RAS oncogenes are the most commonly mutated oncogenes in human cancer, and RAS-mutant cancers represent a major burden of human disease. Though these oncogenes were discovered decades ago, recent years have seen major advances in understanding of their structure and function, including the therapeutic and prognostic significance of diverse isoforms. Targeting of these mutations has proven difficult, despite some successes with inhibition of RAS effector signalling. More recently, direct RAS inhibition has been achieved in a trial setting. While this has yet to be translated to everyday clinical practice, this development carries much promise. This review summarizes the diverse approaches that have been taken to RAS inhibition and then focuses on the most recent developments in direct inhibition of KRAS(G12C). The three RAS genes, Kirsten rat sarcoma viral oncogene homolog (K-RAS), neuroblastoma RAS viral (v-ras) oncogene homolog (N-RAS) and Harvey rat sarcoma viral oncogene homolog (H-RAS), are the most commonly mutated oncogenes in human cancer with approximately one-third of all cancers driven by these oncoproteins, including 40%-50% of colorectal cancer and over 90% of pancreatic cancers,4. The discovery of oncogenes and the elucidation of intracellular signalling pathways heralded an era of targeted therapy that has greatly improved the outlook for many cancers,6. New agents targeting receptor kinases and their downstream mediators demonstrated the ability to stabilise and shrink tumours, with side effects that were frequently milder than those associated with standard cytotoxic chemotherapy. Throughout this enormous paradigm shift in cancer therapeutics, however, RAS stood apart, dominant and seemingly undruggable. The development of direct RAS inhibitors proved very challenging. RAS has a high affinity towards GDP and GTP and a lack of deep hydrophobic pockets which would allow binding of small molecules. Subtle differences in structure and variable activation of RAS proteins added greatly to the complexity and attention largely focused on downstream inhibition of the transduced signalling pathways. In recent years, however, there have been some very promising developments in direct RAS targeting which would suggest that this has real potential as a therapeutic avenue. Here we aim to review current efforts at RAS inhibition in the context of both RAS family biology and the historical efforts which attempted, largely without success, to perturb its role as a major oncogenic driver.Since the discovery of RAS oncogenes as the transforming genes of oncogenic retroviruses. Three RAS genes, H-RAS, K-RAS and N-RAS, encode four protein isoforms: H-RAS, K-RAS4A, K-RAS4B and N-RAS. K-RAS4B is the predominant isoform and is referred to simply as K-RAS in this article. RAS proteins are small GTPases, of about 21kD molecular weight, and are monomeric proteins that have a central role in cell differentiation, adhesion, migration, proliferation and survival. RAS proteins convey signals from growth factors and extracellular components, and are upstream of signalling pathways including the ERK pathway and the PI3K/mTOR survival pathway. As illustrated in The RAS superfamily of genes has about 36 members, which encode for 39 proteins. The consequence of this aberrantly activated RAS is prolonged oncogenic signalling rather than short, controlled bursts of activation. There is subsequent activation of downstream signalling molecules such as PI3K, RAF and Rin1. By contrast, mutations in other RAS codons, or any nonsense mutations, are likely to inhibit rather than enhance the activity of RAS, and do not provide a survival advantage. As well as uncontrolled proliferation, the mutagenic RAS oncogene has been implicated in tumour immune resistance by causing intrinsic - as opposed to adaptive - upregulation of programmed death ligand 1.The normal function of RAS, as described above, can be deranged by mutations which unbalance this equilibrium. Single point missense mutations in codons G12 (most commonly), G13 or Q61 are responsible for converting proto-oncogenes to oncogenes. These mutations favour GTP binding and lead to constitutive activation of RAS, with reduction or loss of GTPase activity. These codons are implicated as their amino acid residues are found in the cavity where GTPase catalytic activity operates. For example, in pancreatic adenocarcinoma, where RAS mutations are virtually ever-present, precursor lesions - pancreatic intraepithelial neoplasia (PanIN) - contain RAS mutations which increase in frequency as they progress stepwise to malignancy. Though generally cooperative with other oncogenes during malignant transformation, these mutations are capable of neoplastic growth in the absence of further genetic abnormalities,14. K-RAS is the isoform most frequently mutated and in addition to being almost inevitably mutated in pancreatic cancers, K-RAS mutations are present in approximately half of colorectal cancers and a third of lung cancers. H-RAS mutations are found in salivary gland cancers , cervical cancers (9%) and urinary tract cancers (9%). N-RAS mutations are found in melanoma (17%), haematologic malignancies (10%) and thyroid cancers (7%). Not only are RAS mutations implicated in carcinogenesis, but also they are strongly associated with treatment resistance,17, and have been shown to be adverse prognostic markers in cancer. RAS-mutated colorectal cancer will not benefit from EGFR-directed treatment with cetuximab or panitumumab. Similarly, in lung cancer RAS status has been shown to be an independent predictor for EGFR tyrosine kinase inhibition.Approximately 25%-30% of cancers contain mutations in one of the RAS isoforms, and they are considered an early genetic event in tumour progression. In addition, given variation in the frequency of isoform mutations within different cancer types, and in the specific mutations involved, there may not be a single effective RAS inhibitor for all RAS-mutated cancers. Historical efforts to target RAS which have made it to the clinic could be broadly summarized as those focusing on RAS plasma membrane localisation and those attempting to indirectly block RAS by inhibiting the downstream effector signalling.Given the central role of RAS both in carcinogenesis and tumour progression, the RAS oncoprotein is an important therapeutic target. . Two farnesyltransferase inhibitors (FTIs), lonafarnib and tipifarnib, were investigated in Phase III trials either as monotherapy or in combination with chemotherapy in a number of different RAS-mutated tumours. Despite Phase I and II clinical trials showing some antitumour activity and low toxicity, no improvement in overall survival was reported in Phase III trials-23. One reason for lack of “pan-RAS” efficacy for the FTI class is that K-RAS and N-RAS membrane localisation can be achieved in the absence of farnesyltransferases, via geranylgeranyl transferases. Attempts were made to target these enzymes with geranylgeranyltransferase inhibitors, but these were ineffective and associated with toxicity. In contrast, H-RAS is not a substrate for geranylgeranyl transferase and therefore its membrane localization could be suppressed solely by FTIs. Tipifarnib has demonstrated preclinical activity against a wide panel of H-RAS-mutated head and neck squamous cell carcinoma xenograft models and is undergoing clinical development in advanced head and neck cancers harbouring activating H-RAS mutations (NCT02383927). These efforts highlight the differences between RAS isoforms and the need for a tailored, isoform-specific approach to clinical trial design.In order to carry out their role, RAS proteins must become membrane-bound. This involves a complex series of post-translational modifications. Three enzymatic steps are necessary for RAS to associate with membranes - (1) prenylation of the CAAX box by farnesyltransferase (FTase); (2) cleavage of the terminal AAX residues by RAS converting enzymes RCE1; and (3) methylation of the cysteine residues of the CAAX box by isoprenylcysteine carboxyl methyltransferase ICMT. Farnesylation, the addition of farnesyl groups to RAS, is a critical step in creating a hydrophobic domain in RAS that allows the protein to associate with the plasma membrane, and therefore to be biologically active. Farnesyltransferases (FTases) are the enzymes responsible for this step, and they were an early target in efforts to inhibit RAS function. A further target is isoprenylcysteine carboxyl methyltransferase (ICMT), an enzyme at the endoplasmic reticulum which increases RAS membrane affinity. It lacks homology with other protein methyltransferases, which adds to its specificity as a target. While agents that target ICMT have been isolated, and have demonstrated promising results in vitro against cancer cell lines, they have not been tested in the clinical setting. Palmitoylation, the modification by the fatty acid palmitate, is necessary for the membrane interactions of H-RAS and N-RAS. Depalmitoylation inhibitors and palmitoyl acyltransferases have been described as having activity against RAS, but uncertainty regarding their specificity and concern about off-target effects, have impeded their further clinical development. Many other proteins are modified by prenylation and farnesylation to ensure their correct subcellular localization, which makes off-target effects unavoidable even for highly specific inhibitors. Moreover, RAS can also signal from endomembranes  in addition to the plasma membrane,30. It is unclear how localization inhibitors affect RAS signalling from different subcellular compartments.Salirasib is an S-trans, trans-farnesylthiosalicylic acid and a novel oral RAS inhibitor which competes with farnesylated RAS for binding sites on membranes. A recent trial demonstrated encouraging activity in patients with advanced solid tumours including a subset with K-RAS mutations. These approaches have largely failed, perhaps mainly due to the inherent complexity and redundancy within these networks, but also due to a lack of specificity in the selection of inhibitors as well as isoform homogeneity within each subpopulation. In addition, the concept of linear pathways is misleading and has given way to that of signalling networks, whereby activated kinases interact via RAS (or other GTPases) with a large variety of signalling molecules resulting in highly interconnected networks.Given the historic difficulties in directly targeting RAS, many efforts focused instead on inhibiting the downstream signal transduction pathways, either at a single point or as a combined approach targeting different nodal points. RAS effector families are involved in cancer initiation and maintenance, and it was hoped that inhibition of downstream proteins within these pathways could be an effective means of countering RAS-mediated oncogenesisEGFR: There is substantial crosstalk between EGFR tyrosine kinase and RAS. With EGFR upstream of RAS, inactivation of these receptor tyrosine kinases can in theory reduce RAS activation, and this linear model explains the lack of clinical activity for EGFR inhibitors in colorectal cancer in the setting of K-RAS or N-RAS-mutant tumours,33. This has also been demonstrated experimentally, through activation of the RAS signalling pathway by introduction of an activated K-RAS allele, confirming that as mutated RAS is constitutively active, disruption of signalling from EGFR impairs the therapeutic effect of anti-EGFR monoclonal antibodies. Similarly in advanced non-small cell lung cancer (NSCLC), EGFR inhibition is insufficient to produce a response in the setting of RAS-mutant