The synthesis of Graphene is critical to achieving its functions in practical applications. Different methods have been used to synthesis graphene, but graphite exfoliation is considered the simplest way to produce graphene and graphene oxide. In general, controlling the synthesis conditions to achieving the optimum yield, keeping the pristine structure to realize on-demand properties, minimum layers with the smallest lateral size, and minimum oxygen content are the most obstacles experienced by researchers. Each application requires a specific graphene model, graphene oxides GO, or even graphene intercalated compounds (GIC) depending on synthesis conditions and approach. This paper reviewed and summarized the most researches in this field and focusing on exfoliation methods. It is known that graphite and diamond are the two natural crystalline allotropic forms of carbon for a prolonged time. The atomic arrangement of carbon atoms in these materials gives different properties. For example, graphite is soft and black, and stable, while diamond is hard and transparent [1]. Diamond and graphite consist of extended networks of spAllotropes are elements that are chemically identical but vary in their physical properties. In 1985, the discovery of fullerenes by Kroto et al. [3] marked the beginning of an era of synthetic carbon allotropes with striking properties. Iijima, in 1991, discovered carbon nanotube (CNT) [4]. While in 2004, graphene, which is a two-dimensional form of graphite, was isolated at Manchester University by Novoselov et al. [5]. 2 graphitic materials, including (0D) fullerenes, (1D) carbon nanotubes, and stacked into (3D) graphite [7].Graphene is a 2-dimensional structure consists of a single atom thick sheet of carbon crystallized as a honeycomb structure monolayer [6]. Graphene has been considered as the fundamental building block for all spn . The physical, chemical and mechanical properties of the allotropes of carbon  are different depending on valence bond hybridization [8].Allotropes of carbon can be defined by valence bonds hybridizations spGraphite has a layered planar structure wherein each carbon atoms layer is arranged in a honeycomb lattice with a separation of 0.142 nm. The interplanar spacing, d, is (0.335nm) [9]. In diamond, the carbon atoms are arranged in a face-centered cubic crystal structure (FCC) system with diamond lattice [10]. Fullerenes are zero-dimensional hollow carbon cage structures with carbon atoms arranged in a lattice similar to the soccer-ball. The most widely publicized fullerene is buckyball (C60). Carbon nanotubes (CNTs) are one dimension structure that can be viewed as a hollow cylinder formed by rolling a two-dimensional graphene sheet into a cylinder with a half fullerene at its ends. Carbon nanotubes are either single-walled CNTs (SWNT) or multiwalled CNTs (MWNT) [11]. Graphene is a flat single atom thick sheet of carbon packed into a honeycomb crystal plane and has a 2D structure [6]. Due to the outstanding features and large applications, Graphene has attracted great interest from scientists and engineers [7]. Moreover, it is the thinnest and strongest material that is ever subjected to measurement [12]. Graphite is considered to consist of huge numbers of graphene layers. There are two kinds of bonds form among the graphene layers. The graphene layers are held together by the weak Vander Waal attraction with a length of about 0.341nm between the adjacent graphene layers [13]. This weak attraction between layers can ease the exfoliation into individual layers. The other bond is between carbon-carbon atoms with 0.142 nm spacing, which is a strong covalent bond entire each layer [14]. Graphenes can exist in different forms: graphene oxide, chemically modified graphenes, bilayer graphenes, etc. These nanomaterials have many medical applications, integrated circuits, transparent conducting electrodes, desalination, solar cells, energy storage, biodevices, etc. [15].2/V.S) [18], which means an electron moves through it without much scattering or resistance. Graphene has a high specific surface area of 2630 m2/g [19]. The unique structure enables graphene of many distinctive properties and promising applications. Graphene is the strongest material ever measured and has excellent mechanical properties with an elastic modulus of 1 TPa and ultimate tensile strength of 130 GPa [20]. Thus, graphene is the strongest material ever discovered to date. It is estimated that graphene is 100 times stronger than the best steel ever for the same thickness [17]. Thus, graphene is a promising material for high-performance nanoelectronics, heat dissipation, sensors, field emission, and transparent conductor. One single-layer of graphene has a transparency of 97.7% of incident light and absorbs only 2.3% and, it is almost completely transparent [21]. Due to its unique thermal, electrical, and mechanical characteristics, Graphene has been utilized in many applications. A Single-layer of graphene has thermal conductivity as high as 5000 W/m.K [16] and higher about 10 times than Cu and superior to carbon nanotubes (CNTs) and diamond [17]. Graphene has high mobility of charge carriers , and electrochemical are the most commonly used methods.4.1. Chemical vapour deposition (CVD)The chemical vapor deposition (CVD) on transition metals  is another method used to prepare graphene, which requires a temperature as high as 1000 °C with a hydrocarbon gas flow as precursor . This method is applied at high temperatures when exposing transition metal to low concentration hydrocarbon gas till the surface is saturated with carbon atoms  .Methane gas is generally used as a source of carbon. Hydrogen and argon are also used with methane as reaction stabilizers and enhancing the film uniformity. Zhan