details using free-text responses. Survey responses were extracted from the survey data into Microsoft Excel.Analysis commenced through familiarisation with the dataset. The textual responses were read, organised and coded using open-label coding independently by SD and JF—both practicing chiropractors and researchers—in Microsoft Excel. These were consolidated to a common code set through discussion by these two authors. Codes were then mapped together, and overarching themes emerged through further discussion with JV, a qualitative research fellow and DN, a professor of integrated musculoskeletal (MSK) care, bringing multiple perspectives to the analysis. A pragmatic approach was taken in this study.This study received approval from AECC University College Ethics Sub Committee on the 13/05/2020 (#E124/05/2020).Of the 534 responses to the survey 175 (32.8%) self-identified as not using RCs and 137 provided textual information as to why this was. Free-text responses varied in length from 3 to 467 words (mean 33 words), with the majority providing sufficient detail to explain their reasons for not providing RCs. Respondent characteristics are described in Table We identified 4 themes in our analysis: (i) professional identity, (ii) diagnostic uncertainty, (iii) perceived patient preferences, and (iv) practical difficulties. Each theme is described in detail below and quotations are used as exemplars with an identifier for respondent number and member association e.g., R1—SCA). Non-standard capitalisations for words such as “Chiropractic” or “Chiropractors” were present in respondent text. Figure —SCA. NonMany respondents reported that provision of RCs did not align with their strong sense of identity as providers of physical hands-on care. Some commented that the essence of chiropractic is encapsulated within the roots of its name: "Chiropractic comes from the Greek done by hand" (R14—SCA). Some participants stated, in general terms, that RCs present a barrier to providing this style of care: "My work is hands on that cannot be done remotely" (R34—SCA). Others pointed to specific treatments that they perceived as essential to chiropractic care yet self-evidently impossible to provide in RCs, such as spinal manipulation to correct subluxations. "You can't talk a subluxation away, neither can you provide Chiropractic (by hand) care over the phone" (R19—BCA).Some respondents explicitly juxtaposed chiropractic with other healthcare professions that they portrayed as more amenable to incorporating RCs. Some, for example, mentioned that RCs may be more appropriate for psychotherapy: "Chiropractic is manual therapy not talk therapy… If someone needs CBT this should be done by a CBT practitioner" (R4—UCA). A few participants explicitly contrasted their work, perceived as inescapably manual, with that of physical therapists who could use RCs to provide advice and/or suggest exercises: "Exercises are the remit of physiotherapists" (R136—UCA) "I don't want to be just an advice and exercise giver" (R78—BCA).Many respondents expressed concerns about reaching a safe diagnosis during RCs. “I believe you should carry out a thorough case history and physical examination before you make any diagnosis” (R40—UCA). Without the ability to perform an adequate physical examination during a RC, a few respondents were concerned about the possibility of missed diagnosis or red flags. “I fear you would miss important red flag since you are unable to do a physical examination” (R76—UCA).There was also a concern that an inaccurate diagnosis may result in incorrect advice being given to the patient. “Whilst I may be able to offer basic self help advice, the inability to perform a thorough assessment of my patients condition could result in incorrect and inappropriate advice with potentially catastrophic outcomes” (R131—BCA).Some respondents perceived a lack of patient demand for RCs. A few suggested that uptake in their clinic would be limited if the service was offered. “my patient base is such I would strongly doubt many would want a remote consultation” (R65—BCA).There was a general perception among many respondents that patients prefer face-to-face consultations and are expecting a hands-on treatment when they have a consultation with a chiropractor. “Patients don’t see a Chiropractor for a chat and advice, they come for a specific Chiropractic adjustment with full focus and intent” (R135—No member association).A significant number of respondents questioned the ethics of charging for RCs resulting in a disinclination for providing that service. “I do not feel as though I could ethically charge for it, and therefore I don’t want to do it” (R42—BCA).Practical difficulties in providing RCs was an important reason as to why chiropractors are not providing RCs. Due to restrictions imposed by the UK government in response to the COVID-19 pandemic many chiropractors were working at home which for some meant a lack of space or uninterrupted time. This was expressed in a few of the respondents’ answers where they found it difficult to find a quiet and confidential place to work away from their family “I do not have a suitable space where staying with family during lockdown” (R24—BCA). The technology involved in RCs created barriers for a few respondents, including problems with access to sufficient quality internet bandwidths and phone signals to ensure good patient care over video or phone, and lack of access to patient notes for chiropractors who did not use digital notes. Whilst others reported being uncomfortable with using technology for such purposes “I am not ‘technology minded’” (R55—No member association) and lacked the training and/or knowledge of utilising and delivering care via these contemporary technologies.The use of RCs provided an opportunity for UK chiropractors to deliver ongoing care during the COVID-19 pandemic, whilst meeting the government and national body guidelines for safe practice –15. ManyProfessional identity is formed from a core set of beliefs and values that are unique to a particular group or sub-group of professionals . The ideThe results of this study show that a substantial proportion of chiropractors hold a professional identity in which ‘hands-on’ therapy sits at the heart of the care they provide for their patients and RCs represent a medium of clinician and patient interaction in which this type of therapy cannot be delivered. The circumstances surrounding the pandemic was a difficult pill to swallow for these chiropractors, where clinician and patient alike cannot engage in their usual care for risk of threat to their health and in some cases their mortality through something as basic as human touch. This led to a situation where the benefits of manual therapy interventions did not outweigh the risks of potential viral transmission, especially for individuals within vulnerable groups with comorbidities. Where urgent MSK care was required, chiropractors conducted risk assessments alongside informed patient consent to justify delivering in-person care for a limited number of patients within these groups .Communication as an intervention is not seen by these chiropractors as being at the core of the chiropractic encounter. These respondents consider talking therapy, exercises and advice as secondary forms of care that supplement rather than replace the physical care provided by chiropractors. However, this perception of the adjunctive nature of non-physical interventions conflicts with the evidence-base for lower back pain (LBP) and neck pain, conditions that constitute the vast majority of presentations chiropractors manage on a day-to-day basis . ComprehA minority within this survey, identified with subluxation theories for the justification of manual therapy interventions, which precluded their use of RCs due to its omission of hands-on care. Despite evidence for the detection and correction of subluxations being unsubstantiated , there rAscertaining clinical and satisfaction outcomes arising from chiropractors delivering RCs would provide key insights into the veracity of beliefs in the primacy of hands-on care as based in clinical reality or merely a historical legacy of the origins of the profession. Indeed, when asked, chiropractic patients undergoing care via RCs remain highly satisfied .The chiropractors’ concerns regarding reaching a safe diagnosis mirror those of other healthcare professionals trained within systems that emphasize the role of physical examination skills. I.e., some healthcare professionals report feeling cautious in reaching diagnostic decisions without being able to use particular skills during a consultation –41. HoweThe concern expressed by respondents of this study is reflected in advice given to general practitioners in the UK where the use of ‘safety netting’ advice is emphasised when performing RCs, with a low threshold suggested for arranging face-to-face assessment in the presence of diagnostic uncertainty , 45. ThiOur study showed that within a group of participants who did not utilise RCs, chiropractors perceived that there was a lack of patient demand for RCs within their patient base, that patients would not expect to receive advice and explanation alone and non-face-to-face approaches when consulting with a chiropractor to manage their pain, and that the value of RCs was not sufficient to ethically charge patients for this service.These perceptions are at odds with prior work exploring patients’ beliefs when attending chiropractors. For example, Sigrell  reportedIn a recent survey of patients receiving care via RCs with UK chiropractors, none reported dissatisfaction with their RCs . Of thosHowever, only the views of chiropractors choosing not to engage in RCs were included in the presented study. It is possible that this subgroup is in part defined by the emphasis they place on manual therapy, which may in turn influence their belief as to the specific component of care that patients value and expect to receive. This therefore may underpin the emergence of a theme suggesting patients don’t value care that does not include a physical component, and may potentially explain their behaviour and decisions not to offer or effectively promote RCs to patients.Practical barriers to providing RCs emerged as a minor theme, yet they are important factors to consider. The concerns of chiropractors, who are also parents, regarding working from home, have been echoed by many parents in the UK . Their vThe limited or lack of infrastructure of their work setup at home whilst clinics were closed clearly made it difficult for chiropractors, and their problems were predominantly centred around access to resources that they would normally have in their clinics but was not available in their home environment. Implementation of strategies such as migration of paper clinical notes to digital notes that allows encrypted access in the home setting outside of private chiropractic clinics may be useful in case of similar future incidents that require the provision of care in non-face-to-face settings.For chiropractors who are less tech-savvy, this aspect could be addressed by offering training support and resources for chiropractors in use of RCs with their patients in terms of adaptation of clinical space, processes, and communication including consent, as well as the practical aspects of choosing suitable software and hardware to effectively deliver RCs . ChiroprClinicians may want to consider how they might adapt their existing clinical processes to an RC setting; this may include the logistics of appointment booking, pre-RC patient information, methods of patient access to the RC, identity checking, remote physical testing, intervention delivery, remotely provided follow-up resources, and processing of payments.Other considerations include compliance to health and safety legislations both for the patient’s and the chiropractor’s environment to mitigate, for example, risks of falling during movements or activities, and to consider workstation assessment and ergonomics for employees such as admin or clinical staff .As far as we are aware, this study is the first to explore chiropractors’ perspectives of RCs and provides an important insight into the key barriers for wider implementation in the chiropractic profession. The study was conducted by a multidisciplinary team of chiropractors and academic researchers, bringing a multidimensional perspective to the analysis and interpretation.Just under 58% of respondents were female which is slightly higher than the equal gender split of chiropractors registered with the GCC (F = 50.02%). Due to heterogeneity of data, it is not possible to comment further on the generalisability of responses received when compared to the population of UK chiropractors.This study carried out qualitative analysis of free text survey responses which is a methodology that has previously raised concerns in the literature on its limitations , 52. FreAdditionally, respondents were self-selecting in choosing to reply to a study about RCs and so may have been more likely to attract responses from those who held strong views about the subject. Our sample contained a slightly disproportionately high response from females with data that was largely provided by those entering the profession over 10 years ago.The COVID-19 pandemic may accelerate already underway changes in the way healthcare is provided going forward, with RCs becoming more commonplace in primary healthcare provision. Barriers have been identified to chiropractors adopting RCs, some of which appear fundamental to their identity whilst others are likely amenable to change with training and experience.