disease. It is possible, however, that the specific RAS mutation plays a significant role in determining whether upstream inhibition of EGFR may be effective. We have seen in K-RAS G12D mutation-specific advanced solid tumours that a response can be achieved with pan-ERBB/EGFR inhibitors Afatinib and neratinib,37. EGFR and pan-ERBB inhibitors have shown promise in preclinical studies in combination with both direct covalent RAS inhibitors and MEK inhibitors. This synergistic effect between EGFR inhibitors and covalent RAS inhibitors appears to be the result of EGFR inhibition leading to a reduced amount of GTP-bound RAS, therefore leaving RAS in the unbound GDP state open to targeting by direct inhibition.ERK pathway: Directly downstream of RAS signaling are the ERK and PI3K signaling pathways. Active GTP-bound RAS results in RAF dimerization and phosphorylation, RAF kinase activity and, ultimately, phosphorylation of its substrates MEK1 and MEK2. The terminal kinases of this pathway, ERK1 and ERK2, act as growth promoting transcription factors. The RAS-RAF-MEK-ERK cascade is targeted with RAF kinase inhibitors, ERK inhibitors or MEK inhibitors. Eleven RAF kinase inhibitors have reached clinical evaluation and four are approved for use by the US Food and Drug Administration (FDA). Vemurafenib and dabrafenib are two ATP-competitive RAF inhibitors that are approved for use in BRAF-mutant metastatic melanoma. Further clinical evidence in both lung cancer and colorectal cancer has shown benefit for these agents when BRAF is mutated,40. The current agents used in clinical practice, dabrafenib and vemurafenib, act on RAF monomers. However, in RAS-mutant cancer, RAF inhibition has been unsuccessful and the clinical experience has been very negative. The reason for this is that clinically used RAF inhibitors enhance RAF kinase homo-and heterodimerization, leading to the paradoxical activation of ERK signalling. Homo- and heterodimerization of the RAF kinases BRAF and CRAF significantly increases their catalytic activities. The binding of RAF molecules to active RAS drives RAF dimerization by inducing conformational changes, dephosphorylation of inhibitory residues, and brings RAF molecules into proximity of each other. Due to allosteric interactions between protomers in the RAF dimer, inhibitor binding to the first protomer in a dimer strongly decreases the affinity of the second protomer to the inhibitor. In this constellation the drug-bound RAF protomer allosterically activates the drug-free protomer causing paradoxical pathway activation and drug resistance. As oncogenic RAS proteins are effective drivers of RAF kinase dimerization, RAS mutations lead to intrinsic or acquired resistance to RAF inhibitors. Overcoming dimerization-induced resistance to RAF inhibitors could lead to effective anti-RAS therapy.. There are a number of newer agents that target RAF dimers rather than the monomer isoform all of which appear to demonstrate less paradoxical upregulation of the ERK pathway,46. Belvarafenib and LXH-254 are pan-RAF inhibitors, effective against the monomer and dimer isoform, which are under clinical evaluation both as monotherapy and in combinations for RAS-mutant advanced solid tumours.Another interesting drawback to targeting wildtype RAF in RAS-mutant disease is that inhibition of wildtype RAF can paradoxically upregulate the ERK pathway in the setting of RAS mutations leading to downstream phosphorylation of MEK and ERK. In RAS-mutant tumours, MEK inhibition as monotherapy has failed to demonstrate meaningful benefits largely due to the induction of feedback loops similar to those when RAF inhibitors are use in this setting. Trametinib, Cobimetinib and Binimetinib are allosteric, non-ATP competitive inhibitors of MEK1 and MEK2, and are used in combination with RAF inhibitors in the treatment of melanoma. A number of trials have failed to show clinical benefit for MEK inhibition in advanced KRAS-mutant pancreatic, colorectal and non-small cell lung cancer-51. One possible explanation for this is that inhibition of the ERK pathway induces autophagy, a process of cellular recycling that protects cells from the cytocidal effects of pathway inhibition. Pancreatic cancer cells in particular utilize autophagy for growth and as a means of resistance to ERK inhibition. Combining downstream MEK inhibition with hydroxychloroquine - an inhibitor of autophagy - displayed synergistic anti-proliferative effects against pancreatic ductal adenocarcinoma (PDA) cell lines and promoted striking regression of PDA xenografts from 2 patients with PDA which was superior to (standard of care) gemcitabine plus nab-paclitaxel chemotherapy. Highly encouraging clinical responses were also seen with this commercially available drug combination and clinical trials are ongoing ,54.ERK upregulation through MEK appears to be the predominant method of resistance to BRAF directed monotherapy and we have seen both in preclinical and clinical studies that concurrent inhibition of BRAF and MEK can decrease acquired resistance and delay progressionERK pathway: With ERK being the final kinase in the RAF-MEK-ERK pathway it would appear to be an attractive target to inhibit in RAS or RAF mutant tumours. Based on previous preclinical studies of the compound SCH-772984, a dual ERK1/2 inhibitor which demonstrated a reduction of phosphorylated ERK in a number of RAS-mutant cancer cell lines, an oral version MK-8353 was developed for clinical testing. In a Phase I study, however, with 26 patients with advanced K-RAS or N-RAS mutated tumours, no objective responses were seen. It is now being tested in combination with a MEK inhibitor (selumetinib) and the anti-PD-1 inhibitor pembrolizumab in patients with RAS-mutant cancers. In K-RAS-mutant tumour models, ERK inhibitors such as GDC-0944 have shown efficacy in combination with the MEK inhibitor cobimetinib. Phase I studies of the combination were stopped prematurely due to toxicity. The ERK inhibitor was studied as monotherapy in a Phase I trial and appeared tolerable. In this study, 14 patients had K-RAS-mutant advanced malignancies and of those 4 had stable disease and 10 had progression. ERK pathway suppression detected with NanoString gene expression was observed more commonly in those with BRAF-mutant tumours compared to those with KRAS-mutant tumours.. This in combination with nab-paclitaxel has been examined in a Phase 1 study in patients with advanced pancreas cancer and results are awaited (NCT02608229). Other studies are underway examining its role as monotherapy and in combination with other agents in patients with genetic alteration in the ERK pathway . LY-3214996 is a selective inhibitor of ERK1 and ERK2. Unfortunately, however, in a Phase I study of this agent, only one patient with advanced RAS-mutant cancer had stable disease and the remaining patients had progressive disease. KO-947, similarly a selective ERK1/2 inhibitor, demonstrated potent and sustained reduction in phosphorylated ERK in vitro in RAS-mutant cell lines. This is now in clinical studies for patients with advanced RAS- or RAF-mutant tumours (NCT03051035).Ulixertinib is another ATP competitive ERK1/2 inhibitor that has shown clinical efficacy in N-RAS-mutant melanomaPI3K pathway: The other major target of RAS effector signalling is the PI3K-AKT-mTOR pathway. PI3K is implicated in RAS-dependent cancer initiation and maintenance. While there are many inhibitors of this signalling pathway under investigation, they have demonstrated little activity as monotherapy in RAS-mutant cancers. KRAS and BRAF mutations are predictive of resistance to mTOR inhibition. Moreover, inhibition of mTOR may lead to the upregulation and activation of MEK-ERK pathways. Therefore, the dual inhibition of the PI3K and the ERK pathway would appear to be a reasonable therapeutic target. With this in mind combination strategies have been explored in RAS mutant disease using both mTOR inhibitors along with RAF/MEK inhibitors. Direct PI3K inhibitors have also been trialled with MEK inhibitors. However, in clinical trials, this combination was both toxic and only minimally efficacious.. Unfortunately no AKT inhibitors are approved for use in clinical practice. The evaluation of AKT inhibition with MEK inhibitors is under investigation, but similar challenging side effect profiles have been seen to that of combined mTOR and MEK inhibition.AKT, which plays a key role in the activation of mTOR following its interaction with one of the 3 main PI3Ks, is amplified in the setting of RAS-mutant cancers,69. Finally, there are selectivity and toxicity challenges. The “switch region”, which changes conformation upon GTP binding and recruits effector proteins, is highly conserved across other G-proteins in the body. Therefore, any agents which target this region in RAS, would be associated with risk of toxicity elsewhere.Attempts at direct RAS inhibition have been hampered by numerous challenges. Firstly, the activity of RAS is tightly governed by GEFs and GAPs, which control the high-affinity, picomolar interactions between GDP, GTP and RAS. Putative inhibitors struggle to overcome this affinity. Secondly, there are structural challenges. An effective inhibitor would typically require a deep hydrophobic pocket for binding. These pockets were long understood to be absent, although recent research demonstrates that they may arise dynamically as RAS goes through the GDP/GTP cycle and compounds that bind to RAS at the RAF binding site and inhibit RAS/RAF complex formation. However, none were sufficiently potent to be considered for further investigation. More recently, however, direct targeting of RAS has been achieved for the G12C K-RAS mutation. This has been realised through the development of a small molecule inhibitor that binds covalently to the cysteine residue that results from the specific G12C mutation and has been shown to inhibit oncogenic RAS,73. The inherently reactive nature of cysteine which is found at codon 12 of K-RAS(G12C) can be exploited for covalent small molecule inhibitors and the idea of targeting cysteine is one that is commonly exploited in drug discovery. Another important feature of targeting this cysteine is that wild-type K-RAS lacks the cysteine in the active site unlike the mutant K-RAS(G12C).A number of agents investigated were compounds that competed directly with GDP for the nucleotide binding site of RASet al. initially identified this novel allosteric binding pocket behind switch II referred to as the switch-II pocket, which led to the development of the first compounds to irreversibly target G12C. These compounds bind to K-RAS(G12C) in the GDP-bound inactive state, blocking SOS-catalysed nucleotide exchange and ultimately inhibiting K-RAS(G12C) association with RAF. This switch II pocket is present in the GDP-bound inactive form of K-RAS only. Therefore, targeting of G12C needs to occur in the GDP-bound state. K-RAS(G12C) in its steady state is in the active GTP-bound state, but the presence of a high level of GTPase activity leaves it open to covalent attack. The identification of this pocket led to a search for covalent inhibitors of K-RAS. The first was a molecule ARS-853, and its development led to the proof of concept that this specific isoform of K-RAS could be targeted using a covalent inhibitor.Ostrem . This particular G12C inhibitor has succeeded where others had not as its potency and selectivity was optimised through an interaction with a previously unexploited groove His95. Preclinical studies have demonstrated responses and regression of K-RAS mutant tumours treated with AMG510. The results of the Phase I study of sotorasib (AMG510) demonstrated promising anticancer activity in patients with advanced solid tumours harbouring K-RAS(G12C) mutations. 