The incidence of human papillomavirus (HPV)-associated cancer is increasing and HPV is now implicated in the aetiology of more than 60% of all oropharyngeal squamous cell carcinomas (OPSCC). In OPSCC, innate immune cells such as neutrophils and macrophages generally correlate with poor prognosis, whilst adaptive immune cells, such as lymphocytes, tend to correlate with improved prognosis. This may, in part, be due to differences in the immune response within the tumour microenvironment leading to the recruitment of specific tumour-associated leukocyte sub-populations. In this study, we aimed to examine if differences exist in the levels of infiltrated leukocyte sub-populations, with particular emphasis on tumour-associated neutrophils (TAN), and to determine the mechanism of chemokine-induced leukocyte recruitment in HPV-positive compared to HPV-negative OPSCC. Immunohistochemical analysis showed that HPV-negative OPSCC contained significantly more neutrophils than HPV-positive tumours, whilst levels of CD68+ macrophages and CD3+ lymphocytes were similar. Using a 3D tissue culture model to represent tumour-stromal interactions, we demonstrated that HPV-negative tumour-stromal co-cultures expressed significantly higher levels of CXCL8, leading to increased neutrophil recruitment compared to their HPV-positive counterparts. HPV-negative OPSCC cells have previously been shown to express higher levels of IL-1 than their HPV-positive counterparts, indicating that this cytokine may be responsible for driving increased chemokine production in the HPV-negative 3D model. Inhibition of IL-1R in the tumour-stromal models using the receptor-specific antagonist, anakinra, dramatically reduced chemokine secretion and significantly impaired neutrophil and monocyte recruitment, suggesting that this tumour-stromal response is mediated by the IL-1/IL-1R axis. Here, we identify a mechanism by which HPV-negative OPSCC may recruit more TAN than HPV-positive OPSCC. Since TAN are associated with poor prognosis in OPSCC, our study identifies potential therapeutic targets aimed at redressing the chemokine imbalance to reduce innate immune cell infiltration with the aim of improving patient outcome. The worldwide increasing incidence of human papillomavirus (HPV)-driven oropharyngeal squamous cell cancer (OPSCC) has raised the profile of these tumours and intensified interest in this area of cancer research . Of partIn many malignant tumours, a pro-inflammatory tumour microenvironment drives the recruitment of tumour-infiltrating leukocytes that in turn have a major effect on tumour progression . The preIt has been known for many years that the increased gene expression and subsequent secretion of chemokines by cells at infected or inflammatory sites is mediated by pro-inflammatory cytokines, such as IL-1, that are present within the local environment . There aThis study utilised patient samples and associated clinical data within the period of primary diagnosis 2002–2012. Patients were selected using the Chemocare database system  and further cross-referenced against the histopathological database held at Sheffield Teaching Hospitals NHS Trust, UK in order to confirm diagnosis and to identify each tissue biopsy reference number. Patients were included in the study if there was sufficient tissue remaining after clinical histopathological diagnosis for TMA generation and if there was a reasonably complete dataset of associated clinicopathological information. All biopsy material was obtained before commencement of anti-cancer treatment. Paraffin-wax embedded tissue samples were retrieved and collated in a blinded fashion with respect to HPV status. HPV positivity using RNAScope® was determined as described in Hendawi et al. , whilst https://qupath.github.io) , embeddin vitro 3D model data. In addition to CXCL8, increased abundance of other neutrophil-specific chemokines such as CXCL1, CXCL5 and CXCL6 is likely, as evidenced by our previous cytokine array analysis [Using these 3D tumour-stromal models we observed that HPV-negative FaDu tumour/NTF co-cultures produced substantially more chemokines than NTF or FaDu MCTS cultured alone, and importantly, much more than UPCI-SCC90 HPV-positive tumour-stromal models. Furthermore, expression of the neutrophil-specific chemokine CXCL8 was produced at significantly higher levels than CCL2 or CCL5 in HPV-negative tumour-stromal models, suggesting that neutrophil-specific chemokines may out-weigh those for monocytes and lymphocytes, potentially skewing leukocyte subset recruitment in these tumours. We were unable to produce reliable and consistent MCTS from other HPV-positive cells lines and so our 3D analysis is limited to FaDu and UPCI-SCC90 cell lines. However, the chemokine profiles detected using these MCTS directly correlated with the profiles observed for other HPV-positive/negative cell lines in our previous 2D study , providianalysis , and thiBlocking IL-1R using the highly specific antagonist anakinra almost completely abolished chemokine production, providing further support to the notion that IL-1/IL-1R paracrine signalling between HPV-negative tumour cells and NTF is of paramount importance for chemokine production. In support of this, increased levels of IL-1β have been found in HPV-negative compared to HPV-positive OPSCC patient samples , 41.Tonsillar  and oralin vitro models to study complex tumour microenvironment interactions with multiple cell types that may negate some of the problems experienced in the differences between human and murine immune systems encountered during in vivo animal experiments and also with 2D culture.Given the high levels of chemokines produced by the paracrine tumour-stromal interactions in HPV-negative OPSCC models it was no surprise that these co-cultures exhibited substantially more overall leukocyte recruitment than HPV-positive 3D models. However, it is remarkable that we observed significantly more neutrophil infiltration into HPV-negative tumour-stromal models than their HPV-positive counterparts, mirroring that observed in human patient tumour samples. Treatment with anakinra significantly reduced the recruitment of both neutrophils and monocytes into both HPV-positive and HPV-negative tumour-stromal models, although the treatment was far more pronounced for HPV-negative tumour-stromal models, once again underscoring the importance of IL-1β in immune cell recruitment to tumours. These data are encouraging and validate the usefulness of human 3D in vitro, it could be argued that this antagonist may be effective for both types of tumour, however, the increased numbers of leukocytes recruited to HPV-negative OPSCC suggests that the treatment would be most effective for this cancer type. Therefore, blockade of the IL-1/IL-1R axis may be a promising adjunct anti-tumour therapy that would particularly affect HPV-negative OPSCC, although there may be inherent side effects in the blocking of this key inflammatory pathway, such as potential changes to inflammatory signalling pathways leading to dysregulated host inflammatory responses to infection or alterations to the activation of important immune cells types such as natural killer and cytotoxic T cells that are crucial in host anti-cancer responses [The use of anakinra to inhibit immune cell infiltration is an interesting concept. Indeed, anakinra was found to reduce circulating levels of CXCL8, tumour growth and the number of infiltrating TAM into mice bearing erlotinib-resistant adenosquamous OSCC  and laryngeal carcinoma (SQ20B) xenografts grown subcutaneously. Overall survival in these mice was increased further when they were treated with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, indicating that this adjunct therapy may be effective at overcoming EGFR inhibitor tumour resistance . Anakinresponses , 64.in vivo and 3D tumour-stromal in vitro data show, for the first time, that HPV-negative OPSCC contain significantly more TAN than HPV-positive OPSCC. Additionally, the mechanism of neutrophil recruitment appears to be via IL-1-mediated CXCL8 release by NTF. HPV infection appears to prevent IL-1 expression by tumour cells thereby suppressing TAN recruitment. Since TAN have been linked with poor prognosis, their apparent reduced recruitment to HPV-positive tumours may partially explain the improved outcome imparted by HPV-infection in OPSCC. Targeting the IL-1/IL-1R axis may be a viable consideration for the treatment of HPV-negative OPSCC.In summary, our human The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by National Research Ethical Committee approval (UK 12/LO/2018). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CM, KH, and RB conceived and designed the research. SA-S, NH, BO, and CM performed experiments, analysed the data, conducted statistical analysis, and interpreted the results. The manuscript was written and figures prepared by CM, SA-S and BO, and further edited by all the authors. PO contributed essential reagents and expert knowledge. All authors are aware of the content and have read and approved the manuscript for publication.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.


Previous cognitive learning interventions have focused primarily on learning one or two novel real-world skills at a time, or utilizing computer-based programs to enhance specific cognitive skills . While these studies yielded immediate cognitive improvements in participants, the long-term benefits of continuing to learn several real-world skills in older adulthood is unclear. In the present two studies, the long-term (1-year post-intervention) benefits of a multi-skill learning intervention were investigated with older adult participants. Study 1 (a pilot sample) included 6 participants  and Study 2 included 27 participants . Following a three month intervention which entailed simultaneously learning at least three real-world skills, such as photography, drawing, and Spanish, participants’ cognitive abilities were assessed using four tasks , as well as RAVLT and Digit Span. Follow-up assessments were completed at three-, six-, and 12-month follow-ups after the interventions. Linear mixed-effects regression models revealed significant cognitive improvements across time points up to one year following the intervention compared to baseline assessments. These promising results support the idea that intense learning experiences may lead to considerable cognitive growth in older adulthood, as they do earlier in the lifespan.


We present a case of bifurcation percutaneous coronary intervention (PCI) of the left main trunk (LMT) using a proximal balloon edge dilation (PBED) technique following a proximal optimizing technique (POT). The procedure of the PBED technique entailed precise positioning of the balloon for SB dilation, with the proximal radiopaque marker lying in the cross-sectional plane of the stent struts at the left circumflex artery (LCx) ostium. The PBED technique might prevent stent deformation induced by side branch (SB) dilation and eliminates the need for the second POT procedure in the re-POT sequence. In fact, three-dimensional reconstruction of optical coherence tomography (3D-OCT) revealed good opening of stent cells overlying the LCx ostium without deformation of stent struts causing incomplete stent apposition at the site opposite the LCx, so the second POT procedure was unnecessary in this case.Learning objective: This is the first case report to describe bifurcation PCI of the LMT using POT-PBED procedures under 3D-OCT guidance. POT-PBED procedures might offer excellent acute results for cross-over single-stent implantation in LMT bifurcation lesions and could eliminate the second POT procedure in the re-POT sequence.>< The default approach in bifurcation percutaneous coronary intervention (PCI) generally involves simple strategies with a single stent or provisional side branch (SB) stenting . We prev2 minimal luminal area of a 23-mm long lesion having a thick cap-fibroatheroma plaque with calcification (2 to 4.4 mm2). OCT demonstrated well-apposed stent struts opposite the LCx  showed total occlusion of the mid-right coronary artery and primary PCI was performed. One month after successful primary PCI, physiological assessment by fractional flow reserve detected residual ischemia at the distal LMT. We thus decided to perform bifurcation PCI  under 3D-OCT guidance. CAG showed 75% stenosis at the distal LMT , A and Bfication , a-e. Prfication . After sfication , a, b anfication , c. LumeAn informative experimental study demonstrated that a re-POT sequence, comprising initial POT, SB inflation, and second POT, significantly improved the final results of cross-over single-stent implantation for bifurcation lesions , and theExperimental comparison data between the POT-PBED procedures and kissing balloon technique that is widely used in real practice are not available. Future experimental investigations are necessary to reveal the advantage of PBED technique against kissing balloon technique.In conclusion, POT-PBED procedures showed excellent acute results for cross-over single-stent implantation in an LMT bifurcation lesion visualized by 3D-OCT.All other authors have nothing to disclose.