Atherosclerosis (AS) is a complex disease caused in part by dyslipidemia and chronic inflammation. AS is associated with serious cardiovascular disease and remains the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) have evolved as an attractive therapeutic agent in various diseases including AS. Human umbilical cord MSCs (UCSCs) have been used in cell therapy trials due to their ability to differentiate and proliferate. The present study aimed to investigate the effect of UCSCs treatment on atherosclerotic plaque formation and the progression of lesions in a high-fat diet rabbit model.6 in 500 μL phosphate buffered saline) after 1 month of high-fat diet, once every 2 weeks, for 3 months. The model group was given PBS only. We analyzed serum biomarkers, used ultrasound and histopathology to detect arterial plaques and laser Doppler imaging to measure peripheral blood vessel blood filling, and analyzed the intestinal flora and metabolism.Rabbits were fed a high-fat diet and then randomly divided into three groups: control, model, and treatment groups. Rabbits in the treatment group were injected with UCSCs (6 × 10N-oxide (TMAO) were down-regulated in the treatment group.Histological analysis showed that the aortic plaque area was significantly reduced in the treatment group. We also found a significant decrease in macrophage accumulation and apoptosis, an increase in expression of scavenger receptors CD36 and SRA1, a decrease in uptake of modified low–density protein (ox-LDL), and a decrease in levels of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α following UCSCs treatment. We also found that anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-β expression increased in the aorta atherosclerotic plaque of the treatment group. UCSCs treatment improved the early peripheral blood filling, reduced the serum lipid level, and inhibited inflammation progression by regulating the intestinal flora dysbiosis caused by the high-fat diet. More specifically, levels of the microbiota-dependent metabolite trimethylamine-UCSCs treatment alleviated atherosclerotic plaque burden by reducing inflammation, regulating the intestinal flora and TMAO levels, and repairing the damaged endothelium. Atherosclerosis (AS) is a systemic disease that is associated with the accumulation of lipid-laden macrophages in the arterial wall, which leads to arterial wall thickening and lumen narrowing, subsequently resulting in heart disease, cerebral infarction, and other severe complications . AS is cThere is overwhelming evidence that gut microbes and their metabolites contribute to AS pathogenesis . Lipid lMesenchymal stem cells (MSCs) have been demonstrated to be effective in the treatment of various diseases, including AS, due to their tissue repair, anti-inflammatory, and immunological properties . MSCs caTo the best of our knowledge, no study has reported the use of UCSCs in the treatment of AS. Therefore, we intravenously transplanted UCSCs in a high-fat diet rabbit model to observe their therapeutic effect and understand their mechanism of action. We found that UCSCs transplantation reduced inflammation in the aortic atherosclerotic plaques by regulating the production and polarization of macrophages, repairing damaged endothelial cells (EC), correcting the intestinal flora imbalance, and preventing harmful metabolite production caused by the high-fat diet. The findings presented here offer new therapeutic insights into to use of UCSCs for treating AS.Japanese big-ear white rabbits  were purchased from Beijing Fu Long Teng Fei Experimental Animal Research Institute Co., Ltd. (SCXK [Jing] 2018-0009). Rabbits were randomly divided into three groups: normal, model, and treatment; each group included 5 females and 5 males. The animals were housed in separate cages in ordinary animal house facilities (SYXK [Jing] 2017-0027) and had free access to food and water. The use of animals was in compliance with the Health Guide of Animal Welfare of the National Institutes for Animal Care and Use, and the experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animal Science (permit number: QC19020). All procedures were conducted according to institutional guidelines.2 at 37 °C. The media was changed every three days until the cells were confluent, and the cells were then digested and passaged using pancreatin .UCSCs were obtained from Cyagen Biosciences Inc. . The UCSCs were placed in a 6-well plate and cultured  in MEM  analysis .Cells at passage 3 (P3) were collected to identify UCSCs surface markers including CD90-fitc , CD29-pe-cy5 , CD73-pe , CD105-apc , and HLA-apc-cy7 . Cells were incubated in 100 μL staining solution at 4 °C for 30 min. We added 1 ml phosphate buffered saline (PBS) and centrifuged the cells at 400×6 in 500 μL PBS, once every 2 weeks for 3 months [Rabbits in the model and treatment groups were fed a high-fat diet containing 0.5% cholesterol, 5% lard oil, 5% yolk powder, 0.2% pig bile salts, and 89.3% basal feed , 150 g per day for 16 weeks. After 1 month of high-fat diet, rabbits in the treatment group were intravenously injected with UCSCs via the ear vein at a dose of 6 × 103 months , 32. RabBeckman AU5800, USA).At 1, 2, and 3 months after treatment, blood samples were collected in normal serum tubes . We used enzymatic detection methods to assay serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels, the alanine substrate method to measure alanine transaminase (ALT), antibody inhibition methods to measure creatine kinase MB (CK-MB), and immunity transmission turbidity with an automatic biochemical analyzer to measure apolipoprotein B (ApoB) .The aortic arch, carotid artery, and abdominal aorta in each rabbit were imaged using a color Doppler ultrasound diagnostic system  3 months after high-fat diet.After treatment, animals were anesthetized and euthanized as previously described . The entBefore the animals were euthanized, fresh feces were taken from each group for intestinal 16S rDNA microbial community analysis. This detection was carried out by Beijing Ouyisaisi Biotechnology Co., Ltd. Diversity analysis of alpha and beta in the intestinal flora and the comparison of relative abundance of the TOP15 flora at the genus level were analyzed.3 and 0.03 g) was collected. TMAO metabolite detection was carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS) . The procedure was done as follows: sample grinding (JXFSTPRP-24/32), purification (SB-5200DT), enrichment, purification, Ultra Performance Liquid Chromatography (UPLC) separation (AB ExionLC), MS/MS Multi-response mode (MRM) detection (AB Sciex Qtrap 6500+), data collection, and qualitative and quantitative analyses of metabolites.Animals in each group were euthanized, and a small piece of fresh liver tissue  and Image J 1.42 . Statistical analyses were performed using SPSS 16.0 software  and Microsoft Excel . Continuous data are presented as mean ± standard deviation (SD). A single-factor one-way analysis of variance (ANOVA) was used to evaluate inter-group differences. Differences were considered significant when the UCSCs at P3 were collected for morphological observation. Phase contrast microscopy showed that the UCSCs had a spindle shape and a fibroblast-like morphology Fig. A. FCM deP < 0.01), TC (P < 0.05), and LDL-C (P < 0.01) levels in the treatment group were significantly lower than those in the model group. At 2 months after UCSCs treatment, serum ALT (P < 0.01) and LDL-C (P < 0.05) levels declined in the treatment group compared to those in the model group. After 3 months of UCSCs treatment, serum ALT (P < 0.01), TC (P < 0.01), LDL-C (P < 0.01), TG (P < 0.01), CK-MB (P < 0.01), and ApoB (P < 0.01) levels in the treatment group were all significantly lower than those in the model group . Correspondingly, the average blood flow filling value of the treatment group was higher compared to the model group (P < 0.05)  was significantly reduced (05) Fig. B.After 1 and 3 months of high-fat diet, we performed vascular ultrasonic analysis. After 1 month of high-fat diet, we did not detect any atherosclerotic plaques in the aorta, carotid artery, or abdominal aorta in the model group and UCSCs treatment group. After 3 months of high-fat diet, the aortic arch, carotid artery, and abdominal aorta in the model group showed different degrees of atherosclerotic plaque burden, with incidences of 80% (8/10), 60% (6/10), and 70% (7/10), respectively. Atherosclerotic plaques were also observed in the treatment group, with incidences of atherosclerotic plaque in the aortic arch, carotid artery, and abdominal aorta of 40% (4/10), 10% (1/10), and 20% (2/10), respectively  , cell proliferation increased (P < 0.01), and EC marker expression increased (P < 0.05) in the UCSCs treatment group  and the inflammatory factors IL-6 (P < 0.01) and TNF-α (P < 0.01) were significantly increased in the model group compared to the UCSCs treatment group. Immunosuppressive factors IL-10 (P < 0.01) and TGF-β (P < 0.05) were significantly decreased in the model group compared to the UCSCs treatment group  Fig. A. PatholP < 0.05 or P < 0.01), while there was no obvious difference between the UCSCs treatment and normal groups  and content (P < 0.05) of TMAO in the UCSCs treatment group were significantly lower compared to the model group  and the expression of macrophage scavenger receptors CD36 (P < 0.01) and SRA-1 (P < 0.01)  Fig. B, C.The use of MSCs has increased in the clinical treatment of various diseases. However, for an MSC population to be effective clinically, it must have low antigenicity . Since aIn this study, we used an in vivo high-fat diet model to induce AS in rabbits. Cholesterol-fed rabbits are widely used for AS studies  because Cholesterol, TG, and lipoproteins are known factors in atherosclerosis pathogenesis. Increased serum LDL and TG concentrations are responsible for the formation of