129 patients were treated on the dose escalation study, 59 with NSCLC, 42 with colorectal cancer and 28 with other solid tumours. Sotorasib appeared to be well tolerated with 11.6% of grade 3 or 4 toxicity. 32.2% of the NSCLC had an objective response with a total of 88.1% having a response or stable disease. The median progression free survival was 6.3 months. In the colorectal cohort 7.1% had a confirmed response with 73.8% having a response or stable disease with a median PFS of 4 months. This study represented the first clinical trial demonstrating objective response to direct KRAS inhibition. On December 8, 2020 the FDA granted Breakthrough Therapy designation for its investigational K-RAS(G12C) inhibitor, sotorasib, for the treatment of patients with locally advanced or metastatic NSCLC with K-RAS(G12C) mutations, as determined by an FDA-approved test, following at least one prior systemic therapy. Preclinical studies have also demonstrated that sotorasib was able to clear colon cancer from mice when given in combination with checkpoint inhibitors. There is a Phase 1/2 study under way of sotorasib in solid tumours which will include a combination arm of sotorasib with an anti(PD-1/L1) (NCT03600883).A number of advances in the area ultimately led to the development of AMG510 (sotorasib). A Phase 1/2 study of adagrasib monotherapy in patients with pretreated NSCLC demonstrated an overall response rate of 45% and a high disease control rate. This multiple-expansion-cohort trial is ongoing, investigating the use of adagrasib combined with pembrolizumab in NSCLC, afatinib in NSCLC or cetuximab in colorectal cancer. Research on this agent in nonclinical models has also demonstrated mechanisms of resistance, including KRAS nucleotide cycling and pathways that induce feedback reactivation or bypass KRAS dependence.Adagrasib (MRTX849) is an additional agent under investigation in this field. It is a potent, highly selective inhibitor of KRAS(G12C)K-RAS(G12C) mutations only account for a small proportion of KRAS mutations that are found in cancer and are primarily found in lung cancer. As these irreversible allosteric inhibitors block RAS signalling by exclusively binding to the cysteine residue that results from the specific mutation, this limits their application to the particular allele they target. To target KRAS(G12D) and KRAS(G12V) different approaches are needed as these mutants lack the cysteines needed in the active state.In vitro studies have shown that Compound 3144, a molecule that binds a conserved residue Asp38 in switch-I, can block RAS effector binding. This compound suppresses the growth of KRAS(G13D) tumours in vivo. A major concern, however, is that pan-inhibition of RAS potentially may lead to considerable toxicity as normal cellular function is reliant on RAS signalling in non-cancerous cells. Models by which the deletion of all three RAS isoforms is carried out are not compatible with life, and therefore a pan inhibition of RAS in humans is likely to result in significant off-target toxicity. Nevertheless, it has been suggested that if such a compound were optimised for greater potency and specificity, this would be a viable approach.Attempting to design and develop drugs specifically targeting each individual RAS mutation would be extremely challenging and time-consuming, so direct targeting of ligand binding sites conserved on all RAS proteins  has been thought to be one potential method of inhibiting RAS across all mutation and tumour types. . GTP binding and activation of RAS leads to conformational changes in it, allowing it to bind effectors in RAS-binding domains. Mutations in RAS that are relevant to cancer usually lead to RAS permanently in the GTP bound active state. Efforts have been made to block this nucleotide exchange in an attempt to stop RAS transitioning to the active GTP bound form.Nucleotide exchange inhibition: In order to cycle between active GTP-bound and inactive GDP-bound states, RAS possesses intrinsic guanine nucleotide exchange and GTP hydrolysis activities. This cycling and exchange are accelerated by GEF and by GAPs, both of which change the activation state of RAS through covalent modifications. Upon activation of GEFs, nucleotide binding is destabilized and GDP is released. As GTP is much more prevalent than GDP in the cell, this loss of GDP leads to a transient formation of RAS-GTP, the active state. Of these, SOS is the best known and studied of the RAS-specific GEFs and this has led to the development of a number of strategies and attempts to block it and ultimately inhibit RAS. SOS1 binds RAS at its catalytic binding site and thereby promotes exchange of GDP for GTP. RAS-GTP can also bind at an allosteric site on SOS1 to enhance GEF activity. Genetic inactivation of SOS1 has been shown to decrease the survival of RAS-mutant tumor cells, but not in RAS wild type cells that are not reliant on RAS signaling. Inhibition of SOS1 has been thought of as an attractive mechanism of RAS inhibition over direct RAS inhibitors as it does not appear to depend on targeting specific mutations.An alternative to blocking the direct nucleotide site is to inhibit the proteins that regulate the nucleotide exchange process. Normal RAS activation requires nucleotide exchange, processing, membrane localization and effector binding. Targeting any of these steps can be used to indirectly inhibit RAS. GEFs are responsible for releasing GDP from RAS allowing it to be replaced by GTP and ultimately leading to activation of RAS. In mammals, three families of RAS-specific GEFs exist: SOS, RASGRF1/Cdc25Mm and GRP/Cal-DAG-GEFin vitro and in vivo. This orally bioavailable agent appears to reduce RAS-GTP level and inhibits ERK pathway signaling, thereby limiting the growth of tumor cells driven by RAS. Whilst most RAS variants appear to show reduction in cell proliferation when exposed to BI-3406, certain variants appear less sensitive. Mutations in codon 61 due to the resultant molecular conformation appear to have low intrinsic GTPase activity and are subsequently less sensitive to SOS1 inhibition. Initial studies of SOS1 inhibitor BI-3406 suggest that it may benefit as many as 80%-90% of RAS-driven cancers.BI-3406 is a potent and selective SOS1:K-RAS interaction inhibitor that potently decreased the formation of GTP-bound RAS and reduced cell proliferation of RAS-driven cancers both SOS1 is phosphorylated by ERK, a kinase in the ERK pathway downstream of RAS, ultimately leading to the reduction of its GEF activity. It is thought that efforts to treat RAS-driven cancers with MEK inhibitors have failed in part as inhibition of MEK reduces the activity of ERK1/2, resulting in the release of a negative feedback loop, thus increasing the activity of SOS1-dependent formation of GTP-bound RAS. Combination therapy of a MEK inhibitor with BI-3406 blocks this negative feedback by reducing levels of phospho-MEK and phospho-ERK leading to sustained pathway inhibition and potentiating the benefit of SOS1 inhibition. A Phase I clinical trial of this combination to assess safety, tolerability and preliminary efficacy, as well as another SOS1 inhibitor, BI-1701963 in combination with Trametinib is ongoing (NCT040111458).. Mutations in PTPN11, which encodes SHP2, cause “rasopathies” and are found in about 50% of patients with Noonan syndrome. Although not fully defined, SHP2 appears to act as a scaffold protein, binding GRB2 and SOS1 in close proximity to RAS and ultimately thereby increasing RAS nucleotide exchange. Research has explored the role of SHP2 inhibitors in the treatment of various cancers. In the preclinical setting, the allosteric SHP2 inhibitor SHP099 has been shown to inhibit myeloid leukaemia cell lines, and to elicit a response in colorectal cancer cell lines. Of note, in the colorectal cancer cells, these responses seemed limited to cells that were sensitive to lapatinib, and therefore dependent on EGFR signalling. By contrast, RAS- or BRAF-mutant cells were generally resistant to SHP099.Although much less defined than SOS, another protein involved in the nucleotide exchange process is SHP2. SHP2 is a non-receptor protein tyrosine phosphatase that is required for the full activation of the ERK pathwayEffective and safe inhibition of RAS was considered a holy grail for cancer researchers decades ago, and it remains so today. While extraordinary advances have been made in our understanding of RAS and carcinogenesis - as well as methods of targeting its downstream effectors - the direct inhibition of RAS does not yet have a role in everyday practice. However, our deeper understanding of molecular pathogenesis is the foundation on which that future progress will be built, and effective treatments are now close.. Therefore, those designing clinical trials should carefully consider the need for isoform and mutation-specific approaches.The unmet need for proven therapies in this setting is clear, given the prevalence of RAS mutations in common cancers, many of which are highly fatal. Our recognition of the structure and clinical significance of diverse RAS isoforms represents a major step forward, of relevance to all future efforts at RAS targeting. Their importance is clear when considering candidate inhibitors of RAS plasma membrane localization, which are now reaching the clinic. Even individual isoforms, like K-RAS, have a range of possible mutations with different responses to targeted therapies.The development of inhibitors of RAS effector signalling, which are in widespread clinical practice, is an example of the concrete achievements of research in this area. This success has been tempered over time by failures, many of which can be attributed to misconceptions of RAS signalling pathways as unidirectional and linear. A more nuanced appreciation of these as delicately balanced signalling networks can help researchers refine their approach, and may warrant increased focus on drug combinations, as seen in trials involving inhibitors of MEK and autophagy. However, these studies also raise questions - why did response rates to Sotorasib vary between cancers despite identical mutations, and what provoked early progression in some patients after an initial response? It is suggested that tumour heterogeneity or driver mutations alternative to K-RAS(G12C) may be the key to understanding this and that, as with inhibition of RAS effector signalling, drug combinations may be the next step. In addition, optimizing monotherapy by developing inhibitors with higher affinity for inactive KRAS(G12C) or the use of pulsatile therapy has been suggested as approaches to maximise therapeutic effect.Possibly showing greater promise than any of these initiatives, however, is efforts at direct targeting of RAS. The past 12 months has seen breakthroughs in this field that have been sought for years, vindicating decades of basic scientific endeavour. Successes here belie outdated dogma describing RAS as “undruggable”, demonstrating that drug exposure associated with preclinical efficacy can be achieved safely in patientsIn summary, a more textured understanding of RAS pathogenesis emerging from decades of basic scientific research has led to a more refined approach to RAS inhibition, which is now beginning to bear fruit. The coming months and years will hopefully take these efforts “over the line” to the routine use of effective therapeutics in the clinic setting, but it will be a long time before we have fully tapped the potential of RAS inhibition.