In December 2020, the Food and Drug Administration (FDA) issued Emergency Use Authorizations (EUAs) for Pfizer-BioNTech and Moderna COVID-19 vaccines, and in February 2021, FDA issued an EUA for the Janssen (Johnson & Johnson) COVID-19 vaccine. After each EUA, the Advisory Committee on Immunization Practices (ACIP) issued interim recommendations for vaccine use; currently Pfizer-BioNTech is authorized and recommended for persons aged ≥12 years and Moderna and Janssen for persons aged ≥18 years  after Janssen COVID-19 vaccination. GBS is a rare neurologic disorder characterized by acute or subacute onset of weakness in limbs or cranial nerve–innervated muscles and by laboratory findings of increased cerebrospinal fluid protein with normal numbers of cells; the clinical presentation and severity vary  Work Group,On July 22, 2021, ACIP met to review currently available evidence of risks associated with COVID-19 vaccination. The findings from VaST and ACIP COVID-19 Vaccine Work Group assessments, including a summary of the data reviewed, were presented to ACIP during this meeting. ACIP’s comprehensive assessment included risks for GBS and TTS after Janssen COVID-19 vaccination and myocarditis after mRNA COVID-19 vaccination in persons aged ≥18 years. To date, there has been no increased risk detected for GBS or TTS after mRNA COVID-19 vaccination, and there has been no increased risk detected for myocarditis after Janssen COVID-19 vaccination. Persons aged <18 years were not included in this assessment because a benefit-risk assessment for persons aged 12–29 years was recently presented to ACIP in June 2021To assess the benefit-risk balance of COVID-19 vaccination in adults, ACIP reviewed an assessment comparing the benefits of vaccination (numbers of COVID-19 cases and severe disease outcomes prevented) to the risks , using methods similar to those described previously.The risks assessed for the Janssen COVID-19 vaccination were 1) the number of GBS patients reported to VAERS that occurred within 42 days of Janssen COVID-19 vaccination per million doses administered through June 30, 2021, and 2) the number of patients with TTS reported to VAERS that occurred after Janssen COVID-19 vaccination per million doses through July 8, 2021. The risks for mRNA COVID-19 vaccination were assessed as the number of patients reported to VAERS with myocarditis after receipt of dose 2 of an mRNA COVID-19 vaccine per million doses. Each benefit-risk assessment was stratified by age group  and sex. The Janssen COVID-19 vaccine analysis assumed 90% vaccine effectivenessAs of June 30, 2021, approximately 12.6 million doses of Janssen COVID-19 vaccine had been administered in the United States to persons aged ≥18 years. Within VAERS,Through July 8, 2021, 38 cases of TTS within 15 days of vaccination and reported to VAERS met the case definition.As of June 30, 2021, approximately 141 million second mRNA COVID-19 vaccine doses had been administered in the United States to persons aged ≥18 years. Within VAERS, 497 reports of myocarditis after the second mRNA COVID-19 vaccine dose were received for persons aged ≥18 years. The reporting rate of myocarditis overall among adults was 3.5 cases per million second doses of mRNA COVID-19 vaccine administered. In subgroup analyses by age and sex, the reporting rate was highest among males aged 18–29 years (24.3 cases per million mRNA COVID-19 vaccine second doses administered). Reports of cases in persons aged 18–29 years were individually reviewed and confirmed to meet case definitions, whereas reports of cases in persons aged ≥30 years were received and processedThe estimated benefits  outweighed the risks  in all persons aged ≥18 years included in this analysis . For exaACIP also reviewed population-level considerations, including that COVID-19 cases are rising in the United States, particularly with the predominance of the highly transmissible B.1.617.2 (Delta) variant. More than one half (61%) of U.S. adults aged ≥18 years are fully vaccinated  the benefits of vaccinating all recommended age groups with either the Janssen COVID-19 vaccine or mRNA COVID-19 vaccine outweigh the risks for vaccination, including the risks for GBS and TTS after Janssen COVID-19 vaccination, or myocarditis after mRNA COVID-19 vaccination; 2) continuing safety monitoring of serious adverse events after COVID-19 vaccination is critical; and 3) providers and the public should be informed about these potential harms and the use of COVID-19 vaccines. The analysis did not include potential benefits of preventing post–COVID-19 conditions, or likely ongoing benefits beyond the 120-day period; for these reasons, the benefits of COVID-19 vaccination are underestimated.ACIP members discussed concerns about the clinical severity of the rare risk for GBS and TTS. In addition, they noted the importance of providing options for the type of COVID-19 vaccines offered, especially in the context of the current COVID-19 epidemiology and current vaccine coverage in the United States. ACIP emphasized the importance of informing vaccination providers, and all persons receiving COVID-19 vaccines about the benefits and risks, including the risks after Janssen COVID-19 vaccination for GBS, particularly in males aged 50–64 years, and for TTS among females aged 30–49; and the risk for myocarditis after mRNA COVID-19 vaccination, particularly in males aged 18–29 years. CDC has provided guidance regarding evaluation and management of GBS, TTS, and myocarditis.CDC has updated patient education and communication materialsBased on ACIP’s conclusion regarding the benefit-risk assessment on July 22, 2021, vaccination with any of the available COVID-19 vaccines licensed under the FDA EUAs continues to be recommended for all persons aged ≥18 years. With the Delta variant, this is more urgent than ever. In addition, the Pfizer-BioNTech COVID-19 vaccine continues to be recommended for persons aged ≥12 years.CDC and FDA will continue to closely monitor reports of serious adverse events and will present any additional data to ACIP for consideration. The benefit-risk analyses for COVID-19 vaccines can be updated to reflect changes in epidemiology of the COVID-19 pandemic and additional information on the risk for serious adverse events after vaccination. ACIP recommendation for use of all COVID-19 vaccines under an EUA are interim and will be updated as additional information becomes available.https://vaers.hhs.gov/index.html or 1-800-822-7967. In addition, CDC has developed a voluntary smartphone-based online tool (v-safe) that uses text messaging and online surveys to provide near real-time health check-ins after receipt of a COVID-19 vaccine. In cases of v-safe reports that include possible medically attended health events, CDC’s v-safe call center follows up with the vaccine recipient to collect additional information for completion of a VAERS report. Information on v-safe is available at https://www.cdc.gov/vsafe.FDA requires that vaccine providers report to VAERS vaccination administration errors, serious adverse events,Rare serious adverse events have been reported after COVID-19 vaccination, including Guillain-Barré syndrome (GBS) and thrombosis with thrombocytopenia syndrome (TTS) after Janssen COVID-19 vaccination and myocarditis after mRNA (Pfizer-BioNTech and Moderna) COVID-19 vaccination.On July 22, 2021, the Advisory Committee on Immunization Practices reviewed updated benefit-risk analyses after Janssen and mRNA COVID-19 vaccination and concluded that the benefits outweigh the risks for rare serious adverse events after COVID-19 vaccination.Continued COVID-19 vaccination will prevent COVID-19 morbidity and mortality far exceeding GBS, TTS, and myocarditis cases expected. Information about rare adverse events should be disseminated to providers, vaccine recipients, and the public.