atherosclerotic lesions . APOB isBifidobacterium spp. can reduce circulating TG and LDL levels and increase HDL levels [Lactobacillus species can reduce plasma cholesterol levels [Bifidobacterium and Lactobacillus, and reduce the level of TMAO synthesis. Therefore, our findings indicate that serum lipid levels are reduced to some extent by UCSCs treatment.It has been reported that gut microbes and their composition can regulate lipid metabolism  , 41. A dL levels . Experiml levels . TMAO, al levels , 46. In Much of the current evidence indicates that macrophages participate in AS pathogenesis , as theiUCSCs have been shown to inhibit the differentiation and maturation of other important inflammatory cells, namely dendritic cells (DCs) , by reduEmerging data suggest that intestinal flora dysbiosis can contribute to AS development by increasing systemic inflammation –63. A hiThe intestinal microbiota and host immune system regulate each other . TherefoTMAO generation is dependent on the gut microbiota, and it can exacerbate inflammatory reactions in the vascular wall and impair cholesterol reverse transport . TMAO caPrevious studies have shown that the formation of atherosclerotic plaques in large-medium size arteries is associated with vascular lesions in peripheral vessels, resulting in reduced blood flow filling or filling obstruction. UCSCs transplantation can improve the morphology and function of peripheral blood vessels in the early stage of AS. Our ultrasound analysis found that the incidence of atherosclerotic plaques in the UCSCs treatment group was lower than that in the model group after 3 months of high fat diet, further supporting the hypothesis that early UCSCs intervention therapy can reduce the formation of atherosclerotic plaques.This study demonstrates that UCSCs transplantation in the treatment of AS at the early stages can not only attenuate atherosclerotic plaque formation and progression in large and medium-sized vessels, but also improve early peripheral blood filling. UCSCs transplantation can alleviate AS by reducing the serum lipid level, inhibiting the production of macrophages and inflammatory cytokines (IL-6 and TNF-α), inhibiting apoptosis, promoting production of anti-inflammatory cytokines (IL-10 and TGF-β) and endothelial cells, further inhibiting inflammatory responses and repairing damaged endothelium. UCSCs can also inhibit inflammation progression and ox-LDL phagocytosis by balancing intestinal flora dysbiosis caused by high-fat diet, as well as reducing TMAO production, which can also lessen atherosclerotic plaque burden. Future work will provide and in-depth investigation of the molecular mechanism by which UCSCs regulate intestinal flora and metabolite production to reduce AS progression.


A 35-year-old woman underwent bilateral lung transplantation for primary ciliary dyskinesia and developed vascular tumors over a slow time course. Initial presentation of non-specific vascular tumors in the lungs and liver for up to 6 years after transplantation evolved toward bilateral ovarian angiosarcoma. Tumor analysis by haplotyping and human leukocyte antigen typing showed mixed donor chimerism, proving donor origin of the tumoral lesions. In retrospect, the donor became brain dead following neurosurgical complications for a previously biopsy-proven cerebral hemangioma, which is believed to have been a precursor lesion of the vascular malignancy in the recipient. Donor-transmitted tumors should always be suspected in solid organ transplant recipients in case of uncommon disease course or histology, and proper tissue-based diagnosis using sensitive techniques should be pursued. Malignancy is a known complication after solid organ transplantation related to the use of immunosuppression and early development of age-associated neoplasms . The preA 35-year-old woman underwent bilateral lung transplantation for primary ciliary dyskinesia in 2013. The donor was a 44-year-old female who became brain dead following neurosurgical complications for a previously biopsy-proven cerebral hemangioma. Chest and abdominal CT, performed prior to organ allocation, excluded concurrent vascular lesions or metastases. The lungs, both kidneys, and liver were allocated for transplantation to four different recipients. Our recipient was treated with tacrolimus, mycophenolate, and steroids as standard maintenance immunosuppressive regimen.18F-fluorodeoxyglucose PET-CT (18F-FDG PET-CT), and further follow-up was provided. A slight increase to 20 mm was detected 6 months later, but again 18F-FDG PET-CT revealed no FDG avidity  and α-smooth muscle actin . The conclusion was undetermined vascular lesions without signs of malignancy, but with an uncertain biological behavior. The lung wedge excision was revised given this information, and one vascular lesion, consistent with a hemangioma, was noted.Follow-up CT 6 months later demonstrated two small nodules of 11 and 15 mm in the costodiaphragmatic sinus of the left lower lobe ; and had a local absence of α-smooth muscle actin  was initiated. After eight consecutive weekly administrations, re-evaluation demonstrated disease progression, and systemic treatment was changed to single-agent doxorubicin (75 mg/m2) every 3 weeks. Further disease progression was documented after three cycles. Oral treatment with an anti-angiogenic tyrosine kinase inhibitor pazopanib (daily dose of 400 and afterward 800 mg) was initiated. However, 3 months later, further disease progression with lymph node metastases was noted, and fourth-line systemic treatment with gemcitabine  was started, yet without avail. Best supportive care was initiated upon radiographic development of brain, kidney, and thyroid metastases, and the patient succumbed 2.5 weeks later, 7.6 years after transplantation.Two weeks postoperatively, because of severe right upper quadrant abdominal pain, massive hepatic tumor progression was observed (® RSC DNA FFPE Kit (Promega), followed by co-amplification and three-color detection of 16 loci (15 STR loci and amelogenin) . Forty percent of cells of non-recipient origin were present in the ovarian tumor tissue. The DNA profile of these foreign cells was compared with the donor tissue profile, confirming the presence of similar alleles  loci. Briefly, DNA was extracted from formalin-fixed paraffin-embedded tissue of the donor cerebrum and recipient left ovarian tumor using Maxwell alleles Table 1.In retrospect, the donor most likely had a low-grade precursor of this vascular malignancy with circulating tumor cells that, in combination with chronic immunosuppression, resulted in late-onset donor-related vascular lung lesions in the recipient after 3 years, followed by liver and ovarian metastases. Eurotransplant, responsible for the allocation of donor organs in this patient, was informed of this serious adverse event. To our knowledge, the other recipients related to this multi-organ donor remained tumor-free: one kidney recipient died from septic shock due to a perianal abscess, the liver recipient died of an unknown cause 2 months post-transplant, and the other kidney recipient is alive and closely monitored after additional investigations were reassuring.Organ transplantation is a well-established treatment modality for many end-stage diseases but, as with most medical interventions, carries a significant risk of acute or chronic complications. One potential complication is unexpected disease transmission from the donor to the recipient, estimated to occur in less than 1% of transplant procedures , 9. Extede novo occurrence or recurrence of malignancy or donor-related malignancy. Donor-related malignancies mostly occur early after transplantation and can be either due to transmission of occult tumor cells at the time of transplantation  or due to tumor arising in donor-origin cells without prior malignancy in the donor   –15. The ignancy) . Our pathistory of malignancy may nevertheless be considered potential donors, depending on the type of cancer, interventions given, and interval between diagnosis/cure and organ donation. The same is true for donors with active malignancies with a minimal to low risk for transmissions, such as low-grade skin cancer, carcinomas in situ of the uterine cervix, and low-grade primary central nervous system tumors (in situ) are usually avoided, especially if there is a risk of the late appearance of metastasis, such as after breast cancer. Transmission rates vary between different tumor types and even with the type of organs transplanted; risk categorizations for specific malignancy types are available , and may impact other organ recipients from the same donor, hence the importance of establishing the exact genome-of-origin; a sensitive method using multiple STR loci or single-nucleotide polymorphisms is recommended in gender-identical transplants, whereby distinction can be made in almost all cases .We describe a unique case of donor-transmitted angiosarcoma, with late post-transplant presentation as non-specific vascular tumors in the lungs and liver for up to 6 years after transplantation, yet with the evolution toward a Krukenberg tumor and finally disseminated lethal disease. Donor origin of tumors detected in solid organ transplant recipients should always be suspected in case of an uncommon disease course or histology, and proper tissue-based diagnosis should be pursued, given the possible implications for treatment and outcome of other organ recipients from the same multi-organ donor.This includes the following important collaborators of our lung transplant program who were directly involved in the care of our lung transplant recipients during the past years:Jonas Yserbyt, PhDArne P. Neyrinck, PhDVeronique Schaevers, MScBruno Desschans, MScDirk Claes, MScKaren Denaux, MScPaul De Leyn, PhDHerbert Decaluwé, PhDHans Van Veer, MDLieven Depypere, PhDMarie-Paule Emonds, PhDThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the University Hospital of Leuven under agreement S51577. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SB: performed the research/study, collected the data, and wrote the manuscript. LD: performed the HLA analysis and critically revised the manuscript. LM, IV, and SV: performed genetic analyses and critically revised the manuscript. FJ and PS: the treating oncologists and critically revised the manuscript. BW, RS, and SD: performed the histological analyses and critically revised the manuscript. DV: the treating lung transplant surgeon and critically revised the manuscript. LC: the assisting lung transplant surgeon and critically revised the manuscript. LG, LJD, and GV: the treating lung transplant physicians and critically revised the manuscript. RV: the delegating lung transplant physician over time, coordinated and designed the research/study, collected the data, and critically revised the manuscript. All authors contributed to the article and approved the submitted version.The following authors are supported by a research fellowship but received no specific funding for the current study: RV is a senior clinical research fellow of the Fund for Scientific Research Flanders (FWO). GV and DV are supported by the Broere Charitable Foundation. LC is supported by a KU Leuven University Chair funded by Medtronic.The authors confirm that the work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form in English or in any other language, without the written consent of the copyright holder.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 yeast Atg8 protein and its paralogs in mammals, mammalian Atg8s (mAtg8s), have been primarily appreciated for their participation in autophagy. However, lipidated mAtg8s, including the most frequently used autophagosomal membrane marker LC3B, are found on cellular membranes other than autophagosomes. Here we put forward a hypothesis that the lipidation of mAtg8s, termed ‘Atg8ylation', is a general membrane stress and remodeling response analogous to the role that ubiquitylation plays in tagging proteins. Ubiquitin and mAtg8s are related in sequence and structure, and the lipidation of mAtg8s occurs on its C-terminal glycine, akin to the C-terminal glycine of ubiquitin. Conceptually, we propose that mAtg8s and Atg8ylation are to membranes what ubiquitin and ubiquitylation are to proteins, and that, like ubiquitylation, Atg8ylation has a multitude of downstream effector outputs, one of which is autophagy. The yeast Atg8 protein and its seven mammalian Atg8 paralogs (mAtg8s) that include LC3A, LC3B, LC3B2, LC3C, GABARAP, GABARAPL1, and GABARAPL2/GATE16 , a proceFigures 1 and 2). Ubiquitylation is associated with disassembly, degradation and removal of misfolded proteins triggered by stressors [Figure 1) is to membranes what ubiquitylation is to proteins.Of note, mAtg8s are related to ubiquitin in sequence and structure . Atg8ylation is a process whereby the Atg8 conjugation machinery is recruited to damaged or otherwise stressed membranes or to membranes undergoing remodeling under various homeostatic or non-homeostatic conditions  and catalyzes mAtg8s' conjugation to phosphatidylethanolamine (PE) [Figure 1C). ATG7 conjugates to the C-terminal glycine of mAtg8s, exposed post-translationally by ATG4 proteases. This is followed by action of the E2-like activity of ATG3 on ATG8s. Finally, the conjugation of mATG8s to PE or PS is mediated by an E3-like complex ATG5-ATG12/ATG16L1  [Ubiquitylation of proteins often occurs on misfolded and aggregated proteins caused by stressors as well as under unperturbed conditions, modulating normal protein activity, localization, and their interactions (ine (PE) –18, or pgure 1C) .Figure 2A-C), underscoring the similarities between the two systems and the biochemical relatedness of ubiquitylation and Atg8ylation consistent with the parallels in the conjugation cascade. The two differ principally in the type of targets that ubiquitin or Atg8s are being conjugated to – in the case of ubiquitin to the stressed and degradation-bound or otherwise modified proteins, whereas in the case of mAtg8s, to the stressed or otherwise engaged membranes. The diverse events downstream or associated with Atg8ylation are individually described in subsections below.Of note, the diverse mammalian Atg8s are all related to ubiquitin  TheCanonical autophagy and its variations manifested in different forms of selective or quality control autophagy, defined by the captured cargo  , 8 depenFigure 3A) [Autophagosomal membrane formation is believed to entail mAtg8 conjugation machinery and Atg8ylation, i.e. Atg8 lipidation on membranes that form autophagosomes (gure 3A) . Howevergure 3A) , 57. Thigure 3A) . Atg8ylagure 3A) –61, sevegure 3A) , autolysgure 3A) , and lysgure 3A)  in the aFigure 3B). LAP per se lacks cytosolic cargo, but some Lapoid processes involve cytosolic cargo. LAP involves Atg8ylation, specifically of LC3 subfamily of mAtg8s [LAP , 62, LANf mAtg8s , on membf mAtg8s , 68–70. f mAtg8s .The key distinguishing feature of LAP, LANDO, and other Lapoid processes that differentiates them from canonical autophagy is the absence of double membranes, i.e., Atg8ylation occurs on the delimiting single bilayer membrane of the stressed endosomes and phagosomes. Further key differences are the dependence of LAP on RUBCN (Rubicon), which is an inhibitor of canonical autophagy, and its independence of FIP200, which is required for canonical autophagy and Atg8ylation of conventional double membrane autophagosomes.In principle, a similar mAtg8 lipidation machinery is involved in Lapoid phenomena as in the case of canonical autophagy. It is the ATG16L1 component of the Atg8ylation E3 ligase (ATG5-ATG12/ATG16L1) that discerns between target membranes that are to be Atg8ylated . Lipid bThe upstream machinery that recognizes stressed membranes remains to be elucidated, but may involve signals form TLRs, stretching of lipid bilayers during osmotic stress, ion transport events, etc. One upstream signal that has been clearly established in LAP is NOX2 (NADPH oxidase) , which iHow do LAP or Lapoid processes terminate, is another open question. Phagosomes that are Atg8ylated (LAP) appear to fuse with lysosomes in a more efficient way . The mecLAP and Lapoid functions include anti-inflammatory processes such as those failing in lupus , eliminaFigure 3C) and its proteins occurs in response to osmotic stress or glucose-starvation, adjusting the size of lysosomes under stress conditions [Selective microautophagy of mammalian lysosomal membranes  mirrors its role in selective microautophagy. Microautophagy directly carried by lysosomes and endosomes can in principle result in sequestration, degradation or secretion of intralumenal vesicles (ILVs). The exocytosed ILVs in the extracellular space are referred to as exosomes, a subtype of EVs . EVs areFigure 3D). Proteomic and RNA profiling of EVs identified diverse RNA binding proteins and small non-coding RNAs requiring LC3 and the Atg8ylation machinery for packaging and secretion [Atg8ylation participates in the formation of exosomes , 98 and ecretion . Among oecretion .Atg8ylation also plays a role in the type of unconventional secretion or excretion/extrusion of cytoplasmic material referred to as secretory autophagy , 102, wiBiological membranes provide a key diffusional barrier and define the physical boundaries of cells and their intracellular compartments; they are endowed with selective permeabilities, channels, pumps, and signaling systems , 119. A Mycobacterium tuberculosis [The plasma membrane is by its boundary nature under continuous risk of being damaged by exogenous agents, and conversely is used for programmed permeabilization events associated with cell death pathways. Recent studies –126 suggrculosis , 126. Whrculosis  or endocrculosis .Intracellular membranes can also come under the physical assault or be subjected to membrane stress, such as osmotic changes. The lysosome is the digestive organelle of the cell and thus receives cargo destined for degradation from both cellular interior and the exterior space, via autophagic, endosomal and phagocytic pathways . By the Lysosomal damage elicits a set of diverse responses to restore lysosomal homeostasis , 143–148Atg8ylation contributes to signaling events that activate TFEB (transcription factor EB) to initiate the lysosomal protein expression program. Starvation and additional signals, e.g., lysosomal damage, induce TFEB translocation form the cytosol to the nucleus where it stimulates lysosomal gene expression, and to an extent several autophagy genes –158. ApaFigure 3E). TFEB interacts directly with GABARAP [TFEB is activated by a variety of membrane and other stress conditions including starvation , 164, ly GABARAP  and the  GABARAP  and lyso GABARAP . Calcium GABARAP , 165. At GABARAP , whereas GABARAP . Further GABARAP , which iAtg8ylation has appreciable effects on TFEB activity during starvation, lysosomal damage of xenophagy , 162, 1, 171. GAThe herein proposed unifying concept of Atg8ylation refers to a process of tagging stressed membranes with lipidated mAtg8s. This includes damaged, or otherwise mobilized and remodeling membranes leading to a plethora of outcomes. Often, these processes occur on endosomal, lysosomal and autophagosomal membranes but may not be limited to these organelles. The spectrum of presentations includes canonical autophagy, LAP and Lapoid processes, selective microautophagy and unconventional secretion, different stages of the MERiT lysosomal QC system, and membrane modification during programmed or signaling events.The elucidation of the canonical autophagy pathway in yeast has provided a clear linear pathway of Atg8-driven steps, from autophagosome formation to their fusion with the vacuole/lysosome. In mammalian systems, this