Working in COVID-19 settings during the pandemic is a stressful experience that has been linked to psychologically traumatic symptoms Thus, supportive measures are proposed for healthcare personnel, even in countries with low COVID-19 burden. Nurses may be at a higher risk of experiencing work-related traumatic stress response during the COVID-19 pandemic compared to other clinicians. This study aimed to investigate the correlations between work-related trauma symptoms and demographic factors, psychosocial hazards and stress response in a census sample of nurses working in COVID-19 settings in Cyprus. In this nationwide descriptive and cross-sectional study, data were collected between April and May 2020 using a questionnaire that included sociodemographic, educational and employment and work-related variables, as well as a modified version of the Secondary Traumatic Stress Scale (STSS) for the assessment of work-related trauma symptoms during the pandemic. Overall, 233 nurses participated (with a response rate of 61.3%) and 25.7% of them reported clinical work-related trauma symptoms . The mean value for emotional exhaustion was 7.3 , while the value for distress that was caused by being avoided due to work in COVID-19 units was 6.98 . Positive associations were noted between trauma symptoms and both emotional exhaustion and distress from being avoided by others due to work in a COVID-19 setting and a negative association was also found between trauma symptoms and satisfaction from organizational support variables (all  The COVID-19 pandemic has been a challenge to healthcare systems and healthcare workers, including nurses, as it has increased work-related psychological hazards and has affected the physical and mental health of clinicians ,3,4,5. WWork-related psychosocial hazards may adversely affect the health and well-being of clinicians by causing stress response; however, although psychosocial hazards may be present in work environments, they may not be experienced as threats and thus, may not cause any stress response ,7. ThereClinicians who are employed in COVID-19 settings are exposed to increased workload; care rationing ,13; the All the above factors are associated with decreased work satisfaction and the intention to quit the job ,16; at tWork in COVID-19 settings may involve exposure to traumatic events . Work-rePost-traumatic stress disorder is defined as a clinical condition that occurs in individuals who have been exposed to severely traumatic events, including work-related events . AccordiVicarious (secondary) trauma includes adverse impacts from indirect exposure to potentially traumatic events in the workplace, such as caring for service users who are coping with traumatic events, reviewing distressing information or witnessing a fatality, as well as knowing about or witnessing the suffering of others, all of which are relevant to healthcare employees during the pandemic . Since nSeveral tools exist for the assessment of the severity of PTSD symptoms, such as the Impact of Events Scale or the Post-Traumatic Symptom Scale, which have been used in research samples of healthcare workers who were assessed for vicarious (secondary) trauma . Although different factors, such as personal characteristics and work-related factors, have been suggested to play a role in developing symptoms of mental distress among clinicians who have been exposed to traumatic events ,26, onlyNevertheless, existing data calls for attention to be paid to the social, relational and environmental context of clinicians to better understand their distress and their risk of developing mental health problems during and after the pandemic . SimilarΤhe aim of the present study was to assess the prevalence and intensity of work-related traumatic stress response in Greek Cypriot nurses who were employed in COVID-19 settings. Our secondary objectives included the following: (i) an exploration of the associations between the presence and intensity of traumatic stress response and (a) sociodemographic variables, (b) work satisfaction and (c) self-assessed work-related psychosocial hazards ; (ii) an exploration of self-assessed work-related stress response .A descriptive and cross-sectional design was applied in this study. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines were followed .The study population comprised nurses working in COVID-19 settings in public healthcare services in the Republic of Cyprus (RC). The Nicosia General Hospital (NGH) is the largest public tertiary hospital in the RC and is based in Nicosia. Its emergency department (ED) (which employs 12 physicians and 72 nurses) provides services to over 140 patients per day and, in parallel, it was the referral center for those reporting COVID-19 symptoms during the first wave of the pandemic. Following assessment in the ED, patients who produced positive SARS-CoV-2 tests were either admitted to an ICU  or they were transferred to (a) one of the two open COVID-19 units in the NGH or (b) the COVID-19 referral hospital in Famagusta, according to the severity of their symptoms. The Famagusta referral hospital ran one step-down unit  and four open wards. Nurses from all of the above settings were invited and participated in the present study .A census sampling approach was applied. Based on power analysis, the sample size had to be 104 nurses for moderate correlation effect (0.3–0.4) to be demonstrated between work-related traumatic stress response and work, employment and demographic variables . By taking previous data on the response rate of Greek Cypriot nurses into consideration , a totalOur inclusion criteria included (a) employment in a COVID-19 setting , (b) the ability to read and write in Greek and (c) the provision of written informed consent. There were no exclusion criteria.Sociodemographic , education (education level) and employment  variables were assessed by using closed-ended questions. The following work variables were approximately reported by responders: the number of patients treated per work setting; the number of deaths per day in the last month in the work setting from any cause; the number of night shifts per month; the number of days in quarantine after producing a positive SARS-CoV-2 test/self-quarantine due to exposure to SARS-CoV-2 (close contact). The degrees of (a) emotional exhaustion, (b) work satisfaction, (c) satisfaction from care provided in the last month, (d) satisfaction from information provided about COVID-19, (e) satisfaction from the provision of personal protective equipment by the work organization and (f) distress caused by being avoided due to work in COVID-19 healthcare settings were assessed by using visual analogue scales (VASs). The VAS scores ranged from 1 to 10, with higher values indicating increased satisfaction/distress. The VAS tools that were used for the measurement of the degree of emotional exhaustion, work satisfaction and satisfaction from provided care have been validated in previous studies in Greek-Cypriot nurses . The VASThe modified 17-item version of the Secondary Traumatic Stress Scale (STSS-M) was used for the assessment of work-related traumatic stress response. The items that were included in the original STSS were modified accordingly to be suitable to experiences of psychological trauma that were related to work in COVID-19 settings. Examples of the modified statements included “Reminders of my work with clients in the COVID-19 unit upset me” and “My heart started pounding when I thought about my work with clients in the COVID-19 unit”. The items were rated on a 5-point Likert scale, from 1 (never) to 5 (very often). The participants were asked to report the frequency at which they experienced the situations that were described in each item during the previous two weeks. The total STSS-M score ranges from 17 to 85, with higher scores indicating more intense self-reported trauma symptoms (TS). The 17 items of the instrument could be grouped into avoidance, arousal and intrusion symptoms, which reflected the three subscales of the STSS-M . The total STSS-M scores were interpreted by comparing the scores of the study participants to the normative scores. Specifically, the responses of the participants were classified into categories based on the percentiles: total STSS-M scores that were at or below the 50th percentile  were interpreted as being indicative of no or low-intensity trauma symptoms; scores between the 51st and 75th percentiles  were interpreted as mild-intensity trauma symptoms; scores between the 76th and 90th percentiles  were interpreted as moderate-intensity trauma symptoms; scores between the 91st and 95th percentiles  were interpreted as high-intensity trauma symptoms; scores that were above the 95th percentile  were interpreted as severe-intensity trauma symptoms. Furthermore, total STSS-M scores that were at or above the cutoff value of 56  were indicative of clinically relevant traumatic stress symptoms that were caused by exposure to COVID-19-related work conditions. The internal consistency reliability coefficient  of the original STSS has been reported to be as high as 0.93, and 0.9 for relevant modified versions ,41. PrevData collection took place in April and May 2020 via a self-administered paper questionnaire that was distributed by the researchers to participants in the study work settings. The response rate was 61.3%. Written informed consent was provided by all participants. Anonymity and the voluntary nature of participation in the study were also assured. Each questionnaire package was provided in an open and opaque envelope with no identifying characteristics. The study was conducted according to the guidelines of the Declaration of Helsinki. Permission to carry out the study was obtained from the National Committee of Bioethics of Cyprus [EEBK/EP/2019/27].t-test and ANOVA parametric measures were applied for comparisons between the groups. For significant differences between multiple groups, post-hoc analyses were carried out by using the Scheffe test. The chi-squared test was used for comparisons between groups regarding the categorical variables. Pearson’s r was assessed to determine the correlations between the numerical variables. Aiming to model the predictors of work-related traumatic stress response , a multivariate analysis was applied by using stepwise logistic regression. Specifically, the total STSS-M score was transformed into a categorical dichotomous variable (dependent variable) as follows: no clinical TS (17–55 STSS-M score); clinical TS (56-85 STSS-M score). The following were also included as independent variables: (a) sociodemographic, educational, work-related and employment variables; (b) VAS variables, as described in the Data Analysis section. The level of statistical significance was set at < 0.05. The IBM SPSS  statistical package was used for the data analysis. Frequencies were assessed for categorical variables and mean values (M) and standard deviations (SD) were assessed for continuous variables. The age and years of work experience were transformed from continuous into categorical variables. The following age groups were formed: up to 35 years; 35–45 