et al. in 2018 [40] showed a strong correlation between substrate morphology and graphene domain density. The use of a pre-annealed Cu foil followed by electro-polishing treatment will give ultra-smooth Cu surfaces. The graphene grows on annealed electro-polished Cu substrates show better quality in terms of lower domain density and higher layer uniformity than those grown on Cu substrates with only annealing or only electro-polishing treatment. Well-ordered graphite films of nanometer thickness have been grown on Ni substrates using chemical vapor deposition (CVD) with a thickness of a few graphene layers with graphite film thickness is about 1.5 ± 0.5 nm [41].Ni as the catalyst has some limitations such that an over a small region of few to tens of microns, single and few-layered graphene is obtained and not homogeneously throughout the entire substrate. The control lack over the number of the layers is partial because, upon cooling, the carbon segregation from the metal carbide happens rapidly within the nickel grains and heterogeneously at the grain boundaries. The use of polycrystalline copper foils as a substrate in CVD gave exceptional results in uniform deposition of high-quality single-layered graphene over large areas [30]. This method can produce high-quality graphene and suitable for mass production, but it is not cost-effective and easy. The availability of high-quality and large-area graphene is a crucial requirement for the production of graphene-based nanoelectronics. As discussed above, the other methods for graphene production have their limitation. The CVD method [38] requires a purification process to eliminate the catalyst particles and transfer Graphene to another substrate; the chemical reduction of GO requires chemicals, which can affect graphene properties; the scotch tape method [5] is not suitable for large scale production of Graphene. The field of epitaxial graphene on silicon carbide is rapidly growing by annealing SiC as an insulator at high temperatures under vacuum to form a graphitic surface by sublimating Si atoms [42]  [43]. The potential of epitaxial graphene as new electronic material is now well recognized [44]. Silicon carbide (SiC) was used as substrates for the epitaxial growth of graphene. The substrate is heated at high temperatures (>1250 °C) and low pressures (~10−6 Torr). Ultrathin epitaxial graphite films were grown by thermal decomposition on the (0001) surface of 6H-SiC. The films are typically composed of 3 graphene sheets [45]. The graphene epitaxial growth on silicon carbide is considered an ideal material for high-end electronics that might surpass silicon in terms of key parameters such as speed, feature size, and power consumption [44]. The epitaxial growth method can prepare high-quality graphene with excellent properties, but it is not easy to obtain a high production rate with this method.Kretschmer et al. [46] were the first that utilized the arc-discharge method to synthesize C60 [47]. The main concept of graphene synthesis by arc discharge is similar when applying an electric field between two graphite electrodes under argon or helium gases. When the electrodes’ distance between the two electrodes becomes close to 1–2 mm, the arc discharge starts, and the evaporated materials from the anode are deposited on the cathode. The anode is almost previously compounded with catalysts . Also, the cathode should be rotated to keep the distance constant [48]. The arc discharge process is illustrated in Figure 4.2SO4 (90%) /HNO3 (70%) [49]. In comparison, Kosynkin et al. (2009) obtained oxidized graphene nanoribbons by suspending MWCNTs in concentrated H2SO4 and then treated with KMnO4. The isolated graphene nanoribbons were highly soluble in water, ethanol, and other polar organic solvents [50]. The nanotubes’ unzipping seems similar to the ‘unzipping’ of graphite oxide [51] and [52]. The opening could occur longitudinally or spirally way, depending on the attacked site and chirality angle. Manganate ester formation (O=MnO-2=O) is the main reason to destroy the alkene structure of CNT, making these initial attacked sites more exposable to the next intensive reaction with permanganate [50]. Figure 5 illustrates the proposed unzipping mechanism of CNTs [50]. Saito et al. (2002) showed that MWCNTs could be cut to several nanometers of CNTs by sonication in mixed strong acids (H4.5.1. Graphene oxide3 and fuming nitric HNO3 + NO2. Staudenmaier developed this method later in 1898 [54]. Another improvement in the Brodie method was made by Hofmann and König (1937) [55]. Hummers and Offeman made further development on the Brodie method [56]. Marcano et al. [57] improved on the Hummers method by eliminating sodium nitrate, thus excluding toxic nitrous gas production.Large amounts of graphene can be produced at a reasonable cost from graphite oxide. Graphene oxide can be prepared by oxidation of graphite using concentrated acids and strong oxidants . The oxidation of graphite to form graphite oxide was introduced in 1859 by Brodie [53] when graphite was oxidized in the presence of potassium chlorate KClOIn these modifications, the key developments concentrate on the use of safer oxidants and more effective procedures. Staudenmaier and Hofmann’s methods use potassium chlorate, and the methods of Hummers and Tour use potassium permanganate as the primary oxidant. Nitric, sulphuric and phosphoric acids or their mixtures are used as solvents in both synthesis methods.4, NaNO3, and H2SO4 to oxidize graphite into graphite oxide within a few hours. The advantages of Hummer’s method are as follows. First, the reaction is completed within a few hours. Second, the oxidizing agent KMnO4 is used to replace KClO3 and improving reaction safety by avoiding the evolution of explosive ClO2 