pathway and its core components are conserved. The presence of multiple Atg8 paralogs in mammals reflects increased complexity, whereas experimental analyses have revealed that mAtg8s function in processes other than autophagy. The proliferation of these ‘non-canonical autophagy-related' processes may at first appear confusing. The Atg8ylation concept proposed here resets the view of what lipidation of mAtg8s represents considering its standalone manifestation and nature – it is a covalent modification of membranes the way ubiquitin covalently modifies proteins. Once this point of view is considered, it is relatively easy to understand how this can participate in a multitude of processes, of which canonical autophagy is just a subset. This could be useful as more and more ‘anomalies' or non-canonical processes are being described, and should spur further investigations based on the principle of Atg8tylation freed from the conceptual confines, however elegant, of the canonical autophagy pathway. Retrospectively, membrane processes such as LAP, LANDO, selective microautophagy, EV/exosome secretion, etc., can be easier to understand from this point of view, while stimulating future systematic discoveries.The crossovers between ubiquitylation and Atg8ylation exist, as glimpsed from emerging examples of ubiquitylation being able to occur on complex glycolipids such as bacterial LPS (lipopolysaccharide) when introduced into the mammalian cell , potentiWith the introduction of Atg8ylation as a concept some old questions remain, and new questions arise: What exactly are the functional and mechanistic consequences of Atg8ylation in general and in specific examples covered here and how and does the lipidation of mAtg8s affect their functionalities? Do Atg8ylated membranes share specific types of lipid modifications, changes in ordering/tension within the bilayer, or other molecular features that are specific to ‘stress' or a ‘stress' signal? When a target protein reaches a threshold of ubiquitin modifications this often leads to its proteasomal degradation; could Atg8ylation similarly operate in the context of membrane remodeling and degradation? Doe Atg8ylation also take place under basal conditions, and, if so, would there be quantitative and qualitative differences between basal vs. ‘stress' conditions? With Atg8ylation expanding the functional roles of mAtg8s to outside of the cells through unconventional secretion, do these processes serve to excrete material from the cells or take active signaling roles such as in the case of EVs in intercellular communication? What are the specific roles of mAtg8s and Atg8ylation in processes of unconventional/secretory autophagy, and do they play a role in cargo selection or vesicle formation or both?As the most celebrated Atg8ylation process, canonical autophagy impacts a spectrum of pathophysiological conditions including cancer, neurodegenerative diseases, metabolic and cardiovascular disorders, autoimmunity, inflammation and infection , 178. LA


Efficient mobilization of hematopoietic stem cells (HSCs) from bone marrow niche into circulation is the key to successful collection and transplantation in patients with hematological malignancies. The efficacy of various HSCs mobilization regimens has been widely investigated, but the results are inconsistent.We performed comprehensive databases searching for eligible randomized controlled trials (RCTs) that comparing the efficacy of HSCs mobilization regimens in patients with hematological malignancies. Bayesian network meta-analyses were performed with WinBUGS. Standard dose of granulocyte colony-stimulating factor (G-CSF SD) was chosen as the common comparator. Estimates of relative treatment effects for other regimens were reported as mean differences (MD) or odds ratio (OR) with associated 95% credibility interval (95% CrI). The surface under the cumulative ranking curve (SUCRA) were obtained to present rank probabilities of all included regimens.+ cells (× 106/kg), among which ID-AraC + G-CSF SD ranked first with a probability of being best regimen of 100%. Moreover, ID-AraC + G-CSF SD and G-CSF SD + Plerixafor SD are associated with significantly higher successful rate of achieving optimal target (collecting ≥ 4–6 × 106 CD34+ cells/kg). For patients with NHL, data are pooled from 5 trials for 4 regimens, the results show that compared with G-CSF SD alone, G-CSF SD + Plerixafor SD  and G-CSF SD plus the new CXC chemokine receptor-4 (CXCR-4) antagonist YF-H-2015005  are associated with significantly higher number of total CD34+ cells collected. These 2 regimens are also associated with significantly higher successful rate of achieving optimal target. There are no significant differences in rate of achieving optimal target between G-CSF SD + Plerixafor SD and G-CSF + YF-H-2015005.Databases searching and study selection identified 44 eligible RCTs, of which the mobilization results are summarized. Then we compared the efficacy of mobilization regimens separately for patients with multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) by including 13 eligible trials for network meta-analysis, involving 638 patients with MM and 592 patients with NHL. For patients with MM, data are pooled from 8 trials for 6 regimens, including G-CSF in standard dose (SD) or reduced dose (RD) combined with cyclophosphamide (CY), intermediate-dose cytarabine (ID-AraC) or plerixafor. The results show that compared with G-CSF SD alone, 3 regimens including ID-AraC + G-CSF SD , G-CSF SD + Plerixafor SD , and CY + G-CSF RD  are associated with significantly increased total number of collected CD34In conclusion, ID-AraC plus G-CSF is associated with the highest probability of being best mobilization regimen in patients with MM. For patients with NHL, G-CSF in combination with plerixafor or YF-H-2015005 showed similar improvements in HSCs mobilization efficacy. The relative effects of other chemotherapy-based mobilization regimens still require to be determined with further investigations.The online version contains supplementary material available at 10.1186/s13287-022-02802-6. For patients with newly diagnosed multiple myeloma (MM), high-risk or relapsed non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL), HDT/ASCT is part of standard care that could significantly prolonged progression-free survival and overall survival –6. For lrequired , 13. Increquired . Therefo+ cells in approximately 5–30% of patients with MM or lymphoma [+ cells yield and reduce tumor cells burden, but with the expense of increased toxicity [Granulocyte colony-stimulating factor (G-CSF) is the most commonly used mobilization agent in clinical practice. G-CSF alone can induce effective HSCs mobilization through complicated mechanisms such as triggering the release of proteolytic enzymes and disrupting the stromal derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR-4) axis , 15. Howlymphoma , 14. Thelymphoma . In additoxicity . The effIn this study, we aimed to perform a systematic review and network meta-analysis to compare the efficacy of mobilization regimens in patients with hematological malignancies, hoping to provide high-level evidence for decision making in clinical practice via synthesizing available direct and indirect evidence from relevant RCTs.This study is conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) extension statement for network meta-analyses . We sear+ cells per kilogram (kg) of body weight, and the proportions of patients achieving optimal mobilization target (collecting ≥ 4–6 × 106 CD34+ cells/kg). We excluded quasi-randomized studies, dose-escalating studies, pharmacokinetic studies, cost-effectiveness analyses, post-hoc analyses, studies on healthy volunteers, and studies investigating the effects of mobilization agents on hematologic recovery. Studies including patients with malignancies of other systems  were also excluded in the review. For network meta-analysis, studies that concurrently included patients with different hematological malignancies without providing subgroup results were excluded to reduce heterogeneity. Since the mobilization strategies and support therapies changed a lot over time, studies that enrolled participants before the year of 2000 were excluded.Two investigators (CXL and GXW) independently assessed the eligibility of retrieved citations. Disagreements were resolved by discussion with a third investigator (SNX). RCTs comparing the efficacy of two or more mobilization regimens in patients with hematological malignancies were included. The inclusion criteria were: (i) included patients with primary diagnosis of hematological malignancies and eligible for ASCT; (ii) randomly assigned patients to receive two or more kinds of HSCs mobilization regimens; (iii) reported data for at least one of the efficacy outcomes, including the total number of collected CD34+ cells (× 106/kg), secondary outcome is the successful mobilization rate (described as the proportions of patients collecting ≥ 4–6 × 106 CD34+ cells/kg). For continuous outcomes, the mean value and standard deviation were directly extracted, or estimated from median, range and sample size using previously validated methods [Two investigators (CXL and GXW) independently extracted data using predesigned data collection forms. Results are cross-checked to reach a consensus. The extracted data include trial characteristics, patient characteristics, dosage and duration of mobilization agents, and efficacy outcomes. The primary outcome is total number of collected CD34 methods . For tri methods . Any dis., which could handle trials with multiple arms and rank treatments with additional code [We conducted Bayesian network meta-analyses following the guidelines of the National Institute for Health and Care Excellence Decision Support Unit (NICE DSU) . Networknal code . Three cnal code , 23. The2/day), reduced