years; more than 45 years. Work experience was grouped as follows: less than 5 years; 5–10 years; more than 10 years. The continuous variables were checked for normality and the The sample consisted of 233 nurses: 86 men (36.9%) and 147 (63.1%) women. The majority were up to 35 years old (58.4%) and 57% (n = 133) had more than 10 years of work experience. These data are presented in Approximately 75.1% of the participants reported that they treated fewer than 10 patients per shift and 89.7% reported fewer than five daily deaths from any cause during the previous month. A total of 129 participants (55.4%) worked more than five night shifts per month during the same period. Approximately 47% were employed in an ICU, 22% were employed in an ED, 25% were employed in an open COVID-19 unit and 6% were employed in a step-down ICU.The mean number of days per month that were spent in quarantine after a positive SARS-CoV-2 test was 10.04 , while the mean number of days that were spent in self-quarantine due to exposure to SARS-CoV-2 (close contact) was 3.10 . The sociodemographic and employment characteristics of the study participants are presented in The average values for the variables “Degree of emotional exhaustion”  and “Degree of distress experienced from being avoided due to work in a COVID-19 healthcare setting during the pandemic”  were above moderate , as was the mean value for “Degree of satisfaction from provided care” . All of the VAS scores are presented in The mean total STSS-M score was 45.38 . According to the STSS-M scoring classification, 23.6% (n = 55) had no or low-intensity TS, 25.8% (n = 60) had mild-intensity TS and 24.9% (n = 58) had moderate-intensity TS , while 15.0% (n = 35) had high-intensity TS and 10.7% (n = 25) had severe-intensity TS  . p = 0.014)) and the age group up to 35 years (61.5%) reported more intense TS compared to the other age groups (p = 0. 016). Participants who reported more than five daily deaths reported more intense TS compared to those who reported fewer than five daily deaths (mean (SD): 50.55 (13.83) vs. 44.90 (12.83); p = 0.05)). There were no associations between these variables and work setting. Additional data on the differences in the severity of work-related traumatic stress response  according to demographic characteristics  are presented in Female participants reported more intense TS compared to male participants (mean (SD): 47.01 (12.53) vs. 42.61 (13.30); p < 0.001). STSS-M score was inversely correlated with the degree of satisfaction from provided care in the last month, the degree of satisfaction from information provided about COVID-19 by the administrative managers and the degree of satisfaction from the provided personal protective equipment against COVID-19 (p < 0.002). No correlations were found between the total STSS-M score and work/employment variables   found thOur results were in accordance with those from previous research on work-related traumatic stress response symptoms in mixed samples of healthcare workers during the first wave of the pandemic ,32. SpecWe also found that staff rank, emotional exhaustion, low work satisfaction and distress from being avoided due to work in a COVID-19 setting were associated with clinically relevant work-related traumatic stress response symptoms. The perception of being avoided by others due to work in a COVID-19 setting could be deemed as an aspect of poor social support and could underline the social implications of the pandemic on nurses, which have also been addressed in other studies . OverallIn terms of staff rank, our study revealed that participants with leadership roles were less vulnerable to work-related traumatic stress response symptoms than those who held a staff nurse position, which was contrary to the finding in a study by Inocian et al. (2021) . One posThe positive association between work-related traumatic stress response symptoms and emotional exhaustion that was reported by this study was in accordance with the finding of a study by Luceño-Moreno et al. (2020) . The psyThe present study did not reveal any predictors of traumatic stress symptoms that were related to sociodemographic or employment variables, although previous reviews have shown that female staff nurses working in EDs and ICUs during the pandemic reported higher levels of depressive, anxiety and burnout symptoms compared to male healthcare professionals (nurses or otherwise) employed in non-ICU/non-ED settings ,36. OtheIn this study we also reported that the degrees of satisfaction from provided care, information about COVID-19 and the supply of personal protective equipment were negatively related to work-related traumatic stress symptoms. Other studies have also underlined the link between organizational support measures and mental health in healthcare professionals. Providing protective equipment, together with supporting the needs of healthcare workers, have also been highlighted as crucial for engagement in work and the provision of optimal care to patients . MoreoveAlthough healthcare systems around the world have reformed their mental health services to varying degrees due to pressure from the pandemic, there is still a lot to be improved . Our stuFurthermore, based on the presented results and relevant literature, leadership approaches that could reduce the traumatic impact of work-related psychosocial hazards on frontline nurses include (a) empowerment to participate in decision making and access to information, (b) the provision of adequate personal protective equipment and (c) education about COVID-19 and relevant copying strategies and the development of a COVID-19 workplace protocol to promote safe and well-organized work environments ,56. The Additionally, nurses who are working on the frontline during the pandemic should be closely monitored for the development of mental health problems. The early identification of traumatic stress response symptoms is important as these symptoms are related to burnout, poor quality of work life and the intention to quit the job . In caseLastly, longitudinal studies are needed to explore the trajectory of work-related traumatic stress responses in nurses who are employed in work environments that have increased psychosocial hazards and a multitude of causes of work-related stress responses.The strengths of the present study included the simultaneous assessment of work-related traumatic stress response symptoms during the pandemic, work-related psychosocial hazards and stress response in a large nationwide study that focused on nurses who were employed in COVID-19 healthcare settings in the Republic of Cyprus. Moreover, the sampling method that was applied supported the generalization of the presented results. There was also a large response rate, which minimized any possible selection bias. However, although the questionnaire was distributed to all nurses, selection bias could not be totally excluded. Additional limitations of this study included its cross-sectional design, which limited etiological interpretations, the self-reported assessment of trauma symptoms and possible impact of cultural factors, as the study was conducted in the multicultural Republic of Cyprus. Moreover, we were not aware of the characteristics of the nurses who eventually did not consent to participation; thus, our results need to be replicated in independent studies and settings. Additionally, although three of the VASs that were applied in this study were developed by the authors, they were based on the relevant literature, and were validated by a group of experts. Nevertheless, additional validation methods could have increased the internal validity of the study. Similarly, it is worth mentioning that the developer of the original STSS did not have any input during the development of our modified version.Finally, additional confounders that could have contributed to the reported traumatic symptoms, such as psychiatric history or previous traumatic experiences, were not taken into consideration, nor were the baseline measurements of the mental health status of the participants prior to the onset of the pandemic. Nonetheless, the present study is among the few that have addressed work-related psychosocial hazards  and stress response in relation to work-related traumatic stress response symptoms specifically in nurses who were employed in COVID-19 settings.The results of this study suggested that nurses who are employed in COVID-19 settings are vulnerable to developing work-related traumatic stress response symptoms. Supportive environments in the workplace need to be assured by administrators, even in countries with low COVID-19 burden, while empowerment interventions and relevant facilities need to be available for those who are most at risk, i.e., those in COVID-19 referral centers and those who have the closest contact with COVID-19 patients.


This paper estimates associations between long-term exposure to ambient particulate air pollution and 13 self-reported health outcomes including poor general health, chronic illness, experiencing long-running difficulties with breathing, and experiencing frequent periods of confusion or memory loss. It examines the extent to which these associations are explained by confounding factors.Longitudinally-linked Census data from the Northern Ireland Longitudinal Study linked to data on annual average particulate (PM2.5) concentrations at the 1km grid-square level over the period 2002-2010, exploiting complete residential histories, are used. The paper controls for potentially confounding factors at the neighbourhood, household and individual level, including for prior health, in a multivariate regression framework. Robustness to the presence of remaining unobserved confounders is assessed in two extensions, first by assuming selection on unobservables is proportional to selection on observables, and second through inclusion of an extensive set of fixed effects in the model.There are strong statistical associations between long-term exposure to ambient particulate pollution and all 13 health outcomes measured by the 2011 Northern Ireland Census. Most of these estimated associations survive conditioning on an extensive set of observable controls. Of these, however, only two associations – with chronic illness and with long-running breathing difficulties, where we might expect the strongest causal effects – survive further analysis designed to elicit robustness to selection on unobservables. The estimated magnitudes of these remaining effects are non-trivial. For example, a 5 µg/m3 difference in particulate exposure averaged over 9 years has a similar magnitude effect on the probability of reporting long-running breathing difficulties as the difference between those aged in their 20s and those in their 40s.This study provides evidence of substantial effects of long-term exposure to ambient particulates on the probabilities of experiencing chronic illness and long-running breathing difficulties. These are qualitatively robust to both observed and unobserved confounders. Associations between particulate exposure and other health outcomes in the study are shown to reflect confounders.