Coronavirus disease 2019 (COVID-19) is a global respiratory disease with unique features that have placed all medical professionals in an alarming situation. Preeclampsia is a hypertensive disorder of pregnancy affecting 8%–10% of India’s pregnant population. Assuming that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells through the angiotensin-converting enzyme 2 (ACE2) receptor, the resulting symptoms are due to vasoconstriction, caused by disturbances in the renin–angiotensin system (RAS). Other features of preeclampsia include endothelial dysfunction due to placental ischemia, leading to imbalances in angiogenic and antiangiogenic factors which result in increased blood pressure, proteinuria, altered hepatic enzymes, renal failure, and thrombocytopenia, amongst others. The increased prevalence of preeclampsia that was seen among mothers with SARS-CoV-2 infection might be due to misdiagnosis, as COVID-19 and preeclampsia have coincidental medical features. The major similarities of SARS-CoV-2-infected and preeclamptic women are a rise in pro-inflammatory cytokines, and increased serum ferritin and thrombocytopenia. Therefore, differential diagnosis might be difficult in pregnant women with COVID-19 who present with hypertension and proteinuria, thrombocytopenia, or elevated liver enzymes. The most promising markers for earlier diagnosis of preeclampsia is soluble endoglin (sEng), pregnancy-associated plasma protein-A (PAPP-A), soluble fms-like tyrosine kinase 1 (sFlt-1), and placental growth factor (PlGF). Due to placental hypoxia, sFlt-1 will be overproduced, thus inhibiting PlGF, and this alteration will be observed in the circulation five weeks or more before the onset of symptoms. The sFlt-1/PlGF ratio may also be modified via infectious states, but unregulated levels of those mediators are related to placental insufficiency. Hence, pregnant women with COVID-19 may develop a preeclampsia-like syndrome that might be differentiated properly by angiogenic markers to avoid unnecessary interventions and induced preterm labor. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possibly the most notable infectious virus encountered worldwide. Due to its rapid progression and the absence of precise remedial measures, it has evolved as a global pandemic.4Pregnant women could be more susceptible to SARS-CoV-2 due to the immunological and physiological adaptive remodeling that occurs during pregnancy.11Preeclampsia is a hypertensive disorder of pregnancy that affects nearly 5% of pregnancies worldwide and is one of the leading causes of maternal and fetal morbidity and mortality. It is characterized by new onset of hypertension after 20 weeks of gestation, with or without proteinuria ≥300 mg/24 hours urine collection. The actual cause of preeclampsia remains unknown, and the only curative action is placental removal.14The presence of HELLP syndrome is the classical feature in high-risk cases of preeclampsia.As a placental disease, preeclampsia progresses at two different levels: (1) Abnormal placentation is noted during the early stage of pregnancy; (2) maternal syndrome occurs after 20 weeks’ gestation and is distinguished by the secretion of excess antiangiogenic factors in the blood.25Biomarkers are becoming increasingly significant in the diagnosis of preeclampsia. A survey of the literature reveals a number of biomarkers that have been shown to be sufficiently specific and sensitive to qualify as possible biomarkers. An effective biomarker is one that has the ability to detect preeclamptic women earlier in their disease progression. Much research has also looked into various systems for predicting preeclampsia.Soluble fms-like tyrosine kinase-1 (sFlt-1) is the splice variant of vascular endothelial growth factor (VEGF) receptor-1, which is secreted in response to placental hypoxia; once sFlt-1 binds to VEGF in the extracellular domain, VEGF becomes soluble in plasma. Because sFlt-1 has the VEGF binding site, it can bind all isoforms of the growth factor, as well as proangiogenic protein PlGF.22in vitro by interfering with transforming growth factor 1 (TGF1) binding to its receptor.Soluble endoglin is a putative antiangiogenic factor that influences the production of nitric oxide (NO), vasodilation, and capillary formation by endothelial cells in vivo, and it can also block capillary angiogenesis in vitro. Serum sEng may rise during the last two months of pregnancy in a normal pregnancy, but it rapidly increases in preeclampsia, surging at onset, and it may be linked to disease severity. Further-more, sEng may work in concert with sFlt-1 in the etiology of preeclampsia. As a result, it is believed that elevated sEng levels in the blood and a higher sFlt-1:PlGF ratio can predict the likelihood of preeclampsia,Soluble endoglin can produce high blood pressure by affecting the permeability of blood arteries Pregnancy-associated plasma protein-A (PAPP-A) is a glycoprotein produced primarily in placental trophoblast cells. Its presence could indicate placental ischemia or hypoxia. Essential for a normal pregnancy, PAPP-A can alter placental trophoblast cell infiltration by regulating the action of insulin-like growth factors. Placental PAPP-A is often used in aneuploidy screening in early pregnancy, and it can be used to detect intrauterine growth retardation (IUGR), whereas it cannot detect preeclampsia.29Although pregnancy is a “healthy” physiological process, the fetus may pose a risk to maternal tolerance and the immune system. A healthy pregnancy necessitates maternal immune system tolerance of the semi-allogenic fetus, and the fetus must be safeguarded against rejection by the maternal immunological response.282O2 and O2•−, resulting in greater endothelial dysfunction as compared to monocytes produced from normotensive pregnant women.13Reactive oxygen species (ROS) are oxygen-containing highly reactive compounds produced during aerobic cell metabolism. During pregnancy there is an improved production of ROS, but it is commonly counterbalanced by antioxidants. In preeclampsia, faulty trophoblast invasion and diminished uteroplacental blood flow lead to intervals of ischemia/reperfusion and the hypoxic surroundings that favor oxidative stress, inflammation, and vascular dysfunction. The antioxidant levels during preeclampsia can be too low to counterbalance the increased ROS. Neutrophils and monocytes are key sources of ROS in preeclampsia. Monocytes from preeclamptic women increase HThe incidence of preeclampsia is 3%–7% in nulliparous pregnancies. One of the leading causes of maternal morbidity and mortality,15Structural analyses have shown that SARS-CoV-2 shares 79% of the sequence found in severe acute respiratory syndrome coronavirus (SARS-CoV) and that they may both have the same host receptor, ACE2.38Placentation is a complicated process that involves fetal cells, alterations in maternal uterine circulation, and immunomodulation. Most RAS components, as well as prostaglandins and nitric oxide, have been identified in the human uteroplacental unit, implying that these systems are complexly regulated throughout placental development.40The SARS-CoV-2 virus enters host cells through the membrane-bound receptor of ACE2 and CD147/basigin (BSG). Following receptor binding, the viral-encoded S protein relies on host proteases for cleavage to attain effective membrane fusion. The primary host protease that mediates S protein priming and initiates viral access is the type II transmembrane serine protease, TMPRSS2.41In normal pregnancy the concentration of plasma Ang-(1-7) increases, whereas the ACE concentration decreases; this is reversed in women with preeclampsia. An increased ACE concentration was also reported in women who delivered small-for-gestational-age babies. As placental cells express increased concentrations of ACE2, TMPRSS2 provides an increased affinity to the spike protein.41Studies that examined maternal deaths in SARS-CoV-2-positive antenatal women showed increased AST, ALT,The major similarities between SARS-CoV-2-infected antenatal women and preeclamptic women are increased pro-inflammatory cytokines and serum ferritin, and thrombocytopenia .6 In typ46The differential diagnosis of preeclampsia during pregnancy from SARS-CoV-2-positive pregnant women is via assessment of the placental vascular development through VEGF, PlGF, and antiangiogenic factor sFlt-1. Dysregulation of this balance can also happen due to inflammatory mediators, including cytokines  and the complement cascade pathway. The sFlt-1/PlGF ratio may also be modified as the result of infectious states, but unregulated levels of those mediators are related to placental insufficiency, placental hypoxia, and poor nutrient transport to the fetus, which lead to poor neonatal outcome.444) intravenously. Due to elevated blood pressure and hepatic enzymes she underwent an uncomplicated cesarean delivery.9Mendoza et al. conducted a study on 42 SARS-CoV-2-positive pregnant women; five of the subjects developed clinical and laboratory symptoms similar to preeclampsia. Of the five, only one was confirmed to actually have preeclampsia, presenting with sFlt-1 and PlGF, elevated platelet distribution, and increased lactate dehydrogenase (LDH). The remaining four pregnant women recovered after starting antibiotic therapy and the preeclamptic symptoms were eradicated, whereas in true preeclampsia delivery is the only curative treatment.The impact of COVID-19 on pregnancy has recently received much attention. However, some data are contradictory and methodological flaws have been noted, such as testing for COVID-19 only in symptomatic pregnancies, a lack of control for confounding factors , limited comparisons with non-pregnant women, and an under-representation of COVID-19-positive pregnancies in early pregnancy.6Decreased ACE2 protein has also been reported in placentas from COVID-19-positive pregnancies, indicating that SARS-CoV-2 infection may alter ACE2 expression and biological functions in the placenta, as well as in the maternal and fetal circulations, either directly or indirectly. Vertical transmission may occur through intrauterine, intrapartum, and postpartum mechanisms. Thankfully, the majority of viral illnesses affecting mothers are not vertically transmissible.50The following are the most common features noted in SARS-CoV-2-infected pregnant womenThe most commonly reported symptoms are fever (34%–84%), cough (28%–71%), dyspnea (18%–40%), and myalgia (16%–38%).Infected pregnant women were less prone to have fever, myalgia, or dyspnea than infected non-pregnant women. However, in infected pregnant women, fever and shortness of breath of any duration were linked to an elevated risk of severe maternal–fetal abnormalities.COVID-19 was found to have a modest maternal mortality rate (1%). However, when compared to uninfected pregnant women, the risk of maternal death increased in the presence of SARS-CoV-2 infection.Odds ratios (OR) of preeclampsia  and premature birth  were significantly greater in SARS-CoV-2-positive than-negative pregnancies.Mortality rates differ between countries, with Brazil having a mortality rate during May 2020 of 36/100,000 population among pregnant women with COVID-19 and 252/100,000 population of SARS-infected pregnant women. This may be explained by the high prevalence of hypertensive illnesses, particularly preeclampsia and obesity in pregnant women, as well as socioeconomic concerns and healthcare system flaws.Pregnancy complication: preterm birth (9%–39%). Having COVID-19 enhanced the chance of both medically recommended and spontaneous preterm birth. Even though the majority of preterm births were medically guided, around 6% were spontaneous.Fetal problems were observed in 0.5% of pregnant women with COVID-19, which included admission to a neonatal intensive care unit (15%), IUGR (9%), fetal distress (8.8%), miscarriage (2%), stillbirth (0.7%), and newborn mortality (0.7%). Indeed, recent meta-analyses evaluating similar outcomes in pregnancies with and without COVID-19 found that the disease increased the chance of newborn intensive care unit admission, stillbirth, and low birth weight.The characteristics of SARS-CoV-2-infected pregnant women and preeclamptic women seem to be more or less similar with regard to proteinuria, elevated liver enzymes, thrombocytopenia, and increased pro-inflammatory markers. Hence, diagnosis of preeclampsia could be quite difficult. In view of the above scenario, verification of VEGF, PlGF, sFlt-1, and sFlt-1/PlGF, along with other biochemical markers, plays a crucial role in the differential diagnosis of preeclampsia, and would help to avoid unnecessary interventions and induced preterm labor among SARS-CoV-2-positive pregnant women.Based on current knowledge, placentas are more vulnerable, placing the fetus at higher risk of SARS-CoV-2 infection due to vertical transmission. Placental ACE2, which is known to be down-regulated in SARS-CoV-2 infection, may contribute to changes in important physiological processes during placental development and vascularization. This is confirmed by the fact that SARS-CoV-2 infection during pregnancy can cause a preeclampsia-like condition. The gestational age of exposure, on the other hand, may play a role in influencing placental vascular responses or consequences. Although some placentas lack co-localization between ACE2 and the transmembrane serine protease 2 (TMPRSS2)—a protease that is equally required for SARS-CoV-2 entry and cell proliferation—vertical transmission is still a topic of controversy.