and KClO3. Third, the use of NaNO3 to replace HNO3 eliminates the forming of acid fog [58].Brodie was the first to prepare graphite oxide by oxidation of graphite in a mixture of potassium chlorate. An oxidizing agent and nitric acid helped to intercalate graphite and expand it [53]. Later on, Staudenmaier et al. [54] used nitric acid: sulphuric acid with a volume ratio of 1:4 in an ice bath. Then, graphite was added (4.37 g for every 100 mL) with continuous stirring. The oxidizing agent is potassium chlorate. The weight ratio of graphite to the oxidizing agent is 1:11. Staudenmaier’s method is considered safer than the Brodie method, but toxic gasses are still created; this is why using the Hummers method more than Staudenmaier and Brodie method. In 1958, Hummer developed a new method [56], which is the most common method for preparing graphene oxide using KMnO2, N2O4). In this method, sodium nitrate (NaNO3) was replaced by potassium permanganate. The mixture of reactions consists of (9:1) H2SO4: H3PO4 for the oxidation reaction [57].Later on, a new method was developed by Tour and his group in 2010 to improve the Hummers method in the degree of GO oxidation as well as to minimize the generation of toxic gases (NO2- and sp3- hybridized carbon (C) atoms decorated with oxygen-containing functionalities. These functional groups include hydroxyl (C–OH) and epoxide (C–O–C) functional groups on the basal plane and carbonyl (C=O)) and carboxyl (COOH) groups at the sheet edges [60]. These functionality groups introduce sp3 defect sites to the nanosheets, distorting the intrinsic conjugated π system and lowering overall strength and conductivity. The saturated sp3 carbon atoms bound to oxygen make GO an insulator. Graphene oxide is an electronically hybrid substance that displays both a large energy gap between the σ-states of its sp3- bonded carbons and conducting π-states from sp2 carbon sites [61]. By reducing chemistry, the tunability of the ratio of the sp2 and sp3 fractions is a powerful way to tune its bandgap and, thus, controllably transform graphene oxide from an insulator to a semiconductor or semi-metal [62].Graphene oxide is a single layer of graphite oxide. The chemical oxidation of graphite and its subsequent exfoliation in water results in single-layered GO sheets [59]. Graphene oxide contains a mixture of spWith the recovery of a conjugated structure, graphene oxide can be reduced to graphene by eliminating the oxygen-containing groups. Reduced GO (rGO) sheets are generally referred to as chemically modified graphene, chemically transformed graphene, or reduced graphene oxide [62].2 conjugated graphene network [62]. In thermal or chemical reduction, the functional groups C=O and O=C-OH could be partially removed or converted to a new chemical species such as (C-OH). The degradation of the electrical properties of the RGO is due to the remaining functional groups C-OH or C-O-C in RGO that could not be reduced easily [66].Graphene oxide is an electrically insulating material but can be converted to a conductor by chemical reduction process, electrochemical reduction, thermal treatment, and green reduction , as shown in Figure 7. However, none of the reported reduction methods yield complete oxygen removal, resulting in only partial restoration of the sp2 gases. These gases create large pressure between graphene oxide stacked layers, which are enough to exfoliate individual GO layers is enough to separate two stacked GO sheets [67].Thermal reduction of GO was made by conventional heating  [67], microwave [68], and photo-reduction [69]. In thermal annealing, GO is heated to a high temperature under inert gases such as argon gas. The rapid increase in temperature makes oxygen functional groups decompose into CO and COMicrowave irradiation (MWI) is another thermal reduction method of GO using a microwave oven. This is a rapid method and gives uniform heating of the GO [68]. Zhang et al. proposed another method: a photo-reduction of GO using femtosecond laser irradiation [69].2NH2.xH2O) and dimethyl-hydrazine, can be used as a chemical reducing agent for GO.Another method of GO reduction is the chemical method, which can be carried out using a chemical reducing agent. This is an effective method for mass production of graphene because of availability and low cost compared with other reduction methods [65]. Stankovich et al. were the first to prepare reduced graphene oxide (rGO) [36] using hydrazine as a chemical reducing agent. Hydrazine and its derivatives, such as hydrazine hydrate  by donation of electrons with acceptance of oxygen while it was converted into an intermediate oxidized polymerized thiophene that losses sulfur atoms and transformed into polyhydrocarbon that heat-treated to obtained reduced GO [71]. M. J. Fernandez-Merino et al. used different reducing agents sodium borohydride, pyrogallol, and vitamin C, in addition to hydrazine and made a comparison among them. They found that vitamin C has highest yield suspension compared with other compounds [72]. T.T. Dang et al. reported the influence of temperature on the reduction of graphene oxide by hydrazine in N, N-dimethylformamide (DMF)/HR. Tarcan et al. reduced graphene oxide in N, N-dimethylformamide without using any reducing agents using the solvothermal microwave-assisted method. So, this method gives stable and highly concentrated dispersions of GO through partial removal of oxygenated groups from graphene oxide surface [74].The electrochemical reduction of GO relies on removing oxygen functional groups using regular electrochemical cells containing aqueous solutions (electrolyte) at ambient temperature . Graphene oxide reduction occurs by the exchange of electrons between the inert electrodes and GO sheets or films. This method eliminates