dose  [Databases searching identified 6398 potentially relevant references, of which 1088 duplicates were removed and 5223 records were excluded based on reviewing title and abstract. Full-text publications of the remaining 87 records were retrieved for further evaluation. After excluded studies of cost-effectiveness analysis and post-hoc analysis, and studies that included other diseases, 44 trials were included for systematic review –67. For /m2/day) , 68. Dif/m2/day) , 53. To /m2/day) . In addiThe characteristics of the 44 trials included in review are summarized in Table 9/L or when PB CD34+ cells > 10/μL, required a median interval from drug administration to apheresis initiation of 7–15 days. The end point of apheresis is the achievement of minimal collection target (≥ 2 × 106 CD34+ cells/kg) or optimal collection target (≥ 4–6 × 106 CD34+ cells/kg). The allowed maximum number of apheresis ranges from 3 to 5. The mobilization target and allowed maximum number of apheresis for all studies included in review are listed in Additional file After the administration of mobilization regimens, apheresis procedure was initiated on day 5 in patients mobilized with G-CSF alone or G-CSF plus CXCR4 antagonists. In patients mobilized with chemotherapy-based regimens, apheresis procedure was often initiated when peripheral blood (PB) white blood cells count recovery to more than 1 × 10+ cells collected and the successful rate of achieving optimal target in patients with MM and NHL. For patients with NHL, the addition of another CXCR4 antagonists YF-H-2015005 also significantly improved mobilization efficacy in comparison with G-CSF alone. As for CY plus G-CSF, the results of mobilization efficacy comparison versus G-CSF alone varies in included studies. Silvennoinen et al. reported that CY plus G-CSF RD significantly increased the number of harvested CD34+ cells versus G-CSF SD in patients with MM, and Narayanasami et al. reported that CY plus G-CSF SD significantly increased the number of harvested CD34+ cells versus G-CSF SD in patients with NHL and HL, but another 3 studies did not report any statistically significant difference [+ cells collected (5.9 versus 7.06) [The main mobilization results of the 44 studies included in review are summarized in Table fference , 58, 61.fference . The mobus 7.06) . Comparius 7.06) , 64.+ cells collected and the rare of achieving minimal/optimal target are reported  and Biosimilar G-CSF are compared with G-CSF, and similar results in the number of CD34ed Table . As for rovement , 65, 67.rovement . Other cThe characteristics of the 13 trials that included in the network meta-analysis are presented in Table + cells (× 106/kg) collected from PB are reported in 8 trials for MM, involving 6 mobilizaion regimens. The network plot for all direct comparisons is shown in Fig. + cells (× 106/kg) collected.. Pegfilgrastim 12 mg and 18 mg are associated with lower number of CD34+ cells collected than G-CSF SD. The forest plot with MD and 95% CrI for all included regimens is shown in Fig. + cells collected in patients with MM are shown in Additional file The total number of CD34+ cells collected are compared among 3 regimens  collected  are compared among 6 mobilization regimens for patients with MM, the network plot describing all direct comparisons within these regimens is shown in Additional file The successful rates of achieving optimal target  is associated with significantly higher CD34+ cells yield in patients with MM when compared with G-CSF alone, which is consistent with our results [. suggested that ID-AraC plus G-CSF prominently increased CD34+ cells yield (median of 20.2 versus 5.9 × 106/kg) compared with G-CSF alone, and produced a rate of achieving optimal target (≥ 5 × 106 CD34+ cells/kg) with a single apheresis of 86% versus 41% [One of the most important findings of this study is that intermediate-dose cytarabine (ID-AraC) may be more efficient than cyclophosphamide (CY) when used for HSCs mobilization in patients with MM. Mobilizing chemotherapy followed by G-CSF is a commonly used mobilization strategy in patients with hematological malignancies. Although longer interval from drug administration to initiation of apheresis, higher risk of neutropenic fever, and increase require of hospitalization associated with chemotherapy-based mobilization are widely reported, patients still could greatly benefit from decreased tumor cells burden and high CD34ls yield . For the results . A RCT rrsus 41% . A retrorsus 41% . Consistrsus 41% , 57. Wel+ cells collected [As for patients with NHL, benefits of CY in HSCs mobilization are uncertain, the two RCTs enrolling participants before the year of 2000 to compare the efficacy of CY plus G-CSF versus G-CSF alone did not reach consistent conclusions , 51. Nowollected . RCTs coollected –77.Otherollected –64, 67. + cells and the rate of successful mobilization in patients with MM. Several retrospective studies suggested that plerixafor plus G-CSF is associated with comparable or lower CD34+ cells yield, but lower risk of neutropenic fever, reduced need of antibiotics use and unscheduled hospitalization compared with cyclophosphamide plus G-CSF SD in patients with MM [. suggested that YF-H-2015005 plus G-CSF can significantly improving mobilization efficacy compared with placebo plus G-CSF in patients with NHL [Moreover, our results demonstrated that G-CSF plus plerixafor or the new CXCR4 antagonist YF-H-2015005 improved mobilization efficiency. Since approved for stem cell mobilization by FDA in 2008, the CXCR4 antagonist plerixafor exhibited favorable mobilization results in patients with NHL and MM , 33. Sup with MM –80. Howe with MM , 81. As  with MM . The effwith NHL . Our reswith NHL . We prevwith NHL . In addi., 15,000 U of rhTPO plus G-CSF and mobilizing chemotherapy led to approximately two-fold increase in CD34+ cells yields and proportions of patients reaching optimal target without increased toxicity compared with the non-TPO group [+ cells resulted from adding these factors are not statistically significant [+ cells yield, increased toxicity and delayed platelet recovery compared with G-CSF [. suggested that EPO in combination with G-CSF and chemotherapy mildly increased CD34+ cells yields and reduced requirement of supportive therapy after transplantation, but the sample size is small [The HSCs mobilization potentials of other hematopoietic growth factors such as TPO, GM-CSF, SCF and EPO are also reviewed in this study. TPO is considered as an attractive agent to be used in combination with chemotherapy plus G-CSF in HSCs mobilization . RecombiPO group . Howevernificant , 45, 60.th G-CSF , 31, 89.th G-CSF , 91. Theth G-CSF , 93. Eviis small . To the is small . Recombiis small , 16. TheAs for the most commonly used HSCs mobilizing agent G-CSF, different forms are available now, including the non-glycosylated G-CSF filgrastim, glycosylated G-CSF lenograstim, pegylated G-CSF pegfilgrastim and G-CSF biosimilars. In this study, we integrated data from the two different forms of G-CSF (filgrastim and lenograstim) together, because RCTs did not show any significant difference between filgrastim and lenograstim in terms of mobilization efficacy in both healthy donors and patients with hematological malignancies , 68, 94.There are several limitations in our study. First of all, as mentioned above, some chemotherapy-based mobilization regimens such as vinorelbine plus G-CSF for MM patients and different salvage therapy regimens plus G-CSF for NHL patients failed to be integrated in our network meta-analysis due to the heterogeneity in study design and the lack of connection to the main network. The HSCs mobilization efficacy of these regimens are carefully reviewed in Table In conclusion, our study summarized the results of 44 RCTs comparing different hematopoietic stem cell mobilization regimens for patients with hematological malignancies and compared the efficacy of mobilization regimens separately for patients with MM and NHL. ID-AraC plus G-CSF is associated with the highest probability of being best mobilization regimen in patients with MM. For patients with NHL, G-CSF in combination with plerixafor or YF-H-2015005 significantly improved HSCs mobilization efficacy, salvage therapy regimen followed by G-CSF is also a widely used mobilization strategy but the optimal salvage therapy regimens for mobilization in different disease subtypes still require to be establish with further research.Additional file 1: Table S1. Detailed information of all mobilizing chemotherapy regimens.Additional file 2: Table S2. The specific dosage of mobilization regimens in all included studies.Additional file 3: Table S3. The characteristics and mobilization results of the 44 trials included in review.Additional file 4: Table S4. Results of risk of bias assessment.Additional file 5: Table S5. Results of meta-analysis regarding the rate of reaching optimal target.Additional file 6: Figure S1. The comparison-adjusted funnel plot. A, G-CSF SD; B, Pegfilgrastim 12 mg; C, Pegfilgrastim 18 mg; D, ID-AraC + G-CSF SD; E, G-CSF SD + Plerixafor SD; F, CY + G-CSF RD; G, G-CSF SD + Plerixafor FD; H, G-CSF SD + YF-H-2015005.Additional file 7: Figure S2. The SUCRA graphs regarding the number of total CD34+ cells collected for patients with MM.Additional file 8: Figure S3. The SUCRA graphs regarding the number of total CD34+ cells collected for patients with NHL.Additional file 9: Figure S4. Network plot depicting all direct comparisons in included trials with data about the successful rate of reaching optimal target for patients with MM (A) and NHL (B).Additional file 10: Figure S5. The SUCRA graphs regarding the successful rate of reaching optimal target for patients with MM.Additional file 11: Figure S6. The SUCRA graphs regarding the successful rate of reaching optimal target for patients with NHL.