Small cell lung cancer (SCLC) transformation is a rare resistance mechanism to anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs), for which cytotoxic chemotherapy is often initiated. However, no case has been reported so far in which the SCLC component disappeared after chemotherapy and the tumor responded to ALK-TKI treatment again. A 41-year-old, never-smoker man was diagnosed with multiple metastatic lung adenocarcinoma harboring ALK gene rearrangements. After tumor re-growth was treated with alectinib, histological analysis of re-biopsy of the primary lesion showed combined small cell carcinoma, and cytotoxic chemotherapy was administered. After resistance to chemotherapy developed, the third biopsy of the primary lesion showed the original ALK gene rearrangements without the SCLC component. Alectinib was re-administered, and partial response was obtained. Biopsy for ALK-positive lung cancer that progressed after chemotherapy for SCLC transformation might be useful for decision-making regarding the therapeutic strategy. ALKanaplastic lymphoma kinaseCEAcarcinoembryonic antigenEGFRepidermal growth factor receptorFISHin situ hybridizationfluorescence IHCimmunohistochemistryNSCLCnon-small cell lung cancerSCLCsmall cell lung cancerSLXsialyl-Lewis X-i antigenTKItyrosine kinase inhibitor1Anaplastic lymphoma kinase (ALK) gene rearrangements are present in 3–5% of patients with non-small cell lung cancer (NSCLC). In the past decade, various tyrosine kinase inhibitors (TKIs) showed a dramatic and durable clinical benefit against ALK-positive NSCLC. Nevertheless, drug resistance and recurrent disease still develop in the vast majority of initial responders. The resistance mechanisms in patients with ALK-positive NSCLC comprised ALK gene alterations, such as ALK point mutations and copy-number gains, bypass signaling activation through the activation of other oncogenes, and small cell lung cancer (SCLC) transformation ,2. AlthoA case of adenocarcinoma harboring ALK gene rearrangements that transformed to SCLC following alectinib treatment is reported. After resistance to cytotoxic chemotherapy developed, the third biopsy of the primary lesion showed the original ALK gene rearrangements without the SCLC component, and alectinib re-challenge showed partial response. This case report shows the importance of repeated biopsy for decision-making regarding therapeutic strategies in ALK-positive lung cancer with SCLC transformation.2In March 2010, a 41-year old man with no history of smoking underwent a medical examination for epigastralgia. The echocardiogram showed a massive pericardial effusion, and an emergent pericardial puncture was performed. Multiple metastatic lung cancer (cT4N3M1c) was diagnosed by computed tomography and positron emission tomography . Cytologin situ hybridization (FISH) showed adenocarcinoma with ALK rearrangement. The drug regimen was changed to 300 mg alectinib twice daily, which is the approved dosage in Japan. He eventually achieved a partial response. After 4 years of alectinib treatment, no metastases appeared, but the primary lesion progressed  and fluorescence ogressed A.Fig. 2CHistological analysis based on re-biopsy showed combined small cell carcinoma, in which the SCLC components were CD56 (+), synaptophysin (+), TTF-1 (−), and ALK-1 (−), and the adenocarcinoma components were CD56 (−), synaptophysin (−), TTF-1 (+), and ALK-1 (+) A. Two reThe third biopsy of the primary lesion showed only adenocarcinoma, with CD56 (−), synaptophysin (−), TTF-1 (+), and ALK-1 (+), and disappearance of the SCLC component B. Alecti3Several cases of EGFR-mutant adenocarcinoma that transformed to SCLC and responded to TKIs again after development of resistance to chemotherapy have been reported ,5. To thRB1 and Tp53, plays an important role in transformation to SCLC during treatment with ALK-TKIs or EGFR-TKIs [Some reports have shown that the loss of tumor suppressor genes, such as GFR-TKIs . In the Serum pro-gastrin-releasing peptide precursor (ProGRP) and neuron-specific enolase (NSE) have been reported to be useful predictive markers for SCLC transformation during the development of resistance to ALK-TKIs . In the In conclusion, the present case is the first to show that re-challenge with ALK-TKIs is effective after chemotherapy failure for SCLC transformation. Repeat biopsy at progression might be useful for decision-making regarding the therapeutic strategy for ALK-positive lung cancer with SCLC transformation.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.AY drafted the manuscript. TY reviewed the manuscript. AY, TY, YF, and TY were responsible for final approval of the version to be submitted.None.


In the middle of the COVID-19 pandemic, students at the University of California, Irvine, reimagined their peer-led, small-group, tutorial sessions into an online format. The virtual sessions improved student-reported understanding of physiological principles and reduced exam anxiety. Peer-led review remains a valuable resource in the era of virtual medical education. The COVID-19 pandemic disrupted many aspects of pre-clerkship medical education at the University of California Irvine, School of Medicine (UCISOM), including peer-led exam review sessions, a form of peer-assisted learning (PAL), for first-year medical students (MS1s). PAL has broad benefits in undergraduate medical curricula . With thIn the fall of 2020, MS1s at UCISOM attended virtual, peer-led and faculty-led review sessions to prepare for upcoming physiology block examinations. Peer-led sessions were taught by six MS2 tutors via a rotating Zoom™ breakout room format. MS1 students were divided into six breakout rooms while MS2 peer tutors rotated between rooms and reviewed faculty-approved exam-relevant physiology topics. Tutors spent 20 min in each breakout room and devoted a third of that time to answering MS1 questions. Faculty-led review sessions consisted of content review and practice questions in a large-group Zoom™ format.To explore whether the previously demonstrated benefits of PAL persist when transformed into a digital platform, we surveyed MS1 perceptions of the online peer-led review session compared to the faculty-led sessions. The survey was modified from the published PAL questionnaire , consistOverall, our findings suggest that peer-led review sessions remain an acceptable alternative to faculty-led review sessions in the era of virtual learning, with students preferring peer-led review in various categories. This is consistent with the theory of cognitive congruence as it applies to PAL, which speculates that narrowing the knowledge gap between student and educator enhances learning. Factors such as usage of similar language between educators and students, as well as educators’ understanding of which concepts are important or difficult for learners to grasp, contribute to better educational outcomes , 4. Our In our previous study, we had inferred that student preference for peer-led review may in part be due to the utilization of a small-group format as it facilitates more direct interaction with the tutors . Interes


Breast cancer rises as the most commonly diagnosed cancer in 2020. Among women, breast cancer ranks first in both cancer incidence rate and mortality. Treatment resistance developed from the current clinical therapies limits the efficacy of therapeutic outcomes, thus new treatment approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy developed from adoptive T cell transfer, which typically uses patients' own immune cells to combat cancer. CAR-T cells are armed with specific antibodies to recognize antigens in self-tumor cells thus eliciting cytotoxic effects. In recent years, CAR-T cell therapy has achieved remarkable successes in treating hematologic malignancies; however, the therapeutic effects in solid tumors are not up to expectations including breast cancer. This review aims to discuss the development of CAR-T cell therapy in breast cancer from preclinical studies to ongoing clinical trials. Specifically, we summarize tumor-associated antigens in breast cancer, ongoing clinical trials, obstacles interfering with the therapeutic effects of CAR-T cell therapy, and discuss potential strategies to improve treatment efficacy. Overall, we hope our review provides a landscape view of recent progress for CAR-T cell therapy in breast cancer and ignites interest for further research directions. For metastatic breast cancer, local therapy approaches along with systemic therapy approaches are typically used to reach the main therapy goals of symptom alleviation and prolonging lifeThe global cancer statistics in 2020 shows breast cancer has overridden lung cancer and become the most commonly diagnosed cancer worldwide, with 2.3 million new cases in 11.7% of all cancer types reportedex vivo to express synthetic receptors that recognize tumor-associated antigens (TAAs). Afterwards, CAR-T cells are cultured ex vivo for amplification and then infused back to patients as an anti-cancer treatmentChimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy developed from adoptive T cell transfer (ACT)In 2017, two CAR-T cell therapies targeting CD19 named tisagenlecleucel  and axicabtagene ciloleucel  were approved by FDA for treating children and young adults with relapsed or refractory acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphomas (NHLs), respectivelyIn this review, we focus on CAR-T cell therapy in breast cancers. We first summarize tumor antigens in breast cancer and their clinical relevance, followed by the development of antigen-specific CAR-T cells and their applications from basic research to clinical trials. In addition, we also discuss the challenges of CAR-T cell therapy in solid tumors, the efforts developed to overcome these changelings, and the strengths and weaknesses of those approaches. We hope our review will provide a timely update on CAR-T cell therapy in breast cancer and insights to further improve therapeutic efficacy.Tumor antigens are divided into three classifications based on the expression pattern, namely tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), and cancer germline antigens (CGAs)Receptor tyrosine kinases (RTKs) modulate the crucial cell activities such as proliferation, differentiation, metabolism, and survival after activation by growth factors or hormonesHER2 gene amplification or HER2 overexpression, correlated with poor clinical outcomes, poor prognosis, and disease progressionHER2 gene also contribute to breast cancer development through activating HER2 signalingHuman epidermal growth factor receptor 2 (HER2), also known as ERBB2, belongs to the HER/ERBB family of the receptor tyrosine-protein kinase (RTK) familyHER2+ cancers demonstrated significant tumor growth inhibitionThe monoclonal antibodies targeting HER2, especially trastuzumab, the first targeted therapy approved by FDA for breast cancer, improved the clinical outcome of breast cancer patients in recent yearsEpidermal growth factor receptor (EGFR) is also known as ERBB1 or HER1, belonging to the ERBB family. Once activated by ligand binding, it triggers the same downstream signaling pathways as HER2Hepatocyte growth factor receptor (HGFR), also named cMET, belongs to the RTK family and is encoded by a proto-oncogene METReceptor tyrosine kinase-like orphan receptor 1(ROR1) also belongs to the RTK family. Highest expression of ROR1 is observed in embryogenesis with reduced expression during fetal development, and eventually disappears in terminally differentiated tissues in vitro resulted in increased cytokine release and direct cancer cell lysis compared with AXL-negative cancer cells (such as MCF-7). The same results were observed in MDA-MB-231 derived xenograft mouse models as