the usage of dangerous reducing agents as hydrazine.The green synthesis approach of graphene reduction is rapidly improved due to its low cost, ease of handling. It is an eco-friendly method without hazards, harsh, and expensive materials [77]. Biomolecules, microbes, and plant extracts are used to reduce and cap agents widely used to synthesize metal nanoparticles. Generally, plants contain a wide variety of bio-molecular substances, including amino acids, proteins, alcoholics, polysaccharides, enzymes, polyphenols, and vitamins. Plant extracts may also be used for graph synthesis as a reduction and stabilization agent . Ascorbic acid or (Vitamin C) is a recently reported reducing reagent for graphene oxide with benefits over hydrazine [72]. Ascorbic acid is nontoxic and has higher chemical stability with water so that the produced RGO does not result in aggregation. Taking into consideration the environment and human health in recent years, reduced graphene oxides (RGOs) were produced by green reduction of graphene oxide (GO) using ascorbic acid [72], glucose [80], fructose and sucrose [68], tea leaves [77], melatonin [79] and alcohols [81].2 in size [83]. This method is called the Scotch tape approach, which is not suitable for large production of graphene.Geim and Novoselov [7] prepared a single graphene sheet by peeling off a sheet of graphite using Scotch tape  [82]. This method involves repeatedly peeling highly oriented pyrolytic graphite (HOPG) using scotch tape. The process has been optimized to produce single-layer graphene (SLG) with high structural quality and more than 100 μm2/g), and enhanced surface activity [84]. The increase in the surface area of material can enhance their chemical and physical reactivity. This is very vital in many applications where the surface activity is important such as catalytic materials, ion exchange [85], fillers materials in composites [86], intumescent  material [84].Layered materials such as graphite consist of two-dimensional platelets weakly stacked to form three-dimensional structures. In graphite, these layers form strong chemical bonds in-plane but display weak out-of-plane bonding. This facilitates the exfoliation process to form so-called nanosheets (about micrometers wide but less than a nanometer thick). Such exfoliation leads to an increase in the material’s surface area, over 1000  is a top-down method of exfoliation of natural graphite into few-layer graphenes dispersed in liquid media [87] and are becoming more desirable because they are cost-efficient, using inexpensive graphite, scalable process, produce defect-free graphene, and can be used to deposit the dispersed graphene in a variety of environments and on different substrates not available in using mechanical cleavage or growth methods [88–90].This process involves graphite dispersal in a solvent, ultrasound exfoliation or other mechanical dispersal procedures, and purification. The involved mechanisms of fragmentation and exfoliation have usually been attributed to the force induced by ultrasound and the interaction with the solvent molecules . Liquid phase exfoliation is a four-step method as illustrated by Shen and collaborators [93–95] that include: 1) graphite immersion into the liquid medium, 2) intercalation of the graphite, 3) the exfoliation of these planes, and 4) the stabilization of the exfoliated materials isolated in the medium. In step one, the liquid medium for graphite immersion should effectively reduce van der Waals forces’ attraction between the layers of the nonexfoliated material. The liquid molecules must be inserted between the layers of the crystal before exfoliation. During the intercalation step of the graphite, the interaction between the graphite graphene planes is decreased. In the third step, the exfoliation of these planes occurs, which can be mechanically assisted or not. When 2D material with the liquid molecules inserted is exposed to a mechanical fragmentation force such as shear, van der Waals’ attractive forces can be overcome, facilitating the individual layers’ exfoliation. Finally, in the fourth step, the exfoliated material isolated in the liquid medium should be remaining stable. A fundamental characteristic of this method is the possibility of producing mono and few-layer graphenes stabilized in different media [93–95]. Different precursors of graphitic material are used in Liquid-phase exfoliation, such as graphite oxide, natural graphite, graphite intercalation compound (GIC), and sonication-free liquid-phase exfoliation. These graphite precursors are directly subjected to a solvent treatment to weaken the van der Waals attractive forces between graphene interlayers using external driving forces such as ultrasonication, electric field, shearing, or heat  [96].Liquid-phase exfoliation of graphite oxide is the most widely used method for the preparation of graphene. In this method, the graphite is intercalated with strong oxidizing agents , followed by graphite layers’ expansion via sonication. The reduction of the obtained graphene oxide to graphene is usually conducted by either thermal or chemical approaches [36]. To exfoliate, it is important to resolve the attractions of the van der Waals between adjacent layers. One way to reduce these attractions by oxidation and chemical intercalating reactions is to increase the distance between adjacent layers .The oxygen functional groups such as hydroxyl and epoxy are inserted and attached to graphite layers during graphite oxidation. The d-spacing between these layers has increased significantly from 3.4 to around 7.0 A .The hydroxyl or carboxyl groups covalently bonded with graphene oxide make graphene oxide dispersable in some organic solvents at concentrations of up to 1 mg/mL [36] and water up to 7 mg/ mL [99]. However, the oxidization process significantly causes defects in graphene. Stankovich et al. [36] used hydrazine to reduce GO, and the chemically reduced graphene product (CRG) still has some defects. Other reduction methods are as follows: the solvothermal reduction method of GO in N, N-dimethylformamide (DMF) with hydrazine [100] or without hydrazine [101] as a reducing agent, the use of metal Fe to reduce exfoliated GO [102]. None of these reduction methods can recover the graphene structure completely, and some oxygen-containing groups are still irremovable.The liquid phase exfoliation method and exfoliation of GO of graphite are technically similar, but the first does not involve the oxidation step. Graphene flakes can be produced by exfoliation of graphite using chemical wet dispersion followed by ultrasonication in water and organic solvents. This method’s principal attraction is a simple and scalable process where pristine graphite or expandable graphite is subjected to a solvent treatment to weaken the van der Waals attractive forces between graphene interlayers. External driving forces such as ultrasonication, shearing, or electric field can be applied to help exfoliation into graphene [92]. Graphene’s exfoliation occurs because of the strong interactions between the solvent molecules and the graphitic basal planes, overcoming the energetic penalty for exfoliation and subsequent dispersion. For successful exfoliation, overcoming the van der Waals attractions between the adjacent layers of graphite is necessary.Graphite intercalation compounds (GICs) are formed by the insertion of atomic or molecular layers of a different chemical species  called the intercalant between host graphite sheets [103], see Figure 10. The graphite intercalation compound (GICs), also known as the expanded graphite, is a synthesized intercalation compound of graphite that expands or exfoliates when heated. The intercalation increases the distance between the graphite layers. In pristine graphite, the distance between the layers is typically 0.34 nm, while in a potassium-GIC, the sheet distance has been measured to about 0.53 nm [103]. Because of the increased distance between the graphite layers in the GIC, the van der Waals attraction between the layers was weakened. Thus, the layers can be more easily separated to acquire the desired graphene [104]. The number of layers separated by an intercalant layer is defined as staging. The stage index n denotes the number of graphite layers between adjacent intercalate layers of structural ordering. Fully intercalated graphite will be a stage 1 GIC [105]. The graphite intercalation compounds are of particular physical interest because they have a relatively high structural ordering degree. Although graphene is a material with a single carbon layer, the definition has been broadened in recent years, and the term ‘’few-layer graphene’’ is often used. If the number of carbon layers is 10 or more, the piece is known as a graphite nanoplatelet (GNP), also known as an exfoliated graphite nanoplatelet (GNP) or a graphite nanosheet (GNS) [106].Güler et al. 2016 [107] prepared multilayer graphene by mixing hexagonal graphite (HG) with an acidic mixture to form graphite- intercalated compound (GIC). The GIC is then heated at a temperature between 800–900 ºC to form expanded graphite (EG), followed by dispersion in an organic solvent through ultrasonication.The exfoliated graphite exhibited excellent properties of flexibility, lubricity, and adsorption. The exfoliated graphite has many applications in various fields, such as absorption of spilled oil in water , sealing material [110], fire retardant [111], and electrode [112]. 2 . Graphite’s surface energy is the minimum energy per unit area sufficient for the van der Waals force when two sheets are peeling apart. Graphene and solvent surface energies have to be very close for a successful exfoliation process. The enthalpy of mixing will be reduced, and the exfoliation will increase. Liquids with surface tensions of about 30–40 mJ/m2 are typically good solvent candidates for graphene exfoliation, such as N-methyl-2-pyrrolidone (NMP 40 mJ/m2) [33], N, N-dimethylformamide (DMF 37.1 mJ/m2) [113].Graphite can be exfoliated into single-layer graphene (SLG) and few-layer graphene (FLG) in a solvent having a surface tension of about 40 mJ/mSonication-assisted liquid-phase exfoliation of graphite to give Graphene was first reported by Hernandez in 2008 [33]. In their work, graphite powder was dispersed in a specified organic solvent, such as N, N-dimethylformamide (DMF), and N-methyl pyrrolidone (NMP), followed by sonication and centrifugation. The LPE yield is estimated by measuring graphene concentration in the dispersion using UV-Vis spectroscopy and exploiting the Beer–Lambert law. The degree of exfoliation was determined by analyzing the number of layers in a graphene flake using TEM, AFM, and/or Raman spectroscopy. Defect-free monolayer graphene, bi-layer, and multilayer graphene are obtained. The solubility of graphene in the dispersion was 0.01 mg/mL after 30 min sonication, centrifugation at 500 rpm for 90 min. The number fraction of monolayer graphene in NMP dispersions was estimated at 28%. It was found that NMP gives the best thermodynamic stabilization due to the good matching of graphite –solvent surface energies. The most important criterion for successful graphite exfoliation is the good matching of solvent surface energies with graphene. The best solvent is that with surface energies close to the graphene surface energy [114–116].Graphite consists of many graphene layers joined together by van der Waals attractions between the adjacent layers. Successful exfoliation of graphite requires the overcoming of the van der Waals attractions