Understanding how neurohormonal gut-brain signaling regulates appetite and satiety is vital for the development of therapies for obesity and altered eating behavior. However, reported brain areas associated with appetite or satiety regulators show inconsistency across functional neuroimaging studies. The aim of this study was to systematically assess the convergence of brain regions modulated by appetite and satiety regulators. Twenty-five studies were considered for qualitative synthesis, and 14 independent studies (20-experiments) found eligible for coordinate-based neuroimaging meta-analyses across 212 participants and 123 foci. We employed two different meta-analysis approaches. The results from the systematic review revealed the modulation of insula, amygdala, hippocampus, and orbitofrontal cortex (OFC) with appetite regulators, where satiety regulators were more associated with caudate nucleus, hypothalamus, thalamus, putamen, anterior cingulate cortex in addition to the insula and OFC. The two neuroimaging meta-analyses methods identified the caudate nucleus as a key area associated with satiety regulators. Our results provide quantitative brain activation maps of neurohormonal gut-brain signaling in heathy-weight adults that can be used to define alterations with eating behavior.  •First functional neuroimaging meta-analysis to generate neurohormonal gut-brain activation maps.•Meta-analyses reveal satiety regulators modulate hypothalamus and caudate activity.•ALE and ABC meta-analysis approaches show similar results for caudate association.•Appetite regulators modulate insula, amygdala, hippocampus, OFC activities.•Satiety regulators modulate hypothalamus, thalamus, caudate, insula, putamen, OFC. Clinical and preclinical studies show that an increase in endogenous ghrelin concentrations after fasting activates appetite-related neurons in the hypothalamus and stimulates appetite sensation and meal initiation . FeedingRecent advances in non-invasive neuroimaging techniques have allowed the study of neurohormonal gut-brain signaling pathways that modulate appetite in health and disease . HoweverThe primary aim of this study was to provide a comprehensive analysis of the functional neuroimaging literature on brain areas associated with changes in appetite and satiety regulators in healthy-weight adults, and to produce a quantitative brain activation map of the neurohormonal gut-brain interactions, using coordinate-based neuroimaging meta-analysis. We hypothesize that the insula, hypothalamus, and caudate nucleus are commonly reported brain areas across studies.22.1A comprehensive search was carried out in the MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials (CENTRAL) databases between November 2019 and January 2021 to identify relevant studies using keywords from functional neuroimaging techniques and appetite and satiety responses including terms related to satiety and/or appetite regulators.2.1.1The full search strategy is described in 2.1.2The quality of the included papers was assessed for potential risk of bias by one author (SA), using the Cochrane collaboration to assess the risk of bias in randomized cross-over and randomized controlled trials (ROBINS-I) . The qua2.1.3For each study, the following information was extracted: authors, year of publication, total number of participants, participant details , time of first brain imaging scan after treatment administration, intervention, administration method , assessed appetite and/or satiety regulators that was directly correlated with brain responses, neuroimaging modality and brain stimulation method  and correlation results between brain areas and appetite and satiety regulators. Extracted data were grouped into 1) brain areas correlated positively 2) and/or correlated negatively with appetite regulators, 3) brain areas correlated positively and/or 4) correlated negatively with satiety regulators. In the appetite analysis, data were analyzed during the fasting state or for contrast fasted>fed. In the satiety state analysis, data were derived from a direct contrast between fed state versus fasted/hunger state (fed>fasted) or data assessed postprandially within 1.5 h following the last consumption. Brain areas from each of the sub-grouped data were then pooled and common brain areas across studies were evaluated. To illustrate the concurrence of brain areas generated from the systematic review, anatomically defined masks for the overlapped brain areas were generated using WFU PickAtlas toolbox  in SPM s2.2Neuroimaging studies included in the meta-analysis were pooled from those included in the systematic review. Studies that did not report coordinates for brain activations in response to appetite/satiety regulators in the article or There are multiple algorithms for performing coordinate-based neuroimaging meta-analysis, each has different empirical parameters and assumptions, and each can produce different results conditional on the assumptions. Therefore, to obtain robust results of brain areas associated with appetite and satiety regulators, two different tools were employed: the ALE and ABC meta-analysis methods. The ALE-approach is the most popular method of performing neuroimaging meta-analysis. The algorithm takes into account the number of participants in each study to apply a relevant smoothing, resulting in a higher specificity of the actual overlap between studies . However2.2.1ALE meta-analyses were performed using Ginger ALE version 3.0.2 . ALE analysis uses the reported activation peaks from the individual studies as a three-dimensional Gaussian probability distribution (kernel) centered at the given coordinates to create a modeled activation (MA) map for each study. Individual MA-maps are then combined to calculate statistical ALE maps and ALE values for each cluster. These calculations are confined to a grey matter mask provided by the Ginger ALE software. The ALE maps indicate areas of the brain where convergence between activation foci is greater than would be expected by chance . We adhered to the recommendations of 2.2.2ABC methodology  was performed using the ABC toolbox implemented in the NeuRoi image analysis software . The algorithm of this recently developed model-based method uses the density of coordinates from independent studies as its statistic and requires only the human grey matter volume (one parameter). Statistical thresholding is performed by requiring a minimum proportion of the studies contributing to a cluster and is generally more conservative than false discovery rate (FDR < 0.05). Importantly, this method, in contrast to the ALE-approach, does not require the empirical choice of Gaussian smoothing kernel to extrapolate coordinates to voxel-wise activation maps or the randomization of the coordinates in the empirical space to define the statistical threshold.33.13.1.1Of the 1390 studies identified in the initial search, 81 were selected for full text assessment. Eligibility criteria were based on the PICO (Population-Intervention-Comparator-Outcomes) model, and the inclusion and exclusion criteria are listed in 3.1.2Of the 25 studies included, twenty-two studies used functional magnetic resonance imaging (fMRI), of which eight used food picture task-fMRI , five stAcross the included studies, brain responses for the hungry state were assessed following fasting that ranged between 4 and 14 h, whereas for the satiety state brain responses were assessed within 1.5 h postprandially. Seven studies administered standard meals with different amounts of protein, fat and fiber, containing ingredients such as soya bean, beef or milkshake . Eight s3.1.3As a first step, the data extracted from the 25 studies were grouped into brain areas that correlated: 1) positively and/or 2) negatively with appetite regulators, 3) correlated positively and/or 4) negatively with satiety regulators. Data from brain areas from each of the sub-groups were then pooled and common brain areas across studies evaluated. A full list of overlapped brain areas is reported in 3.1.3.1Eight studies  reported3.1.3.2In terms of satiety regulation, eight studies reported positive modulation in response to satiety regulators with concurrence in the anterior cingulate cortex (ACC) (three studies)  and puta3.2In a second step, we examined the concurrence/overlap in brain regions activated in response to changes in appetite and satiety regulators quantitatively using neuroimaging meta-analysis. Studies were initially grouped following the same methods used for the systemic review: 1) brain areas correlated positively with appetite regulators (4 studies), 2) and/or correlated negatively with appetite regulators (1 study), 3) brain areas correlated positively with satiety regulators (8 studies), 4) and/or correlated negatively with satiety regulators (12 studies). However, due to the small number of studies , for each of these the sub-groups, we could not perform separate meta-analysis. Instead, we performed two primary meta-analyses: one for appetite regulators and the other for satiety regulators, each combined across studies reported negative and positive correlation with brain responses.3.2.1Of the five studies eligible for the meta-analysis with appetite regulators, four assessed positive correlation , and a s3.2.2In terms of the satiety analysis, the ALE and ABC meta-analyses across 14 independent studies (20 experiments combined across increased/decreased brain activation to satiety regulators) included 212 healthy-weight participants and 123 foci provided convergent results, revealing the same cluster see , Fig. 4 4This is the first systematic review and quantitative meta-analysis of functional neuroimaging data to determine which brain regions were most consistently activated in response to appetite/satiety regulators in healthy weight adults. We included studies that directly assessed brain activation in response to appetite/satiety regulators endogenously released, following acute food ingestion, and/or exogenously administered regulators. In addition, we employed two different coordinate-based meta-analysis approaches (ALE and ABC) to reveal convergent brain areas of neurohormonal gut-brain signaling to appetite/satiety regulators.To our knowledge, there is only one previous systematic review that investigated the effects of appetite/satiety regulators on brain regions involved in appetite and satiety . However4.1In support of our hypothesis, the systematic review revealed that the insula was one of the most reported regions across studies, with an overlap in 50% of the studies in response to appetite regulators. These results are in agreement with those of the previous systematic review . The insThe systemic review also identified the amygdala, and OFC as key brain areas modulated with appetite regulators in 62% of the studies. The amygdala, and OFC encode motivation value of food cues . More sp4.2, which may cause discrepancies in findings.The hypothalamus is widely recognized as the gatekeeper to control food intake, highly influenced by nutrients, it is physically connected to other areas involved in maintaining homeostatic energy balance and receives projections from the gastrointestinal tract via the brainstem . The venZanchi et al. (2017)The caudate nucleus is associated with perception of food stimuli, reward processing, and cognitive appetite control . In thisThe thalamus is another key area revealed by this systematic review to modulate with satiety regulators. Thalamic brain activity has been reported to vary as a function of hunger or satiety , ghrelin4.3To our knowledge this is the first study to employ functional neuroimaging meta-analysis to quantitatively define overlap of brain areas associated with satiety regulators across studies. The results from the generated activation maps of the meta-analysis  are more robust than those of any individual imaging study. The generated activation map from the healthy weight participants can be used as a reference or baseline to compare alterations with obesity, or in people with altered eating behavior. Another strength is the stringent and well-defined inclusion and exclusion criteria, which enabled an unbiased assessment of the central mechanisms regulating satiety and appetite. The present systematic review provides data and a clear overview of appetite neuroimaging findings in healthy weight participants.One of the main limitations of this work is the relatively small number of appetite/satiety studies included in the meta-analysis, a consequence of the strict inclusion selection criteria. This did not allow us to perform sub-analyses on appetite/satiety regulators positively/negatively correlated with brain responses, or to investigate possible differences in neurohormonal gut-brain signaling in response to endogenously released appetite and satiety regulators compared with exogenously infused regulators. In addition, the small number of studies might be responsible for the absence of other key brain areas related to appetite and satiety regulations including the insula, thalamus and amygdala, OFC and ACC. The location of brain clusters/foci for the OFC and ACC activations varies widely across studies and this might also explain the lack of their concurrence in our analysis. Moreover, we stress that although a strict selection criterion was employed in this study, the presence of some unknown source of heterogeneity within the selected studies is possible, including individual differences in food preferences. We acknowledge that there are variations in the imaging modalities , study designs (endogenously released or exogenous administration), and food-related paradigms/stimulations  in the studies included, which might introduce some heterogeneity. Ideally, we would have separated the studies based on stimulation type and/or study design and/or imaging modality and performed separate analyses but this was not possible due to the small number of studies. Despite these variations and drawback, the robustness of our findings is supported by similar results obtained with two different approaches (ALE and ABC), particularly for the caudate convergence. In addition, variations/heterogeneity in the included study may lead to reduce the sensitivity of the meta-analysis, making it likely to be conservative, rather than causing false positive results/activations.Finally, another limitation of this review was that the risk of bias analysis was performed using the Cochrane risk of bias guideline . As this5In conclusion, our systematic review and quantitative meta-analyses add to the growing body of evidence describing brain areas involved in appetite and satiety processing. The generated brain activation maps for the gut-brain interactions in healthy weight participants can be used for comparison in future studies to define alterations with obesity and in pepole with altered eating behavior. We present robust evidence from the systematic review and two different coordinate based meta-analysis approaches/methods (ALE an ABC) for the importance of the hypothalamus and caudate nucleus in appetite and satiety processing. However, more work is needed to fully elucidate the complex interactions associated with the central regulation of appetite and satiety.