tumor growth was inhibited by CAR-T therapyin vitroin vitro and reduced tumor size in MDA-MB-231-derived xenograft mouse modelsAXL is a member of the TAM family of RTKsCell surface proteins are presented on the surface of tumor cells, which acts as tumor antigens for CAR-T cell recognition and aids the antitumor effects of T cells. There are 11 surface proteins with aberrantly increased expression in breast cancer that might be suitable for CAR-T cell therapy.Mucin 1 (MUC1), a transmembrane glycoprotein, is usually expressed on the apical side of epithelial cells, secreting mucus to create a chemical barrier to protect host cells from pathogen infectionin vitro and inhibited tumor growth in 9 TNBC cell lines derived xenograft mouse modelsIn TNBC preclinical studies, the second generation of tMUC1-CAR-T cells significantly increased the release of cytokines and chemokines MSLN gene does not interfere with the development, reproduction, and growth in mouse modelsMesothelin (MSLN) is a cell surface protein and is normally expressed on the surface of mesothelial cells in a few tissuesin vitro. Moreover, MSLN-CAR-T cell therapy inhibited tumor growth and metastasis in MDA-MB-231 xenograft modelsThe CAR-T cells targeting MSLN in breast cancers mainly focus on TNBC. The third generation of MSLN-CAR-T cells triggered immune responses by releasing cytokines, exhibiting potent cytotoxicity in both MCF-7-Luc and MDA-MB-231-Luc breast cancer cells CD70 is a costimulatory factor encoded by tumor necrosis factor ligand superfamily member 7 (TNFLS7) and presented on the surface of activated T cells, B cells, and dendritic cellsCD133, also named prominin 1, is a pentaspan transmembrane glycoprotein normally expressed on the protrusions of the plasma membraneCD44 containing variant exon v6 (CD44v6) is the major variant of CD44, which is a cell surface glycoprotein involved in proliferation, motility, and survivalEpithelial cell adhesion molecule (EpCAM) is a glycosylated cell surface protein overexpressed in various epithelial carcinomas, including breast cancerin vitro and in vivoIndeed, extensive studies demonstrated the great effects of EpCAM targeted strategies in solid tumor treatments. The antibody-derived EpCAM-targeted methods manifested antitumor effects. For example, adecatumumab targeting EpCAM inhibited breast cancer metastasis in a dose and target-dependent manner, thus catumaxomab targeting EpCAM has already been approved for the treatment of cancersin vitroChondroitin sulfate proteoglycan 4 (CSPG4) is a transmembrane protein overexpressed in several tumors, including breast cancer in vivo studies using TNBC cell-derived mouse models, ICAM1-CAR-T cells significantly inhibited tumor growth and resulted in prolonged overall survival and long-term remissionIntercellular adhesion molecule-1 (ICAM1) is a surface glycoprotein that belongs to the immunoglobin superfamily. As a molecular adhesion receptor, ICAM1 plays a role in regulating cell activities including signaling transduction, cell adhesion, and migrationTEM8 impaired tumor growth and metastasisin vitro and inhibition of tumor initiation in MDA-MB-468 xenograftsTumor endothelial marker 8 (TEM8), also known as an anthrax toxin receptor 1(ANTXR1), is a group of cell surface glycoproteins. It was identified in terms of its overexpression on epithelial cells of the tumor vasculature and its role in tumor angiogenesisin vitro and in vivo by inhibiting signaling molecules involved in cell survivalTrophoblast cell surface protein 2 (TROP2) overexpression has been detected in breast cancers, especially TNBCs (approximately 90%)Folate receptor alpha (FRα) is a cell surface protein involved in the biosynthesis of nucleotide bases, amino acids, and methylated compounds, playing an important role in cancer developmentin vivoin vivo and in vitro studies+ breast cancer cells and lysed them in vitro, while T cells successfully expanded and trafficked to tumor sites or metastatic tissues to arrest tumor growth and metastasis in MDA-MB-231 derived orthotopic xenograft mouse modelsDisialoganglioside (GD2) is a b-series acidic glycosphingolipid containing two sialic acid residues and facilitates the interaction between tumor cells and extracellular matrix proteinsin vitro study using miRNA silencing NKG2D ligand showed that decreased NKG2D ligand improved the NK cell-mediated cytotoxicity and avoided immune escape of breast cancer cellsin vivo and stronger antitumor effects in TNBC cell cultures and mouse modelsNatural killer (NK) cells are lymphocytes in the innate immune system, eliminating the target cells and secreting cytokines to establish an adaptive immune responseSerum tumor markers are a group of proteins that plays an essential role in the early detection of tumor recurrence, metastasis, and screening of many malignanciesin silico analysis compared gene expression patterns to identify potential targets for breast cancer immunotherapy, resulting in 36 potentially tumor-surface antigens being discovered, including integrin beta-6 (ITGB6), fibroblast growth factor receptor-4 (FGFR4), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1)The antigen selection is one of the determining factors of CAR-T cell therapy. In addition to the targets discussed above, other targets have also been explored as potential antigens for CAR-T cell therapy. A Nineteen antigens targeted by CAR-T cells in breast cancer have been well-studied in preclinical studies, resulting in 12 specific antigens progressing into clinical studies for safety and efficacy tests. 22 CAR-constructed T cells targeting 12 antigens have been investigated in recent years . In 2020, Shanghai PerHum Therapeutics conducted an open-label, single-arm, and dose-escalation phase I clinical study to evaluate the safety, tolerability, and major therapeutic outcomes of CAR-modified autologous T cells in HER2 positive solid tumors including breast cancer (NCT04511871). Besides, there are 2 cMET-CAR-T cell therapy clinical trials granted by the University of Pennsylvania. One was terminated according to funding issues (NCT03060356), while the other one has been completed (NCT01837602) with satisfying clinical results. The results showed that breast cancer patients were well-tolerated to intratumorally injection of cMET-CAR-T cells and none of the patients developed drug-related adverse effects greater than grade 1. In addition, cMET-CAR-T cells elicited antitumor effects by triggering the release of the inflammatory cytokines within the tumorAlthough 12 cell surface proteins have been well-studied in preclinical research, only 6 of them progressed into further clinical studies. 3 CAR-T cell therapy clinical trials for mesothelin are currently under investigations in phase-I to test the safety issues . In contrast, the rest CARs are undergoing phases-I and II clinical trials to test the safety and efficacy (NCT02414269). Besides, 3 CAR-T cell therapy targeting MUC1 were differently constructed in terms of targets, namely the extra domain of the cleaved form of MUC1 (NCT04020575), the aberrantly glycated MUC1 (NCT04025216), and the whole MUC1 (NCT02587689). Moreover, Shenzhen Geno-Immune Medical Institute starts to recruit individuals for a multicenter phase I/II clinical trial to test the safety and efficacy of CD44v6-specific CAR-engineered T cells in CD44v6 positive cancers (NCT04427449). The 3rd generation of EpCAM-CAR-T cells is ongoing a phase I clinical trial for safety and efficacy determination (NCT02915445). Additionally, a phase I/II clinical trial of CD133-CAR-T cell therapy for investigating safety and T cell duration in patients with relapsed and/or chemotherapy-refractory advanced malignancies was completed recently (NCT02541370) but the results were unrevealed. Apart from that, there was a phase I/II clinical trial of CD70-CAR-T cell therapy for breast cancer patients suspended without reason reported (NCT02830724).Unlike RKT and cell surface proteins, only a few other targets have entered the stage of clinical trials. 2 CEA-specific CAR-T cell therapies are in phase I clinical trials  and 1 in phase I and II clinical trials (NCT04348643). Besides, 2 phase I clinical trials of CAR-T cell therapy targeting NKG2D (NCT04107142) and GD2 (NCT03635632) are ongoing to investigate the safety, efficacy, and tolerability of those treatment strategies.To sum up, although various CAR-T cell therapy clinical trials have been conducted in recent years, only a few results of those clinical trials are public. To better understand the safety and efficacy of CAR-T cell therapy in breast cancers, the results of those ongoing clinical trials are important, and more clinical trials of CAR-T cells targeting different antigens are warranted.Although CAR-T cell therapies showed some promising results in preclinical studies and in clinical trials for treating breast cancers, challenges remain to restrict the clinical applications and limit therapeutic outcomes. In general, these challenges include insufficient recruiting infiltration of CAR-T cells into tumors, immunosuppressive microenvironment in breast tumors, tumor heterogenicity, and the on-target/off-tumor side effects for CAR-T cellsCAR-T cells must successfully infiltrate into solid tumors such as breast cancer to specifically target tumor cells, which largely depends on the specific binding between chemokine receptors on the surface of CAR-T cells and the chemokines presenting on tumor cells or tumor microenvironment. The chemokines secreted by cancer cells are varied among tumors; hence it is crucial to identify unique chemokine(s) to guide T cells to recognize a given tumorIn addition, local administration increases the number of CAR-T cells in tumor sites and is suitable for various cancersThe inhibitory tumor cytokines especially transforming growth factor β (TGFβ) and immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) accumulate together in tumor sites contributing to the immunosuppression of the tumor microenvironmentTumor heterogeneity means antigen expression variability on the tumor cell surface in terms of type and level, contributing to another layer of challenge for CAR-T cell therapy. Nevertheless, the successful development of multitarget CAR-T cells has partially tackled this obstacle, showing improved antitumor activities in preclinical studies. One of them has moved forward into clinical trials in breast cancer treatment (NCT04430595). In breast cancer, bispecific CAR-T cells targeting HER2 and MUC1 were successfully constructed and exhibited cytotoxic activitiesToxicity is one of the major challenges limiting the application of CAR-T cell therapy. The common toxicities are classified into six types, namely on-target on-tumor toxicity, on-target/off-tumor toxicity, off-target toxicity, neurotoxicity, genotoxicity, and immunogenicityBreast cancer is currently the most commonly diagnosed cancer around the world, ranking first in both incidence and mortality in womenThe present review discusses the development of CAR-T cell therapy from basic research to clinical trials in breast cancer. We reviewed 19 antigens targeted by CAR-T cells in breast cancer, namely HER2, EGFR, HGFR/cMET, ROR1, AXL, MUC1, MSLN, CD70, CD133, CD44v6, EpCAM, CSGP4, ICAM1, TEM8, TROP2, FRα, GD2, NKG2D, CEA, most of which belong to RTK family and cell surface proteins (Table CAR-T cell therapy in breast cancer gains a big achievement till now; however, due to a lack of supporting evidence, it is still a long way from being applied to breast cancer patients. Therefore, to throw CAR-T cell therapy into clinical as early as possible, more clinical trials are needed, and more in-depth investigations are required to improve the safety issues and overcome the challenges of CAR-T cell therapy.