Kidney transplantation is the most preferred type of renal displacement therapy for end stage renal disease (ESRD) patients. More patients developed ESRD. The most important source is the donations from unrelated spouses. In this study, we aimed to compare the transplantation data obtained from the spouses of the patients with the transplantation data obtained from other relatives.The data including 167 living kidney transplantations performed between January 2006 and December 2019 were retrospectively collected. The patients were divided into two groups; spousal donor group (n: 53) and living-related donor group (n: 114).There was no significant difference in delayed graft function in both groups. There were no patients with acute rejection proven by biopsy or considered biochemically in the spousal donor group. With regard to 3-year results in the living-related donor group the patient survival rate was 100%, while it was 98.2% in terms of graft survival.In conclusion, similar patient and graft survival rates between spousal donor kidney transplantation and living-related kidney transplantation has made spousal donor kidney transplantation, with possible problems in terms of tissue compatibility, an acceptable alternative to donor supply. The number of patients diagnosed with end-stage renal disease (ESRD) is increasing, in which kidney transplantation is the most common type of renal replacement therapy. As more patients develop end-stage renal disease (ESRD) , the issData related to 169 living kidney transplants performed between January 2006 and December 2019 were collected retrospectively, and the cases were divided into spousal donor transplant (n = 53) and living-related donor transplant (n = 116) groups. The garnered demographic data included age, sex, HLA mismatch, length of preoperative dialysis and body mass index (BMI), while the medical data included post-transplant graft and patient survival, serum creatinine levels, delayed graft function and the presence of biopsy-proven acute rejection. The applied immunosuppressive therapy protocol included the preoperative initiation of corticosteroids and mycophenolate mofetil, and intraoperative basiliximab induction, and the recipients were administered 500 mg of intravenous methylprednisolone before reperfusion. In the cases in which transplants were made from living donors, an additional standard dose of tacrolimus therapy was administered to the recipient in cases of creatinine levels of < 3. The same standard therapy was continued as a maintenance treatment protocol for the patient. The target for tacrolimus was to maintain an FK 506 trough level of 8–10 ng/ml. Prednisolone was initiated at a dose of 100 mg on the postoperative 1st day, and was reduced by 10 mg every day until a dose of 20 mg/day was reached.A written informed consent was obtained from the patients and from healthy participants. The study protocol was approved by the Pamukkale University Ethics Committee. The study was conducted in accordance with the principles of the Declaration of Helsinki.SPSS 22.0  was used for performing istatistical analysis. Analytical characteristics were given as percentage, mean and SD, or median. The chi-square test was used for univariate analysis of categorical variables. Values of p < 0.05 were considered to be statistically significant.Table The advanced immunosuppressive therapy approaches adopted over the last two decades have led to a rapid increase in the success rates of kidney transplantation . UnfortuIn the present study, children who received kidneys from their parents accounted for the majority of transplant patients in the living-related donor transplant group, explaining the lower mean age of the recipients and the higher mean age of donors in this group. Young age is considered a risk factor for a higher incidence of rejection in young people with a stronger immunological structure than in others . AddressThe findings of the present study suggest that the 3-year survival rates of both the patient and graft were quite high in both groups. The most important factor contributing to this result is the stringent decision-making mechanism applied by our center in regards to transplants with marginal criteria, as well as the stable immunosuppressive therapy protocol with basiliximab induction applied, which is uncommon in many centers. Similar patient and graft outcomes among spousal and related allografts have been reported also in Caucasian  and JapaIn conclusion, the similar patient and graft survival rates in the spousal and living-related donor kidney transplant groups suggest that spousal donor kidney transplants, in which tissue compatibility issues may arise, are an acceptable alternative for donor supply.


Chronic traumatic brain injury (TBI) can cause permanent disability and thereby negatively affect patients, families, and society. Currently, there is no effective treatment for patients with chronic TBI. One possible option is granulocyte colony-stimulating factor (G-CSF), which has potential neuroregenerative and neuroprotective effects through its ability to mobilize hematopoietic stem cells and increase neurogenic growth factor levels. Previous studies have shown that G-CSF administration is safe for patients with neurological diseases such as stroke and dementia. The present study aimed to explore the safety and efficacy of G-CSF use in patients with chronic TBI. Methods: 38 patients with chronic TBI were administered 3-day rounds of G-CSF (10 μg/kg per day) once a month for 6 months. These patients were clinically evaluated using the modified Rankin scale (mRS) and Karnofsky Performance Score (KPS). Laboratory measures of the leucocyte counts and differential count percentage were also assessed. Results: At the 6-month follow-up, further assessment showed that patients tolerated the treatment well with only mild and transient side effects being observed. Further clinical evaluation showed significant improvements in mRS and KPS after G-CSF treatment. Laboratory results also confirmed the action of the medication, with increased leukocytosis and band forms. Conclusions: The results suggest that 6-month chronic G-CSF treatment is safe for patients with chronic TBI and may provide clinical benefits and neurological improvements. The adverse effects of the treatment, however, are transient and usually tolerable. Thus, these preliminary findings suggest that future clinical trials of G-CSF use in patients with chronic TBI are warranted. Traumatic brain injury (TBI) is one of the leading causes of mortality and disability worldwide. At the subacute stage, rehabilitation usually plays an important role in improving the neurological disability and functional status of patients with TBI; however, for those with chronic TBI, i.e., having suffered the injury more than 6 months ago, the extent of the functional recovery can be limited even after intense rehabilitation. Indeed, patients with chronic TBI usually suffer from severe cognitive and motor deficits that reduce their ability to perform daily activities and thereby their quality of life. Several pharmacological treatments have been studied with the aim of enhancing recovery during chronic TBI; however, such trials have thus far failed to show significant benefits or amelioration of disability . An alteGranulocyte colony-stimulating factor (G-CSF), a member of the cytokine family of growth factors, mobilizes hematopoietic stem cells CD34+) from the bone marrow into the peripheral blood. G-CSF is used routinely to treat neutropenia and for bone marrow reconstitution. However, the mobilized hematopoietic stem cells have demonstrated that G-CSF might have neuroprotective and regenerative effects . The mec4+ from tThis study was approved by the institutional review board of Tzu Chi General Hospital, Hualien, Taiwan. All methods were carried out in accordance with the relevant guidelines and regulations. A total of 38 chronic TBI patients in the study were interviewed over a period of two years (May 2016–March 2018) and had successfully completed the rounds of G-CSF treatment. We hope this preliminary report helps to explore the safety and potential efficacy of G-CSF for chronic TBI.Our patient inclusion criteria were as follows: (i) they had suffered the TBI event at least 6 months before the study; (ii) they were between 40 and 80 years old; and (iii) their disability symptoms were stable without significant improvement under regular rehabilitation within two months before and after enrolment. Our patient exclusion criteria were as follows: (i) there was contraindication for the administration of G-CSF; (ii) they had unstable underlying medical comorbidities (ex: DM and hypertension); and (iii) their white blood cell count was >75,000/μL after G-CSF administration.G-CSF  is available as a commercial product in the form of a colorless fluid . G-CSF was administered through a subcutaneous injection. The patients received six rounds of three daily G-CSF doses at 10 μg/kg/day. Patients were allowed 3 weeks of rest between these rounds; thus, the six rounds of injections were administered within a 6-month period.The primary outcome measures were the modified Rankin scale and the Karnofsky Performance Score and the secondary outcome measures are the laboratory measures. The modified Rankin scale is a widely used measure to report global disability. The grading ranges from category 0 to 6, with 0 indicating no symptoms and 6 being death . Given it-test was used to compare the clinical measures and laboratory values of patients between and after G-CSF administration, with significance set at p < 0.05.Student’s paired p < 0.05). The patients’ mean KPS also improved significantly from 45 ± 14 pre-G-CSF to 58 ± 14 post-G-CSF . White blood cell counts at baseline were 7316 ± 2426 μL (mean ± SD), but these increased significantly by around 6-fold at day 5 after G-CSF injection to 44,096 ± 14,236 μL. The band forms of leucocytes accounted for 27.4 ± 7.9% at day 5 of G-CSF treatment. A biochemistry study including liver and renal functions were within normal values during and after treatment.The chronic TBI patients’ characteristics are summarized in n = 2), diffuse soreness over four limbs (n = 2), and joint pain (n = 1). Mild dizziness, nausea, and gastrointestinal discomfort were also reported during the treatment period . All symptoms subsided spontaneously or were quickly ameliorated following administration of nonsteroidal anti-inflammatory medications.There were no serious side effects related to the use of G-CSF during the study. Among the observed adverse effects, however, the most common included transient myalgia over the lower back  was used as a repeated chronic (over a 6-months period) treatment for patients with chronic TBI. After the final G-CSF injection round, the KPS of patients improved significantly, which suggests that G-CSF has potential benefits as a treatment for TBI at the chronic stage. Chronic G-CSF administration was also apparently safe as most adverse effects were mild and transient. (Safety)Clinical studies in which G-CSF is used to treat neurological diseases are increasing. In most of these studies, G-CSF was administered as 5-day rounds to treat diseases such as stroke, amyotrophic lateral sclerosis, and dementia ,14,15. TStudies involving a rodent model of TBI have previously revealed the benefit of chronic G-CSF treatment ,19. The G-CSF has shown the ability to promote and maintain endogenous neurotrophic factors, such as BDNF, and thereby improve TBI . This suWhile the benefit of using G-CSF as an acute or salvage treatment method has previously been reported in animal studies , the chrWe acknowledge that our preliminary study has several limitations. First, this is a cohort study with a small case number, which significantly undermines the reliability of the effectiveness of G-CSF. However, we aimed to explore the safety of an old drug, G-CSF, with its off-label use for patients with chronic TBI. Future studies to include more patients and even another control group of patients to confirm the effectiveness is warranted. Second, we did not perform sub-group analysis to identify those patients who will possibly get better improvement from G-CSF. Last, but not least, both the modified Rankin scale and Karnofsky Performance Score are easy to administer but give only a very raw and general picture of the patient status without a specific description of patients’ neurological disability. Future studies should include more specific and comprehensive measures of the motor and cognitive status of the patients in order to clarify the areas that mostly benefitted from the treatment.Although a 6-month period of monthly G-CSF administration has not previously been reported, our study provides preliminary evidence to support the safety of using G-CSF monthly over 6 months for the treatment of chronic TBI. A future study including a randomized and controlled trial of G-CSF will shed further light on the efficacy of chronic G-CSF use for improving neurological outcomes in patients with chronic TBI.