Despite their importance to current and future patient care, medical students’ hygiene behaviors and acquisition of practical skills have rarely been studied in previous observational study. Thus, the aim of this study was to investigate the potential impact of the COVID-19 pandemic on medical student’s hygiene and practical skills.This case-control study assessed the effect of the COVID-19 pandemic on hygiene behavior by contrasting the practical skills and hygiene adherence of 371 medical students post the pandemic associated lockdown in March 2020 with that of 355 medical students prior to the SARS-CoV-2 outbreak. Students’ skills were assessed using an objective structured clinical examination (OSCE). Their skills were then compared based on their results in hygienic venipuncture and the total OSCE score.p < 0.001) and after patient contact  as well as disinfecting the puncture site correctly . Prior to the pandemic, students were more proficient in practical skills, such as initial venipuncture , patient communication  and structuring their work process .During the SARS-CoV-2 pandemic, medical students demonstrated an increased level of compliance regarding hand hygiene before (prior COVID-19: 83.7%; during COVID-19: 94.9%; Overall, the COVID-19 pandemic sensitized medical students’ attention and adherence to hygiene requirements, while simultaneously reducing the amount of practice opportunities, thus negatively affecting their practical skills. The latter development may have to be addressed by providing additional practice opportunities for students as soon as the pandemic situation allows.The online version contains supplementary material available at 10.1186/s12909-022-03601-6. - The COVID-19 pandemic sensitizes medical students to hygienic aspects of venipuncture.- The hygiene compliance of medical students is proportional to the duration of the COVID-19 pandemic.- During the COIVD-19 pandemic, medical students performed worse in non-hygienic tasks.The online version contains supplementary material available at 10.1186/s12909-022-03601-6. Over the last century, the world population has repeatedly been confronted with pandemics primarily targeting the respiratory system, such as Influenza A, SARS, MERS-CoV as well as the current COVID-19 pandemic. As these pandemics are associated with high mortality, reducing the transmission of the virus is crucial [“Social distancing” and the implementation of hygiene standards have proven to be reliable precautionary measures to reduce the spread of SARS-CoV-2 –5. Thus,Due to the nature of their work, healthcare professionals are at a particularly high risk of exposure to SARS-CoV-2, as well as susceptible to increased stress levels which result from a pandemic related increased workload , 9. To aThis unicentric case-control study examined the venipuncture skills and level of hygiene-compliance of third year medical students who participated in OSCE at the University of Cologne before and during the COVID-19 pandemic. The data was collected during OSCE, which is a practical test consisting of seven five-minute test stations and usually takes place in the third year of medical school in Cologne. The examined practical skills were assessed by medical students during their practical year. Failure to pass this exam was of no academic relevance to the tested students. Students are prepared for this test in a practical course, which is designed to teach skills such as hygienic venipuncture and hand disinfection. In total 910 medical students underwent OSCE from February 5th, 2019, to February 19th, 2021, in Cologne. 184 medical students participating in the OSCE in February of 2020 were excluded from this study, since they were assessed after the first SARS-CoV-2 case and before the implementation of pandemic containment measures in Germany. Therefore, the 184 medical students could not be assigned to either the control group or the lockdown group. In conclusion, only the data of 726 medical students were included in this study. Medical students who participated in OSCE prior to the first diagnosed SARS-CoV-2 case in Germany provided the control group (cohort 1). On contrary medical students who took part in OSCE after the first lockdown in March 2020 provided the investigated cohort (cohort 2). A subgroup analysis of cohort 2 allowed the comparison of medical students’ hygiene behavior at different stages of the pandemic   was used.All medical students partaking in this study were of legal age. Neither the students nor the examiners knew the purpose of this study prior to the data collection. The local ethics committee reviewed and approved this research project on August 20, 2021, prior to its initiation . Upon enrollment in the medical program at the University of Cologne, students consented in writing to data collection and analysis. The examined retrospective data were analyzed pseudonymously.The aim of this study was to compare medical students’ compliance with standard hygiene protocol regarding venipuncture and other hygienic tasks before and during the COVID-19 pandemic using their results in OSCE.p < 0.001) and venipuncture .Out of 726 medical students partaking in OSCE, 355 (48.9%) participated before (cohort 1) and 371 (51.1%) after the first lockdown in Germany during March 2020 (cohort 2). The relative score of both cohorts showed only a slight deviance regarding their relative score for OSCE .After the lockdown, 352/371 medical students (94.9%) disinfected their hands before patient contact and 212/371 (57.2%) after patient contact, whereas prior to the lockdown only 297/355 students (83.7%) did so before and 69/355 (19.4%) after patient contact (p < 0.001). Moreover, the number of students disinfecting the puncture site increased post lockdown. . No significant difference was found between the groups regarding the observance of the 30-second exposure time of the disinfectant. Here, both demonstrated a high level of hygiene compliance .After lockdown, significantly more students disinfected their hands prior patient contact (94.95%) compared to after patient contact (57.2%) (p = 0.691), the use of sterile and unbent puncture needles  as well as discarding the puncture needle .There was no statistically significant difference between the two cohorts regarding the preparation of the materials , doctor-patient communication  and structure in the work processes  were less frequently demonstrated by the students partaking after the first lockdown. Thus, applying a tourniquet was the only practical skill that medical students were more proficient at after the first lockdown than before  , 180 (48.5%) participated after the first lockdown in March 2020 (cohort 2a) and 191 (51.5%) after the start of the second lockdown in December 2020 (cohort 2b).p = 0.003), while no significant statistical difference could be found between the relative score in venipuncture after the first (74.86 ± 11.3) compared to the second lockdown . No statistical difference was also evident in regards to hand disinfection before patient contact , disinfection of the puncture site , consideration of the 30 second disinfectant exposure time , correct tourniquet usage ,the use of sterile and unbent needles ,discarding the puncture needle . Although the cohorts did not differ significantly in their communication , the number of medical students informing the patient about the procedure decreased by 8.1% during the second lockdown compared to the first.Medical students performed slightly better in OSCE after the first compared to the second lockdown  and successful venipuncture , the cohort who participated in OSCE after the first lockdown in March 2020 performed better than their peers partaking during the second lockdown.In terms of structured work . Materials needed for venipuncture were also more frequently adequately prepared during the second than during the first lockdown   Table .Table 2HSince the World Health Organization (WHO) classified COVID-19 as a pandemic, SARS-CoV-2 has impacted life around the world . WithoutAlthough the world’s population has been threatened by pandemics in every decade of the last 30 years, none had such a strong impact on daily life but also on the hygiene behavior of medical students. For example, during the H1N1 influenza pandemic, neither the awareness of H1N1 influenza increased nor the compliance to hygiene protocols by medical students  , 20, 21.During the H1N1 influenza pandemic, the perceived individual risk of infection appeared to be a strong indicator for the level of pandemic awareness and observance of hygiene behavior among medical students . Due to Overall, however, rates of hand disinfection after venipuncture were inadequate in all studied cohorts. This may be attributed to the utilization of noninfectious simulation manikins in this study. Hence, hand disinfection after venipuncture in this study served only a minor role in self-protection and self-cleaning, which serve as the main motivating factors for medical students’ hand disinfection after patient contact .Moreover, it remains unclear if and how how the hygiene behavior of medical students exposed to COVID-19 changes after the pandemic or the course of their studies. In the literature, medical students with more experience are associated with higher awareness and compliance to SARS-CoV-2 containment measures , 38. ThiSurprisingly, the pandemic-related improvement in hygiene compliance was not reflected in the overall venipuncture score, as medical students performed worse in terms of work structure, successful venipuncture, and patient education during the COVID-19 pandemic. The decreased doctor-patient communication could be a result of pandemic-induced psychological distress or stress due to a lack of hands-on practice opportunities since stress reportedly correlates with decreased doctor-patient communication , 45–49. Even though the pandemic positively impacted the hygiene behavior of medical students, their impaired practical skills must be addressed to restore the former standard of practical medical education and ensure patients’ well-being.This study found an overall increase in compliance with hygiene measures by medical students during the COVID-19 pandemic. Hand disinfection after patient contact was performed more frequently during the second lockdown as opposed to the first lockdown. It can be assumed that the COVID-19 pandemic and its containment measures increased medical students’ awareness of hygiene. However, it remains to be seen whether the hygiene compliance of medical students will persist after the pandemic. Regardless of this, measures should be taken to reinforce this behavior and pass it on to future generations of prospective physicians.The observed shortcomings of medical students during the pandemic in terms of structured work, doctor-patient communication, and venipuncture should be further investigated to identify possible strategies to compensate for these deficiencies going forward.Since this unicentric study was only conducted with medical students at the University of Cologne, a sampling bias cannot be ruled out. Thus, the results cannot be generalized to medical students from other locations.Furthermore, this study was realized in an examination setting, so the collected data may deviate from the behavior in the clinical setting. Also, medical students’ hygiene behaviors and practical skills are a dynamic process, so a single observation time points might not capture these skills accurately.It must also be assumed that the teachers as well as the data collectors could not escape the effects of the COVID-19 pandemic. Sensitization of the data collectors to hygiene during the COVID-19 pandemic, and thus an exaggerated assessment of medical students’ hygiene compliance by the same, thus cannot be ruled out. Another risk of a case control study could be that confounding factors might not have been identified, which in turn could have led to confounding bias.At the same time, the retrospective nature of this study only allows conclusions about the correlation of hygiene, practical skills, and the COVID-19 pandemic. Thus, possible causalities need to be investigated by further prospective studies.Additional file 1: Appendix 1. Rating of the 11-item questionnaire.Additional file 2: Appendix 2. Test of normal distribution prior and after the first COVID-19 lockdown.Additional file 3: Appendix 3. Test of normal distribution for the different student cohorts.