Triple-negative breast cancer (TNBC) has poor prognosis with limited treatment options, with little therapeutic progress made during the past several decades. DNA damage response (DDR) associated therapies, including radiation and inhibitors of DDR, demonstrate potential efficacy against TNBC, especially under the guidance of genomic subtype-directed treatment. The tumor immune microenvironment also contributes greatly to TNBC malignancy and response to conventional and targeted therapies. Immunotherapy represents a developing trend in targeted therapies directed against TNBC and strategies combining immunotherapy and modulators of the DDR pathways are being pursued. There is increasing understanding of the potential interplay between DDR pathways and immune-associated signaling. As such, the question of how we treat TNBC regarding novel immuno-molecular strategies is continually evolving. In this review, we explore the current and upcoming treatment options of TNBC in the context of DNA repair mechanisms and immune-based therapies, with a focus on implications of recent genomic analyses and clinical trial findings. Triple-negative breast cancer (TNBC) is defined by the absence of estrogen and progesterone receptors (ER and PR) and human epidermal growth factor receptor 2 (HER2). This aggressive variant, which accounts for 15-20% of all breast cancers (BC), exhibits a high propensity for early recurrence and metastasis .Understanding of specific heterogeneity in TNBC has served as the basis for certain targeted therapies based on particular molecular subtypes previously identified through genomic and transcriptomic profiling , 4: The Pervasive therapeutic resistance in TNBC is another significant challenge, contributing to higher recurrence rates and decreased survival as compared to other BC subtypes . TherapeNevertheless, based on the frequency of DDR deficiency in TNBC, investigation of novel strategies targeting DNA repair defects have generated hope for improved outcomes. PARPi’s aimed at DDR-deficiency in TNBC have been approved for patients with metastatic HER2-negative BC with an inherited BRCA1 or BRCA2 mutation previously treated with chemotherapy (NCT02000622 using Olaparib), and those with deleterious or suspected deleterious germline BRCA-mutated HER2-negative, locally advanced, or metastatic BC . However, these have restricted application and demonstrate modest albeit intriguing clinical benefit at present –16. A reAnother promising therapy for TNBC exploits the immune system. Given the immunogenic characteristics of TNBC, immunotherapy represents a promising treatment strategy for this aggressive breast cancer with few efficacious systemic options at present. The most successful immunotherapeutic agents to date consist of immune checkpoint inhibitors (ICIs), which block immune co-inhibitory receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), or associated ligands such as programmed cell death ligand 1 (PD-L1), to dis-inhibit TILs and permit tumor-specific cytotoxicity. However, highly immunosuppressive tumor microenvironment (TME) competes with ICI-enhanced anti-tumor immunity and significantly contribute to inconsistent clinical responses. Immunotherapies, particularly combination strategies, represent a refined approach to treating cancers with immune modulating DDR defects, high tumor mutational burden (TMB), and intact anti-tumor immunity, which are all characteristics frequently observed in TNBC. Tumors with intact interferon-gamma (IFN-γ) pathway signaling, robust TILs, increased immune co-inhibitory receptor expression, and high TMB/neoantigen expression have been shown to respond better to immune checkpoint inhibition than weakly immunogenic tumors with inadequately established anti-tumor immunity . As suchGiven the immunosuppressive phenotype associated with TNBC , 11, 13,This review summarizes the promising role of DNA repair deficiency as a surrogate biomarker to guide the use of ICI therapy in TNBC, discusses underlying mechanisms that link DDR signaling to anti-tumor immunity, and outlines the emerging evidence describing the relationship and potential cooperative therapeutic potential between DDR-pathway targeting agents and immunotherapy.Cells routinely undergo DNA damage as the result of cytotoxic stress. In normal physiology, mechanisms of DNA damage detection and repair are critical to preserve genomic integrity and thwart malignancy when DNA damage exceeds the cellular repair threshold. DDR accomplishes this by arresting proliferation and facilitating removal of damaged cells through activation of senescence or apoptosis. As such, defects in DDR genes permit mutations and chromosome rearrangements advantageous for tumor initiation and progression. In TNBC, with alkylating chemotherapies and radiation as major components of therapy, aberrant DDR signaling represents a dominant mechanism of tumorigenesis and treatment resistance, while also yielding potential therapeutic synergies with platinum chemotherapies or targeted therapies. An overview of the DNA damage response and repair pathways is detailed below and shown in via downstream targets Checkpoint Kinase 1 (CHK1) and Checkpoint Kinase 2 (CHK2), respectively, play a vital role in DDR signaling by maintaining replication fork stability and the regulation of cell cycle control checkpoints  and catalyze the synthesis of PARP chains (auto-poly (ADP-ribosylation), resulting in activation of intracellular signaling pathways that enable chromatin remodeling and recruitment of DDR-related protein machinery, thereby preventing accumulation of SSBs  polymerases (PARP1/2) are important DNA-damage sensors and regulators of BER-mediated SSB repair as well as other DDR pathways . These e of SSBs –36. In tClinical use of PARP inhibitors (PARPi’s) is an important example of DDR-specific targeting of HR defective cancers , 37. PARAlthough the greatest efficacy of PARPi has been observed in tumors with BRCA1/2 mutations, consensus is that synthetic lethality insufficiently explains PARPi-related anti-tumor activity. For example, the degree of PARP catalytic inhibition is poorly correlated to PARPi-induced cell-killing in HRD cells . In addiIn the context of TNBC, there is a higher degree of ‘BRCAness’ as compared to other breast cancer subtypes , 5, As svs 24%)  . In a revs 24%) . Conversvs 24%) , 51. ThiOther strategies to exploit HR include inducing a synthetic lethality by generating a BRCAness phenotype. These promising preclinical studies include combinations with inhibitors of EGFR, PI3K, BET, and others –54. We r+ T cells in the TNBC TME versus poor correlation in other subsets of breast cancers. Altogether, these findings indicate the immunotherapeutic efficacy potential in TNBC with MMR deficiency  initiate NHEJ, and these complexes have been shown to be regulated by EGFR amplification and/or p53 mutation-induced overexpression of long non-coding RNA in the NHEJ pathway 1 (LINP1), resulting in NHEJ-mediated chemo- and radiation resistance , 59. DoxMost breast cancer patients receive ionizing radiotherapy (RT) as part of their treatment to improve locoregional control by inducing tumor cell death predominately through the generation of DSBs, which in turn can elicit either protective anti-tumor immune responses or immunosuppression . Unfortuvia dendritic cell cross-presentation of tumor-derived antigens to T cells  of immune resistant tumor cells , 75, 76.Upon activation, T cells begin to express co-inhibitory cell surface receptors that control T cell function, such as CTLA-4 and PD-1. The balance between co-stimulatory and co-inhibitory signals is crucial for cytotoxic T cell activation and immunologic tolerance. Tumors can exploit this balance to escape T cell-mediated, tumor antigen-specific immunity. Importantly, therapeutically targeting these co-inhibitory pathways with immune checkpoint inhibitors (ICIs) is capable of unleashing anti-tumor activity , 82. In CTLA-4, an immune checkpoint constitutively expressed on Tregs and transiently upregulated on activated T cells, inhibits early T cell priming by antigen-presenting cells (APCs) in the lymph nodes . The exp+ tumor antigen-specific CD8+ T cells  cells, and APCs, and yields inhibitory signals through binding of its two ligands, namely PD-L1 and PD-L2 . PD-L1 i T cells , represe T cells , 88. Blo T cells , 89, res+ anti-tumor immune cells, tumor PD-L1 also mediates diverse cell-intrinsic functions that increase cancer virulence, including mTORC1 promotion and autophagy suppression . Studies of Atezolizumab in advanced solid cancers, including heavily pretreated TNBC, demonstrate limited but impressive outcomes, as only 10% of patients experienced clinically meaningful response, with 100% survival rate at 2 years in these responders, and median PFS of PD-L1+ TNBC patients treated with Atezolizumab plus nab-PTX was significantly increased by 50% (7.5 months vs. 5 months) , PD-L1 , and CTLA-4 (Ipilimumab), have generated durable responses across many tumor types . Clinica months) . KEYNOTE months) , 105. Pa months) . CurrentBased on the remarkably durable responses in a small subset of TNBC responders in ICI monotherapy studies, many studies using combination ICI with conventional therapies are currently ongoing and have shown early signs of benefit. Interim data from Impassion130 trial, using nab-paclitaxel in combination with Atezolizumab showed a 40% ORR in metastatic TNBC, and early data suggests a clinically meaningful overall survival benefit in patients with PD-L1 immune cell-positive disease (NCT02425891). Trials investigating the combination of Eribulin and Pembrolizumab in heavily pretreated metastatic TNBC are ongoing, with interim analysis demonstrating a 41.2% ORR to first-line treatment and a 27.3% ORR to later-line treatment . HoweverIn early TNBC, preliminary results from the neoadjuvant I-SPY 2 trial demonstrated that pCR rates increased from 22.3% to 62.4% by adding neoadjuvant Pembrolizumab to paclitaxel followed by anthracycline-based chemotherapy, which represents an approximately 40% improvement in pCR compared with standard chemotherapy alone . The KEYClinical success using immune checkpoint inhibitors has led to the identification of additional checkpoints that mediate tumor immunosuppression, such as the lymphocyte-activation gene 3 (LAG3), T cell immunoglobulin and mucin-domain 3 containing-3 (TIM3), Siglec-15, indoleamine 2, 3-dioxygenase 1 (IDO1), and glucocorticoid-induced tumor necrosis factor receptor (GITR). Targeted therapies for these are undergoing clinical trials in TNBC patients. For example, Siglec-15 is an immune checkpoint that inhibits antigen-specific T cell responses, and is expressed, independent of PD-L1 status, on both tumor and tumor-infiltrating myeloid cells , and a mAlthough clinical trials for immunotherapy in breast cancer have not shown that same high efficacy as in other carcinomas, TNBC is likely to have increased benefit as compared to other types of breast cancer given high mutational load, DDR-deficiency and increased PD-L1 expression. This may be especially true in early-stage TNBC with potentially more favorable tumor immune microenvironments, as studies thus far have mostly evaluated immune checkpoint inhibitors in advanced staged TNBC. However, innate, and adaptive resistance to immunotherapy remains a challenge, and targeted therapies that synergize with the immune-activating potential of immune checkpoint inhibitors is a promising strategy to maximize immunotherapeutic potential in TNBC patients.Recent work has highlighted the important interaction between genomic instability and the immunogenicity and activation of anti-tumor immunity . Highly In support of the notion that tumor cells with extensive genomic instability orchestrate a high octane anti-tumor immune response that is smothered by coexisting immunosuppression, DNA damage and DDR-defects associated with increased TMB and neoantigen production correlate with STING-induced PD-L1 expression and improved ICI response . In somevia cGAS/STING pathway activation or ATM/ATR/CHK1 kinase activity    . Althoug for OS) , 33. In vs. 11%) . A phaseEvidence that unrepaired DNA damage induced by PARPi expands the anti-tumor activity of the ICI has prompted investigation of other key mediators implied in DNA replication and repair, such as ATM, ATR, CHK1, CHK2, DNA-PK, and WEE1 , 121. GiThese promising preclinical studies have led to several early phase clinical trials. A clinical study in patients with advanced or metastatic cancers, use of Ceralasertib, a potent and selective ATR inhibitor in combination with Durvalumab is being evaluated (NCT02264678). A selective ATR kinase inhibitor, AZD6738, is undergoing a phase II study with Olaparib for metastatic TNBC patients with BRCA1/2 mutations or HRD (NCT03330847). The phase Ib BISCAY study, Durvalumab and Olaparib or the WEE1 inhibitor Adavosertib in patients with metastatic cancer with any detected HR-deficiency (NCT02546661). A phase I study combining the CHK1 inhibitor, Prexasertib, with a PD-L1 mAb demonstrated the potential for enhanced therapeutic activity and increased cytotoxic T cell activation .Further highlighting the indication that DDR-inhibitors and DNA-damaging agents may enhance immunotherapeutic response, a phase II clinical trial is evaluating the efficacy of Atezolizumab with stereotactic RT for advanced TNBC (NCT03464942), and a phase I study for the feasibility of adjuvant Durvalumab with a peptide vaccine is underway for patients with stage II and III TNBC after completion of standard adjuvant therapy (NCT02826434). Altogether, early studies indicate a potential therapeutic benefit of DDR-pathway targeting/inhibition in combination with immunotherapy, and ongoing trials will provide new insights into and establish clinical efficacy of the immune potentiating efficacy of DDR-inhibitors.via PD-L1 upregulation. DDR deficiencies represent a frequent aberration in TNBC, and exploitation of immunologic consequences offers potential therapeutic leverage that combines favorable immune effects of DNA/DDR-targeted therapies with restoration of cytotoxic anti-tumor immune cells. The role for endogenous as well as therapy-induced DNA damage signaling in PD-L1 induced expression, and the possibility of circumventing DNA targeted therapy-induced immune suppression with concomitant immunotherapy provide rationale for combining agents targeting the DDR and the immune system. Immunotherapy, chiefly ICI, represents an opportunity to flip the switch back to immune activation, particularly in the context of concomitant DDR pathway targeting therapies, such as PARP inhibitors and others.TNBC represents a highly diverse set of breast cancers with complicated molecular and immunologic landscapes, and thus remains a challenging oncologic entity to tackle effectively. However, advances in genomic profiling and our understanding of the interplay between DNA damage response and cancer immunity has resulted in exciting immuno-molecular therapeutic opportunities. Of these, DDR-deficiencies including BRCAness have been shown to promote immunologic vulnerability through DNA damage-induced high TMB, immune-stimulatory and suppressive features, as well as adaptive immune resistance via PD-L1 induction and complementary restoration of PARPi sensitivity via PD-L1 inhibition, with the added possibility of enhanced anti-tumor immunity, many unanswered questions remain regarding the potential benefit of combined targeted therapies and ICIs in TNBC. In addition to PARPi, other repair pathway mediators such as ATR, and CHK1, are being investigated in combination with immune-based strategies, and thus careful consideration of promising therapeutics as well as other immunotherapeutic strategies in the pipeline should not be overlooked. Optimization of treatment schemas for combined immunotherapeutic strategies remains a challenge, as does validation of biomarkers that will identify which patients will most benefit from either PARP inhibitors in combination with immunotherapy, radiotherapy, or other targeted therapies.PARP inhibitor monotherapies, as well as therapeutic combinations, have demonstrated promising clinical benefit, and their effects on enhancing lethal DNA damage vulnerabilities have been shown. Nonetheless, the underlying mechanisms of PARPi-mediated sensitization of tumors to immunotherapy and/or radiotherapy remain to be fully elucidated. Furthermore, rapid translation of these potential breakthroughs in TNBC treatment will require thoughtful incorporation and thorough dissection of clinical trial outcomes and their implications into everyday clinical practice. Despite preclinical and clinical studies that have demonstrated PARPi-mediated immunosuppression Lastly, identifying additional key mediators of DNA damage-associated immune modulation that regulate disease progression, therapeutic response and resistance will require further preclinical investigation and careful analysis of clinical samples to assess DDR deficiencies in certain tumor subsets, with the ultimate goal of personalizing DNA targeting and immune-based therapies in combination with conventional DNA- and immune-augmenting therapies, such as chemotherapy and radiation, to maximize the combined benefits of each approach and effectively target immunosuppressive pathways that contribute to immune escape and tumor progression. It will also be important to identify mediators of poor response to ICIs and improved prognostic markers for existing therapies to select patients that may benefit from alternative therapeutic strategies and explore options for TNBC refractory to ICI or PD-L1 negative TNBC. Furthermore, the role of less studied DDR mechanisms related to ICI is still unclear, and future work is needed to better predict which DNA damage response and repair pathways are most suitable for therapeutic targeting in specific subsets of patients.Both authors initiated the concept and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the UAB Department of Radiation Oncology.EY: Advisory Board: Astrazeneca, Bayer, Clovis, Strata Oncology; Research Support to Institution: Eli Lilly, Novartis, Puma Biotechnologies.The remaining author declares 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.


Near-infrared photoimmunotherapy (NIR-PIT) is a newly-developed, highly-selective cancer treatment, which utilizes a monoclonal antibody conjugated to a photoabsorbing dye, IRDye700DX (IR700). The antibody conjugate is injected into the patient and accumulates in the tumour. Within 24 h of injection the tumour is exposed to NIR light which activates the conjugate and causes rapid, selective cancer cell death. A global phase III clinical trial of NIR-PIT in recurrent head and neck squamous cell cancer (HNSCC) patients is currently underway. Conditional clinical approval for NIR-PIT in recurrent HNSCC has been granted in Japan as of September 2020. Not only does NIR-PIT induce highly selective and immediate cancer cell killing, but it also stimulates highly active anti-tumour immunity. While monotherapy with NIR-PIT has proven effective it is likely that combinations with immune-checkpoint inhibitors or additional NIR-PIT targeting immune suppressive cells in the tumour microenvironment will further improve results. In this review, we discuss the translational aspects of NIR-PIT especially in HNSCC, and potential future applications. The phase I/II trials showed NIR-PIT was at least equal if not superior to existing second- and third-line therapies for recurrent HNSCC with fewer adverse events, which suggests that NIR-PIT could be an effective new treatment for HNSCC. A global phase III human clinical trial was initiated in 2018 and is currently underway . Recently, NIR-PIT using cetuximab-IR700 was conditionally approved for recurrent HNSCC and was registered for clinical use by the Pharmaceuticals and Medical Devices Agency in Japan. Here, we focus on specific advantages and applications of NIR-PIT.Near infrared photoimmunotherapy (NIR-PIT) is a newly-developed, highly-selective cancer treatment that induces necrotic/immunogenic cell death. NIR-PIT is a two-part therapy which utilizes 1) a monoclonal antibody (mAb) conjugated to a photoabsorbing dye, IRDye700DX (IR700), which is then 2) activated by NIR light  generated by removal of the Fc region of an anti-CD25-IgG was used for local Treg depletion. The smaller size of the F(ab′)2 also resulted in more rapid clearance and less blocking of IL-2/IL-2R binding on effector cells, thus, did not interfere with the desired immune response [+ T cells, NK cells and antigen-presenting cells and greatly enhanced host immunity against both targeted and non-targeted lesions [+ T cells was observed in the treated tumour beds Immunosuppressive cells within the TME are promising targets for NIR-PIT. Tregs are crucial for immunological self-tolerance and help maintain immune homeostasis and prevent autoimmune disease in HNSCC ,58. AlthNK cells ,61. Thusresponse ,27. LocaNIR-PIT is capable of simultaneously targeting two or more kinds of cells with a single shot of NIR light exposure simply by co-injecting two or more different APCs followed by exposure to NIR light ,64. In o4.5Due to the limited number of available murine monoclonal antibodies against syngeneic tumours, CD44, a cancer stem cell marker, was initially used as a tumour target. However, because CD44 is also expressed on effector T cells there are disadvantages to its use. Therefore, as an alternative, a human EGFR-expressing mouse oral squamous cell cancer model, mEERL-hEGFR, human EGFR-expressing murine oropharyngeal cancer cell, was established and was used for combination therapy of human EGFR-targeted NIR-PIT with CD25-targetd NIR-PIT In vivo therapeutic efficacy and immune response to NIR-PIT in orthotopic mouse models of a syngeneic head and neck tumour have been demonstrated Additionally, in order to further simulate the clinical setting, orthotopic mouse models would be superior to xenografted tumour models so that the TME is more realistically recapitulated 4.6Gene transfer technology can be used to make cancer cells express artificial targets suitable for NIR-PIT. For example, HER2-targeted NIR-PIT has promising anti-tumour effects in HER2-positive cancers 4.718F-fluorodeoxyglucose positron emission tomography (18FDG-PET) can be a rapid response marker of treatment success because glucose metabolism in treated tumours was greatly reduced in the early phases after NIR-PIT Monitoring the therapeutic effects in NIR-PIT in real time or immediately after treatment is important in assessing tumour response and determining whether additional treatment is necessary. Preclinically, bioluminescence imaging can evaluate efficacy of NIR-PIT in the acute phase after treatment, but it cannot be translated clinically 5Although most clinical experience has been gained in patients with inoperable HNSCC, NIR-PIT has great potential to become a widely applicable treatment for a variety of cancers. It has the advantages of rapid response with minimal off target effects. When used in combination with immunostimulatory therapies, NIR-PIT may have broader anti-cancer effects throughout the body and may prevent recurrence. Although NIR light does not penetrate deeply in tissue, NIR light can be delivered using both direct surface lighting or via cylindrical diffusing fibres or endoscopes to accommodate various anatomical sites and tumour sizes where a needle or endoscopic device can be inserted. Alternatively, intraoperative or interstitial light delivery can be used for malignancies for which NIR-PIT would otherwise be difficult. Thus, NIR-PIT is a promising candidate for the treatment of HNSCC which serves as an example for other cancers.6NIR-PIT induces a highly selective necrotic/immunogenic cell death for targeted tumour cells, which promotes maturation of DCs and primes cytotoxic T cells to react with cancer-related antigens released from destroyed cancer cells. When NIR-PIT is combined with an immuno-checkpoint inhibitor or NIR-PIT targeting Treg, not only does NIR light expose local tumours but also distant metastatic tumours respond due to the enhanced host tumour immunity induced by the initial anti-tumour NIR-PIT. This “vaccine-like” property of NIR-PIT remains to be explored in human trials.Another issue is that the optimal treatment regimen of NIR-PIT for different types of tumours is currently under investigation. The degree of targeted molecule expression on the cell surface in treated tumours or distribution of immune cells that have anti-tumorigenic effects and other TILs that have pro-tumorigenic effects in TME could be important factors in selecting ideal patients and ideal combinations of NIR-PIT. However, further investigation is needed to determine an optimal combination strategy with NIR-PIT in each tumour type.Data search and selection were identified through searches from PubMed with the following search terms: “near-infrared photoimmunotherapy”, “head and neck cancer”, “monoclonal antibody”, “mouse model”, “epidermal growth factor receptor”, “immune checkpoint inhibitor”, “regulatory T cell”. Only articles published in English between 1989 and 2021 were included.This research was supported by the Intramural Research Program of the National Institutes of Health, ZIA BC011513.YM and HW wrote the manuscript and designed and produced the figures. PLC and HK outlined and supervised, and edited the draft and approved of the final version to be submitted. All authors approved the final version of the manuscript.The authors declare no conflicts of interests.