forces between the adjacent layers. When graphite is immersed in a liquid, the interfacial tension between graphite and solvents should be equal to the graphene-graphene interaction energy [117]. A very close graphite- solvent surface energies resulted in small mixing enthalpy, and the exfoliation occurs more easily [118]. Good solvents are used to disperse graphene minimizing the interfacial tension between the solvent and graphene flakes. The liquid’s surface tension is the force required to resist an external force due to liquid molecules’ cohesive nature. If the interfacial tension between graphitic flakes and the solvent is high, there is poor dispersibility of the solvent’s graphitic flakes [114]. The flakes tend to adhere to each other due to the flakes’ cohesion force and hindering their dispersion in the solvent [119]. Hernandez et al.  reported that solvent with surface tension 40 mN/m is the best solvents for dispersing graphene and graphitic flakes. This will minimize the interfacial tension between the solvent and graphene [119]. It has to be noted that direct LPE in organic solvents like NMP normally produces graphene flakes with lateral dimensions typically < 1 μm, which are too small for applications such as mechanical reinforcement in composites.The solvent molecule-graphene interactions were investigated by Liu et al. [120] using electrophoresis and zeta potential measurements. The charge transfer between the graphene and solvent molecules is responsible for the graphene’s surface charge. Graphene can be positively or negatively charged in the dispersion exhibiting different zeta potential values depending on the different donor and acceptor numbers of a solvent. The electrostatic repulsion of charged graphene sheets will ease the stable dispersions. 2 are the solvents’ toxicity. NMP organic liquid is an eye irritant and also toxic to the reproductive organs [121]. DMF may have toxic effects on multiple organs [122].The main disadvantages of using a solvent with a surface tension of 30–40 mJ/mThe other disadvantages of this method are the low stability, low single-layer content (0.01 mg/mL), and the solvents’ high boiling points make it very difficult to remove the solvent after exfoliation [33]. Therefore, the search for additional solvents is highly recommended to overcome the low yield problem associated with graphene dispersions in organic solvents.Utilizing ionic liquids (ILs) in graphite exfoliation is considered green chemistry due to it is nontoxic, stable chemically, no need for vapor pressure, recyclable by distillation or ion change. Exfoliation by ionic liquids is also considered a one-step procedure (direct exfoliation). Ionic liquids give graphene unique properties, unlike chemical modification that destroys the graphene electronic structure. Ion liquids are completely organic or partially inorganic salts with a melting point below (100 °C). The most significant factor in this technique is the surface tension of ILs, which has to be very close to that of graphene .Fukushima et al. were the first to use ILs to untangle carbon nanotubes using imidazolium-based ILs as solvents assuming interactions between the positive charge of the imidazolium rings and p electrons of the nanotube [125]. Liu et al. prepared graphene nanosheets using imidazolium ILs assisted electrochemical synthesis [126]. Zhou et al. incorporated polymerized ionic liquid with 1-butyl-3-methylimidazolium hexafluorophosphate to disperse graphene. Nonoxidized few-layer graphene highly concentrated and stable colloidal (0.95 mg/mL) was prepared by ultrasonication of natural graphite flakes in this ionic liquid [127]. Nuvoli et al. prepared high concentration few-layer graphene sheets using LPE by grounding and then sonication of graphite in 1-hexyl-3-methylimidazolium hexafluorophosphate (HMIH) as an ionic liquid. The graphene concentration (up to 5.33 mg/ mL) is obtained. Ionic liquids can be useful when green chemistry and/or a nonvolatile, stable solvent medium is required [124].In 2014, Shang et al. prepared large quantities of solvent-free graphene nanosheets and nanodots with low oxygen content have by mechanical grinding exfoliation of natural graphite in a small quantity of ionic liquids  [128]. Electrochemical exfoliation of graphite rods was used to synthesis carbon dots (CDs) by dissolved cheap salts (NaCl and KCl) in distilled water as an electrolyte [129]. Generally, exfoliation using ionic liquids has been associated with other techniques as electrochemical exfoliation, mechanical grinding, or sonication to be more producible.Liquid phase exfoliation by a surfactant is considered an ideal method to prepare high stable concentrated graphene dispersion despite the surfactant’s defects [130]. Six surfactants were utilized involving ionic and nonionic: sodium deoxycholate SDBS, sodium dodecyl sulfate HTAB, Ssodium dodecyl benzenesulfonate SDS, hexadecyltrimethylammonium bromide, and Triton X-100 and Tween 80. The theory explains the different ionic and nonionic surfactants’ different mechanisms in stabilizing graphene dispersions for colloidal stability [130]. Commercially, there are around 20–30 available surfactants that have been used to exfoliate graphite. The main types of surfactants are cationic, anionic, and nonionic surfactants. Sodium dodecyl sulfate (SDS) is an anionic surfactant with a small molecule with a hydrophobic tail and a polar head group [131]. In addition to previous classes, pluronic and tetronic block copolymers were used to produce graphene dispersions with concentrations exceeding 0.07 mg mL-1 via sonication and centrifugation [132].Ionic surfactants such as cetyltrimethylammonium bromide (CTAB) [133], sodium dodecylbenzene sulfonate (SDBS) [33], sodium cholate (SDOC), and other