Pheochromocytoma, neuroendocrine tumor, single cell RNA-sequencing, transcriptome, heterogeneity, SDHB, RET, paraganglinoma; Pheochromocytomas (PC) and paragangliomas (PG) are rare neuroendocrine tumors with varied genetic makeup and are associated with high cardiovascular morbidity and a variable risk of malignancy. The source of the transcriptional heterogeneity of the disease and the underlying biological processes that determine the outcome of PCPG remain largely unclear. We focused on PCPG tumors with germline SDHB and RET mutations, which represent distinct prognostic groups with worse or better prognoses, respectively. We applied single-nuclei RNA sequencing (snRNA-seq) to tissue samples from 11 patients and found high patient-to-patient transcriptome heterogeneity in neuroendocrine tumor cells. The tumor microenvironment also showed heterogeneous profiles, mainly contributed by macrophages of the immune cell clusters and Schwann cells of the stroma. By performing non-negative matrix factorization, we identified common transcriptional programs active in RET and SDHB, as well as distinct modules, including neuronal development, hormone synthesis and secretion, and DNA replication. Similarities between the transcriptomes of the tumor cells and those of the chromaffin- and precursor cell types suggests different developmental stages at which PC and PG tumors appear to be arrested. Pheochromocytomas (PC) and sympathetic paragangliomas (PG) are rare neuroendocrine tumors that originate from chromaffin cell-related populations located inside or outside the adrenal glands, respectively. PCPG is associated with significant morbidity and mortality . The curA plethora of genes have been reported to be responsible for a diverse hereditary background in up to 40% of PCPG , 4. PCPGIn this study, we exploited recent advances in single-nuclei RNA-seq to compare the gene expression landscapes of PCPG with SDHB and RET germline mutations, explore transcriptional heterogeneity, and gain insight into the molecular basis of their different prognoses.Previously selected tissue blocks were transferred to the RadboudUMC biobank and stored at -80°C. Nuclei were prepared from the frozen tissues under RNAse-free conditions. Briefly, samples were cut into ~7 mm pieces and kept on dry ice. The pieces were minced in a pre-cooled douncer in 500uL ice-cold Nuclei EZ lysis buffer 5x with pestle-A and 10x with pestle-B. The suspension was passed through a 70 µm cell strainer, washed with 1.5 mL cold Nuclei EZ Lysis, and incubated on ice for 5 min. The lysate was washed in Nuclei wash/resuspension buffer  and passed through a 40 µm cell strainer. The nuclei were stained with DAPI. To exclude doublets and debris from the final mix and to precisely determine the number of loaded nuclei, we used FACS. A total of 15000 nuclei were sorted into a pre-cooled tube containing the RT-mix (RT-reagent + TSO + Reducing agent B) Immediately before loading the mix to one lane of the Chromium chip, 8.3 ul RT-enzyme was added to the mix, according to the standard protocol of the Chromium Single Cell 3’ kit (v2). All steps for library preparation were performed according to the manufacturer’s protocol. Paired-end sequencing was performed to sequence the prepared libraries using an Illumina NextSeq sequencer.Seurat package in R. To filter out low-quality cells, we first removed cells (nuclei) with less than 10% or more than 250% of the mean gene count (nFeature_RNA) within each individual library. The cell count and gene count information for the single-cell datasets of the PCPG samples are listed in Raw sequencing data were converted into FASTQ files using bcl2fastq. Reads were aligned to the human genome reference sequence (GRCH38) and counted using STAR. The CellRanger (10X Genomics) analysis pipeline was used for sample demultiplexing and single-cell gene counting to generate the gene-cell expression matrix for each library. The gene expression matrix was then processed and analyzed using the FindNeighbors and FindClusters functions with the resolution parameter set to 0.3. Marker genes that differentiated between clusters were identified using the FindAllMarkers function.Data were normalized, scaled to 10000 counts and log-transformed using the NormalizeData function of the Seurat package. Principal component analysis was performed on the scaled data with the 4000 most variable genes. Using the 15 first principal components, we calculated a UMAP representation of the data for visualization and calculated clusters using the FindAllMarkers function and were required to be expressed in at least 25% of the cells in a cluster with a minimal log expression difference of 0.25 between clusters. The gene sets were further filtered for p-values (< 0.05) and log2FoldChange >|1.2|.To identify the cell types, we used sets of well-established markers and annotated each cell type based on their average expression. Differentially expressed (marker) genes were determined using the inferCNV package  were inferred from single-nuclei gene expression profiles using the  package  using thRunNMF function of the swne  using the the swne  package To measure the similarity of a target single-cell transcriptome to a reference single-cell dataset, we used the logistic regression method described previously . BrieflyWe performed single-nucleus transcriptomic profiling (snRNA-seq) on resected tumor tissues from 11 treatment-naïve patients to generate a comprehensive PCPG atlas Table S1Stringent quality filtering yielded 50 868 nuclei, with an average of 1 800 genes per nucleus Methods, Table S2Based on canonical marker genes, we identified three major groups of cell types: neuroendocrine ), immune , and stromal (COL4A1 and COL1A2) cells (arrows). However, adrenal macrophages (colored green) derived from the same location but from different tumor samples were very different. (blue and red arrows). This suggests that the macrophage transcriptome not only has a strong locational component but also a tumor type-specific component. The T and B cells originating from different locations and mutation groups appeared rather similar as they clustered together. Finally, the annotation of the stromal group  to determine large-scale somatic chromosomal changes Figure 2In contrast to the extensive inter-individual and tumor-specific genomic aberrations, the inferred genomic profiles of tumor cells within each tumor population were largely homogeneous, suggesting that the genome remained largely stable following an initial catastrophic event.To assess the inter-tumoral heterogeneity between RET and SDHB PCPG tumor cells, we selected and re-clustered tumor cells. With this finer-grained resolution, we identified UMAP clusters that consisted of cells mostly from one patient. This impinged on both the UMAP plots annotated by patient IDs Table S3We aimed to determine the transcriptional programs that are active across tumor cells and then identify the programs that are differentially enriched between RET and SDHB tumors. We applied non-negative matrix factorization (NMF)  to the sHierarchical clustering of metaprogram-scored cells revealed two major clusters separating RET from SDHB tumor cells (The most pronounced differences in the average enrichment scores between RET (cluster 1) and SDHB (cluster 2) clusters were evident in the M2, M3, M4, and M5 metaprograms  were enriched in RET tumors. We also observed that ‘neuronal development’ was a highly represented transcriptional program in both PCPG tumor cells.The NMF analysis revealed several metaprograms that were associated with neuronal development but showed different enrichment scores among the mutation groups. This implies that the developmental signature is an important element of the tumor cell transcriptome; however, the differences between the mutation groups were not reflected in the ontology terms. To shed light on the developmental aspects of SDHB and RET-PCPG tumors, we compared the transcriptome of the cell types identified in the developing human adrenal gland . This is the first study to reveal PCPG heterogeneity and the consequences of germline mutations at the single-cell level.Neuroendocrine cells, the largest population in the dataset, were identified as tumor cells based on marker genes, and in particular, by inferring copy number variations from gene expression levels . The iCNTo identify the patterns of the single-nuclei transcriptomic profiles based on tumor cells, we applied NMF, an unsupervised learning approach that is employed to approximate high-dimensional datasets in a reduced number of meaningful components , 20, 21.To explore the developmental status of the tumor, we used published datasets of the developing adrenal gland as a reference. Logistic regression analysis revealed that RET-PCPG tumor cells are transcriptionally more similar to developed adrenal chromaffins, whereas SDHB-PCPG tumor cells appear to be in an earlier phase of adrenal development. Our results suggest that PCPG tumor cells had a primarily chromaffin-like phenotype, suggesting that the chromaffin cell development state may be related to mutation-associated prognosis.In summary, we revealed extensive levels of heterogeneity among PCPG tumor cells and identified transcriptional programs related to neuronal development as key processes in these tumor cells. We speculate that in RET-PCPG, the mutation caused a development block during late chromaffin development as compared to the ‘more immature’ SDHB-PCPG tumors. To differentiate this developmental block from alternative transformative events that could also lead to modified transcriptomes of tumor cells, investigation of larger cohorts is needed. Understanding the origin of the tumor and sources of its heterogeneity may help in the development of targeted therapies.https://ega-archive.org .The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by CMO Regio Arnhem-Nijmegen (Gen-omgevinginteracties in de pathogenese van urologische zieken), CWOM-nr: 9803-0060. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.PB, CR, WM, HS and HT drafted the manuscript. HT provided access to samples. PB and CR processed the samples and prepared single nuclei RNAseq libraries. PB, CR, WM and HS contributed to data analysis and interpretation. All authors contributed to the article and approved the submitted version.Radboud University, Nijmegen, The Netherlands). P.B. was supported by the Princess Maxima Center and the Paradifference Foundation. C.R.M. was supported by the Princess Maxima Center and the European Union’s Horizon 2020 Skłodowska-Curie Actions (project AiPBAND) under grant #764281. W.M. was supported by the Italian National Operational Programme on Research 2014-2020 (PON AIM 1859703-2). H.G.S. was supported by the Princess Maxima Center and KiKa (Kinderen Kankervrij).version.The present work was funded by the Paradifference Foundation under the coordination of Peter M T Deen (The authors thank the patients and their families for supporting this research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.