Drug repurposing can be an interesting strategy for an emergency response to the severe acute respiratory syndrome-coronavirus-2, (SARS-COV-2), the causing agent of the coronavirus disease-19 (COVID-19) pandemic. For this, we applied the Connectivity Map (CMap) bioinformatic resource to identify drugs that generate, in the CMap database, gene expression profiles (GEP) that negatively correlate with a SARS-COV-2 GEP, anticipating that these drugs could antagonize the deleterious effects of the virus at cell, tissue or organism levels. We identified several anti-cancer compounds that target MDM2 in the p53 pathway or signaling proteins: Ras, PKBβ, Nitric Oxide synthase, Rho kinase, all involved in the transmission of proliferative and growth signals. We hypothesized that these drugs could interfere with the high rate of biomass synthesis in infected cells, a feature shared with cancer cells. Other compounds including etomoxir, triacsin-c, PTB1-IN-3, are known to modulate lipid metabolism or to favor catabolic reactions by activating AMPK. Four different anti-inflammatory molecules, including dexamethasone, fluorometholone and cytosporone-b, targeting the glucocorticoid receptor, cyclooxygenase, or NUR77 also came out of the analysis. These results represent a first step in the characterization of potential repositioning strategies to treat SARS-COV-2. The severe acute respiratory syndrome-coronavirus-2, SARS-COV-2, emerged in December 2019, causing a viral outbreak declared as a pandemic by the World Health Organization (WHO) [While most infected people will remain asymptomatic, some will start to develop, after an incubation period of 0–14 days, a mild infection of both the upper and lower respiratory tract. The situation can worsen for 20–25% of the patients who develop a strong response to the virus and secondary bacterial/fungal infections, generating the release of inflammatory cytokines, chemokines, and alarmins by both the innate and adaptive immune systems . This “cIn an intense race to stop this pandemic and its associated death toll, all possible forces must be mobilized to have a better understanding of COVID-19 pathological features in order to improve patients’ care and develop new treatments to prevent the onset of severe cases.Vaccines have been developed at high-speed , especiaBesides the continuation of vaccines development, many clinical trials are still ongoing. As of november 2021, 7031 trials testing for potential anti-COVID19 drugs had been registered, including 706 active ones.We used the CMap bioinformatics resource from the Broad Institute , to screAmong several candidate molecules, we were able to highlight anti-inflammatory molecules, anti-cancer agents, signaling and metabolite inhibitors, neuromodulators. These molecules could potentially represent new repositioning resources to fight SARS-COV-2 infection, and interestingly, some of them have already been associated with SARS-COV-2/COVID-19 literature or are being tested in COVID-19 clinical trials.We used the publicly available GSE147507 dataset that studied the transcriptional response to SARS-COV-2 infection . We extrWe also selected data obtained from primary human lung biopsies. Uninfected biopsies were from two individuals and used as replicates. A virus-infected biopsy corresponded to a patient deceased from COVID-19 .https://string-db.org/), we queried our top 50 upregulated genes to create a network that highlights both functional and physical protein associations. We selected interactions with a minimum required score of 0.9 (most strict confidence filter).For gene expression analysis, we used the web-application Phantasus . We prochttps://www.gsea-msigdb.org) and the Reactive database (https://bioconductor.org).To identify enriched pathways from these DEG we used the R package fGSEA in the Molecular Signature Database  and used the Clue.io graphical interface (https://clue.io/). According to the CMap guidelines, we used the top 150 up- and down-regulated DEGs. Compounds identified with the CMap algorithm were filtered based on their enrichment scores . We selected molecules/perturbagens with negative ES, meaning that they reversed the SARS-COV-2 DEG signature and as a consequence could harbor anti-COVID-19 properties. The 22 molecules with the highest negative ES are presented and discussed in this study.We ran the second version of the Connectivity Map dataset referred as “build 02” revolves around TNF and includes IL1A, IL1B, TNFSF14, TNFAIP3, and CSF2 that likely mediates the inflammatory response associated with COVID-19 progression. It is conceivable that these genes could be used as biomarkers to evaluate the effects of potential anti-COVID-19 drugs.We ran a Connectivity Map analysis using the top 150 up- and down-regulated genes. After filtering based on the enrichment scores of compounds (Score<-90), we selected 22 compounds displaying the highest negative enrichment score . A negatAs the CMap database is composed of transcriptional signatures that have been generated on different cellular models by molecules/perturbagens, some being already FDA-approved or involved in pre-clinical studies, it is not surprising that our selection contains five compounds: olanzapine, terbutaline, atorvastatin, fluorometholone and dexamethasone that are already used in clinic and still investigated in various clinical trials . Two triEach identified putative active compound is associated with a specific MOA and a cellular target. The knowledge of these two parameters could shed interesting light on the mechanisms involved in the cellular responses to the SARS-COV-2 virus and the COVID-19 disease. Several compounds target molecules/enzymes that transmit activation signals. The farnesyl transferase enzyme is important to modify the function of many cellular proteins including Ras that mediates proliferation signals. PKCbeta and ROCK1 transmit activation signals in particular for immune cells. The nitric synthase is regulating the levels of the nitric oxide (NO) gaseous signaling molecule that exerts autocrine and paracrine actions, particularly on endothelial cells. Three compounds, with different chemical structures appear to target the p53 regulator MDM suggesting it could be an interesting target. The cytochrome P450 2E1 (CYP2E1) metabolizes many polar molecules, not only clearing carcinogens but also producing naturally signaling molecules such as 20-Hydroxyeicosatetraenoic acid (20-HETE) that acts on the vascular system. Several compounds are known to interfere with the metabolism, in particular fatty acid metabolism, by acting through inhibition of fatty acid synthase (FASN), carnitine palmitoyltransferase (CPT-1), glycogen synthase (GSK-3), protein tyrosine phosphatase N1 (PTPN1) to stimulate the AMPK catabolic pathway. An interesting target is nucleophosmin (NPM1) a nucleolar ribonucleoprotein not only involved in the biogenesis of ribosomes but also in cell division, chromatin remodeling, DNA repair.Several anti-inflammatory compounds not only highlight the role of the glucocorticoid receptor but also put attention on the cyclooxygenase and the NUR77 receptor, respectively involved in the production of inflammatory prostaglandins, and in the balance between proliferation, inflammation and apoptosis.The development of vaccines and drugs against an emerging virus is a several-year-long process. The repurposing of already available and clinically active drugs appears to be a valuable parallel strategy in an emergency scenario.in silico and showed promising properties in vitro/in vivo in a variety of cancers such as renal cancer [The CMap database of perturbagen-induced gene signatures was developed to discover potentially active compounds using gene expression profiles (GEPs) associated with disease states . As mostl cancer , colorecl cancer , bladderl cancer , leukemil cancer , neurobll cancer .We used CMap on SARS-CoV-2 published signatures obtained from human lung cell lines infected with the SARS-CoV-2 virus and from primary lung biopsies from a COVID-19 deceased patient to obtain 22 potential drug candidates representing six functional categories.Our selection identified four anticancer drugs. It has been assumed that virus-infected cells, similarly to cancer cells, are in an anabolic state reflecting their intense biomass synthesis and could therefore be sensitive to anti-cancer agents . None ofFour signaling inhibitors may have been selected for their capacity to block growth and division signals. They represent an interesting category as two of them have already been studied for COVID-19. The farnesyl transferase inhibitor of farnesyl transferase tipifarnib, appeared in a virtual screening of 6218 FDA-approved drugs, and was able to block SARS-COV-2 replication in Vero cells . WhetherOur CMap analysis highlighted several drugs known to affect cell metabolism. They represent an interesting approach as infected cells adapt and stimulate their metabolism to their viral needs. Triascin-C, an inhibitor of fatty acid synthase (FASN) has been shown to interfere with the replication of SARS-COV-2 in Vero E6 cells . A seconFinally, NPM1 inhibitors, such as avrainvillamide-analog-3, have been proposed as a broad anti-viral therapy because NPM1 interacts with various viral proteins in different cellular compartments to support virus replication . NPM1 thregs [As expected from the physiopathology of COVID-19, the CMap analysis uncovered four anti-inflammatory molecules, including dexamethasone, which has been proven to be effective in reducing mortality for patients under respiratory support . Dexametregs .Our analysis also highlighted terbutaline, an agonist of adrenergic receptors with lung bronchus dilatation properties. Terbutaline improved the condition of COVID-19 patients with asthma. Moreover, as it is used via subcutaneous injection, it presents a lower risk of virus dispersion compared to aerosol-based rebreathers . PsychotApplying CMap to COVID-19 data has already allowed different research groups to discover many potentially active molecules that could not only interfere with virus replication but also counteract its damaging effects in infected tissues. The results we present here are a first step in the path to discovery of active new drugs to target the dysregulated mechanisms of cell growth, metabolism and proliferation in SARS-COV-2-infected cells.S1 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.