surfactants [134] were used to exfoliate graphite flakes. Ultrasound treatment of graphite dispersion in N-methyl pyrrolidone solution yields a dispersion with a concentration of 2.21 mg/mL [135]. Graphene dispersed in a monomer (tetraethylene glycol diacrylate), and the latter’s subsequent polymerization yields a dispersion of 9.45 mg/mL [136]. For graphene dispersions in two organosilanes, 3-glycidoxypropyl trimethoxysilane (GPTMS) and phenyl triethoxysilane (PhTES), the concentrations were 0.66 and 8.00 mg/mL for PhTES and GPTMS, respectively [137]. Guardia et al. examined the differences between ionic and nonionic surfactants and showed nonionic surfactants’ dominance [138]. Niu et al. reported a low-cost and environmentally friendly method to directly exfoliate graphite powders into few-layer graphene sheets using inorganic salts to expand graphite for easy exfoliation by low-power ultrasonication [139]. The addition of ethanol to water/surfactant solutions can lead to a high concentration (∼0.46 mg/ml) of graphene dispersions. About 10 wt% of ethanol addition for ionic and nonionic surfactants was found to enhance the exfoliation efficiency and the graphene concentration three times. This enhancement is due to decreasing mixing enthalpy and enhancing the stability of surfactants .A simple and inexpensive green route for large-scale production of exfoliated graphene dispersions using direct sonication of graphene and aqueous solution of perylene tetracarboxylate (PTCA) aromatic semiconducting surfactant. This produced a high yield of single and few-layer graphene sheets with minimal basal plane defects [142]. A surfactant-assisted ultrasonic exfoliation of mildly oxidized graphite has been successfully demonstrated using cationic surfactants and one anionic surfactant. The surfactant charge was found to be a significant factor in affecting the exfoliation efficiency [143].One of graphene’s solvent exfoliation problems is that the good solvents tend to have high boiling points that present problems for flake deposition, so they also are difficult to remove. Low boiling point solvents such as chloroform, isopropanol, and 1-propanol were explored by many researchers for LPE production of graphene because of the removal difficulty of high boiling solvents like DMF NMP, etc. . Catheline et al. prepared individualized graphene flakes as single and double layers with one micron as average lateral size by dissolving the graphite intercalation compounds KC8 in low boiling points ether solvents such as methyl-tetrahydrofuran , tetrahydrofuran , and cyclopentyl methyl ether . This method is expected to significantly benefit composites and thinner film preparation due to high dispersity, stability, or double graphene levels [146].2SO4 solution. The exfoliated graphene sheets exhibit lateral size up to 30 μm [150]. Although this method is easy to implement and prepare graphene, the obtained graphene still has many oxygen functional groups and structural defects due to the oxidation reactions at the graphite anode. When a voltage is applied in a conductive solution for the graphite electrodes, the graphite electrodes can corrode and produce the solvent-functioning graphite nanosheets  [147]. The use of electrochemical exfoliation of graphite anode to prepare graphene was first reported by [126] using a mixed solution containing ionic liquid and water as the electrolyte. Structure with up to 10 graphene layers is called few-layer graphene, while the structure with more than 10, and less than 100 graphene layers is called thin-film graphite [148]. Liu and his group [149] believed that electrochemical exfoliation would play more critical roles in synthesizing graphene materials, but many exciting challenges are still waiting to be explored. These challenges include a better understanding of graphite exfoliation reaction mechanism, parameters that affect large-scale graphene production, such as complex fluid mixing, heat, and mass transport. Finally, the obtained graphene material is suitable for many applications with unique features. For example, in membrane applications, graphene materials must behave tailored size, interlayer spacing, and porosity. Su et al. prepared high-quality graphene sheets by electrochemical exfoliation of graphite. The electrochemical cell consists of graphite anode and platinum cathode in a 0.5M of HMorales et al. used an electrochemical route to produce few-layer graphene from electrochemically-produced graphite intercalation compounds in aqueous perchloric acid. Anodic intercalation is more efficient in terms of time. To prevent the formation of graphite oxide, cathodic pretreatment is preferred. Experimentally, it was found that using electrochemical-potential control is very promising to obtain graphene with different degrees of oxidation [151].An electrochemical route for producing graphene sheets by electrochemical intercalated sodium dodecyl sulfate (SDS) into graphite followed by electrochemical exfoliation of an SDS-intercalated graphite electrode was reported by Alıoğlu et al. [152]. The structural order, size, and a number of layers of synthesized graphene sheets depend on the potential value for SDS intercalation into graphite. Graphene sheets with the highest structural order and lowest number of layers can be obtained using relatively high intercalation potentials. The average size of 500 nm and a monolayer of graphene sheets were prepared at high SDS intercalation potentials into graphite. After successive electrochemical intercalation and exfoliation processes, UV–vis spectra of graphene/SDS suspensions showed a large amount of the reduced form of graphene flakes was obtained [152].4)2SO4 aqueous solution was reported by Chen and Xue [153]. The proposed electrochemical exfoliation