Sci., 2020, 11, 494–498, DOI: 10.1039/C9SC05111B.Correction for ‘Enantioselective synthesis of isochromans and tetrahydroisoquinolines by C–H insertion of donor/donor carbenes’ by Leslie A. Nickerson  The electron pushing arrows in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


We reported unique molecular features of cerebrospinal fluid (CSF) of nonsmall cell lung cancer (NSCLC) patients with leptomeningeal metastasis (LM), suggesting establishing CSF as a better liquid biopsy in clinical practices. We performed next-generation panel sequencing of primary tumor tissue, plasma, and CSF from 131 NSCLC patients with LM and observed high somatic copy number variations (CNV) in CSF of NSCLC patients with LM. The status of EGFR-activating mutations was highly concordant between CSF, plasma, and primary tumors. ALK translocation was detected in 8.3% of tumor tissues but only 2.4% in CSF and 2.7% in plasma. Others such as ROS1 rearrangement, RET fusion, HER2 mutation, NTRK1 fusion, and BRAF V600E mutation were detected in 7.9% of CSF and 11.1% of tumor tissues but only 4% in plasma. Our study has shed light on the unique genomic variations of CSF and demonstrated that CSF might represent better liquid biopsy for NSCLC patients with LM. The incidence of leptomeningeal metastasis (LM) of nonsmall cell lung cancer (NSCLC) is increasing due to improved treatment and ultimately prolonged survival of NSCLC patients , 2. HoweThe individual targeted therapy based on molecular genotyping has increasingly improved the survival of NSCLC –10. EpidOur current study, revealing the genetic alterations of CSF and comparing the genetic difference of CSF with matched plasma and tissue samples, provides stronger evidence of the clinical utility of CSF as an alternative liquid biopsy for NSCLC patients with LM.An overview of the design of the study is shown in via lumbar puncture for cytology examination and next-generation sequencing (NGS), and meanwhile, 10 ml of plasma was collected for NGS. Available primary lung tumor tissues were obtained from the formalin-fixed paraffin-embedded (FFPE) specimen of CT-guided biopsy or biopsy through bronchoscopy. The diagnosis criteria of LM were based on imaging of brain MRI and CSF cytology. MRI findings suggest LM are the pathological enhancement of the leptomeninges of the brain, cranial nerves, and spinal cord. Some patients had no MRI findings, but symptoms indicated LM and CSF cytology confirmed the diagnosis of LM. CSF cytology was reviewed by two independent and experienced pathologists. All patients provided signed informed consent, and the study protocol was approved by the Research Ethics Committee of Nanjing Drum Tower Hospital.One hundred thirty-one NSCLC patients with suspected LM who underwent lumbar puncture between February 2015 and February 2018 in Nanjing Drum Tower Hospital and other medical centers were enrolled in this study. The baseline patients’ characteristics have been described in number (percentage). Seventy-two matched plasma samples and 24 matched tumor tissue samples were also collected in this study. Briefly, approximately 10 ml of CSF was collected According to the manufacturer’s protocol, a minimum of 20% tumor content was required for FFPE specimens, from which genomic DNA was isolated using GeneRead DNA FFPE Kit . Cell-free DNA from fresh CSF and plasma were extracted by QIAamp Circulating Nucleic Acid Kit  according to the manufacture’s instructions. Quantification of genomic DNA from FFPE, CSF, and plasma samples were analyzed using Qubit 3.0 with dsDNA HS Assay .Panel sequencing libraries were prepared using KAPA Hyper Prep Kit  according to the manufacturer’s protocol with modifications for each case . In briehttps://github.com/lh3/bwa/tree/master/bwakit) (https://software.broadinstitute.org/gatk/) (http://adtex.sourceforge.net) with default parameters. Somatic CNVs were retained using paired normal/tumor samples for each exon with the cutoff of 0.65 for copy number loss and 1.50 for copy number gain  . Next, r/bwakit) . Local rg/gatk/) . The somg/gatk/) . Copy-nuber gain .We describe baseline patients characteristics using numbers or percentages. We also describe the genetic alterations in different samples using numbers or percentages. The genetic alterations between different groups using the Chi-square test and all analyses were based on R (V.3.4.4).We screened and enrolled 131 NSCLC patients with LM for analysis. Among them, 24 patients have matched CSF, plasma, and tissue samples for analysis. Seventy-two patients have matched CSF and plasma samples, and 124 patients have CSF analyzed. The study design was as follows Figure 1All of the patients were Chinese and diagnosed with nonsmall cell lung cancer. Most patients were diagnosed with lung adenocarcinoma (127/131). Only four were diagnosed with squamous carcinoma. The median age was 53 years old , and among them, 10 patients were less than 40 years old, 69 were between 40 and 64 years old, and 23 were more than 65 years old. Sixty-seven patients were male and 64 were female. The majority of patients (128/131) were diagnosed with stage IV disease, only two were stage III, and one was stage II before LM diagnosis. All baseline characteristics were listed as following Table 1.p = 0.2339). To investigate whether CSF is a better cell-free DNA (cfDNA) method to detect mutations of the tumor as liquid biopsy, we compared 72 samples of CSF and plasma. We found cfDNA positive in 52 samples of CSF and in 59 samples of plasma, and the detection rate of cfDNA is not statistically different in CSF and plasma as Liquid biopsy is an alternative method to detect mutations for patients if tumor samples are not available. For LM patients, CSF and plasma can be easily obtained. To clarify whether CSF can also be a good method to test the mutations, we compared the CSF and plasma molecular features in 72 samples. Consistent with previous data, EGFR and TP53 were still common both in CSF and plasma, indicating both samples are suitable for mutation testing. However, in CSF, most mutation types were CNV. In plasma, most were missense variants. Except for EGFR and TP53, the most common mutations were MYC, RB1, and NOX2-1 in CSF, but ERBB2, GNAS, and PIK3CA in plasma, as shown in To investigate the molecular mechanism of LM, genetic alterations of the primary tumor, LM-CSF, and LM-plasma were compared with each other. The primary tumors were collected before LM diagnosis. LM-CSF and LM-plasma were collected simultaneously and analyzed with the primary tumor. We investigated 24 paired samples of CSF, plasma, and tumor tissues. We found that unique molecular features of CSF compared with tumor tissues and plasma. IL7R, NOX2-1, and NTRK1 were more frequent in CSF but not that frequent both in tumor tissues and plasma. Consistently however, EGFR, TP53, and RB1 were most frequent in CSF, plasma, and tumor tissues, as shown in Since EGFR and ALK are the most common and critical actionable targets in NSCLC, we also used panel sequencing to detect EGFR and ALK in CSF. As p = 0.005 for EGFR/TP53 and p = 0.0069 for EGFR/TP53/RB1, respectively). Other gene alterations included NTRK1, MET Proto-Oncogene, and ROS1, and we listed the uncommon driver genes, as show in We analyzed the CSF samples of 124 patients with LM. The panel sequencing revealed that the most common mutation that occurred was EGFR, which occurred at 58.4% of the CSF samples. This is consistent with previous data because most of the patients are adenocarcinoma and all of the patients are Asians. Interestingly, TP53 and RB1 were also very common in these samples. TP53 occurred in approximately 50% and RB1 in 20.8% of these samples see Table 3.NSCLC patients with LM have increased significantly as the survival of NSCLC patients is extended. Some studies suggested that liquid biopsy of CSF is more sensitive than liquid biopsy of plasma to detect targetable alterations . In our Targeted therapy has dramatically changed treatment approaches for NSCLC . TargetaExcept for EGFR and ALK, we also found other interesting, unique gene features in CSF samples. NTRK1 copy number variation but not fusions were found in 5.6% of CSF samples. MET and ROS1 have also occurred in CSF samples of NSCLC with LM. We, however, showed that most of the variants are copy number variants, not fusions or rearrangements that have been shown targetable in NSCLC. Therefore, the significance of CNV of these genes has to be explored in the future.Co-occurring genetic alterations have been extensively studied in EGFR-mutant NSCLC . Recent RB1 gene is the first tumor suppressor gene found as the cause of retinoblastoma  than that remains in NSCLC  and poor survival . In our This study has several limitations. First, not all samples are paired with tumor tissues and plasma samples, which cautions the interpretation of these results. Second, the CSF and plasma samples are not large enough. Third, there was no survival time of patients in this study, which means that whether certain gene alterations can impact survival is unclear.CSF samples demonstrate unique molecular features and are concordant with tumor tissues regarding the molecular analysis. Thus, it may represent a better surrogate for NSCLC patients for molecular analysis when no tumor tissues are available.http://www.gov.cn/zhengce/content/2019-06/10/content_5398829.htm, http://english.www.gov.cn/policies/latest_releases/2019/06/10/content_281476708945462.htm), no additional raw data is available at this time. Data of this project can be accessed after an approval application to the China National Genebank . Please refer to https://db.cngb.org/ or email: CNGBdb@cngb.org for detailed application guidance. The submission ID (sub021661) and project accession (CNP0001938) should be included in the application.According to the national legislation/guidelines, specifically the Administrative Regulations of the People’s Republic of China on Human Genetic Resources  2015), Science and Technology Grant (BE2019758), and the Six Talent Peaks Project (TD-SWYY-007) of Jiangsu Province and High-Level Talents Program of Nanjing Medical University; Xisike Clinical Oncology Research Foundation ; and The Medical Interdisciplinary Research Funding of Henan University (No. CJ1205A0240011).Author YS was employed by Nanjing Geneseeq Technology Inc.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. 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