mechanism showed that SO42− and H2O could be intercalated into those graphite sheets. Monolayer and few-layer graphene were obtained due to the SO2 and O2 gases formed within graphite sheets. The exfoliated colloidal graphene can be perfectly stabilized in DMF solvent for more than 1 week. The colloidal graphene can show promising applications in flexible printed electronics and energy storage devices [153]. Anodic exfoliation of graphite in  and a graphite rod were employed in the 3-electrode system, consisting of a platinum wire counter electrode and silver wire (with glass frit) or platinum wire as the pseudo- reference electrode. Highly orientated pyrolytic graphite (HOPG) gives greater advantages in terms of the exfoliate size, but there are difficulties in the electrode set up and require the use of sonication. The use of graphite rod electrode does not require ultrasonication or any secondary physical processing of the resulting dispersion. Few-layer graphene flakes with 2 nm thickness are formed directly with flake diameters from 100–200 nm [154]. 4)2SO4, followed by sonication to separate the oxidized graphene sheets, and then reduce oxidized graphene to graphene. With this strategy, it is possible to convert around 30 wt % of the initial graphite into graphene with short processing times and obtain high yields. The graphene sheets are well dispersed in water, have a lateral size of about 0.5–1 μm, and contain only a few graphene layers, most of which are bilayer sheets [155]. Seville et al. [155] presented a facile and environmentally friendly synthetic strategy to produce stable and easily processable Graphene dispersions in water. This method is an alternative to classical chemical exfoliation methods such as the Hummers method, which is more complex, dangerous, and harmful. The new strategy is based on the electrochemical exfoliation of graphite. It includes three simple steps: anodic exfoliation of graphite in . Using different intercalation potential values of 1, 3, 5, and 7 V, the SDS was intercalated into graphite anode electrode. Then, the SDS-intercalated graphite electrode was exfoliated in the same medium by acting as a cathode. Stable graphene dispersions were obtained after these two electrochemical steps. A new glassy carbon electrode (GCE) was modified with graphite, graphene/GCE. This new graphene/GCE electrode has good electrocatalytic activity and has been used for amperometric determination of nitrite in both standard laboratory and real samples [156]. 2SO4, and a high voltage DC is applied, which exfoliates graphite into graphene. Thus, graphene is collected and then oxidized into GO by chemical oxidation using MHM [157]. The process is easy and manageable for large-scale production. The quality of GO prepared can be controlled by sweeping the voltage and type of electrolyte [97].The synthesis of GO by the electrochemical method is utilized recently. This involves the electrochemical conversion of a graphite electrode (as an anode) into graphene under high potential, followed by its chemical oxidation. The graphite anode is inserted in the electrolyte solution, i.e. H2SO4 [158], and aqueous solutions of different inorganic salts ((NH4)2SO4, Na2SO4, K2SO4, etc.) [159]. A high yield of FLG (>70%) was prepared by electrochemical charging of graphite electrodes in a Li+/propylene carbonate electrolyte. This high yield of FLG obtained from the graphite electrode is significantly higher than that produced by most current liquid-phase exfoliation methods [160]. Generally, the electrolyte for this process can be ionic liquids [126], aqueous acids such as HThe electrolyte plays a crucially important role in the yield and quality of electrochemically synthesized Graphene. A lower amount of graphene is produced by exfoliation in ionic liquids with a small graphene’s small lateral size. The electronic properties of the produced graphene are distorted due to functionalization with ionic liquids [126]. However, high-quality graphene with a large lateral size is produced via exfoliation in acidic electrolytes. Nevertheless, over-oxidation of graphite by acidic electrolytes leading to the presence of oxygen-containing functional groups .2O2/H2O) system that can produce high-quality, anodic few-layer graphene nanosheets in 95% yield at ambient reaction conditions [162].A facile, mild, and environmentally-friendly approach for the efficient electrochemical exfoliation of graphite was developed using a sodium hydroxide/hydrogen peroxide/water (NaOH/HExfoliated graphene oxide/multilayer-graphene (GO-MF) flakes were obtained by electrochemical exfoliation of industrial graphite using tungsten (99.5% W) as the counter electrode and graphite as the working electrode [163]. The analytical techniques and XRD results showed 67 wt.% graphene oxide (GO) and 33 wt.% multilayer-graphene (MG). The mechanism of electrochemical exfoliation of graphite in ionic liquid solution involves (i) anodic oxidation of water, (ii) hydroxyl and oxygen radicals’ generation, which attacks the edges of the various graphite sheets (iii) intercalation by ionic liquid anions between graphite layers, forming the graphite-intercalation-compounds (GICs), and (iv) oxidative cleavage and precipitation of the GICs [164]. Lin et al. showed the electrochemically exfoliated graphene flakes have higher crystallinity and fewer defects than graphene flakes prepared by liquid phase exfoliation [165].2SO4. The size of SO42- ion in solution is ~4.60 Å, which is about ~1.37 times higher than the interlayer spacing of 3.35 Å between graphitic layers in graphite. These SO42- ions under applied voltage intercalate between graphitic layers, and they push the graphitic layers farther apart, making the spacing greater than 3.35 Å. This leads to graphite exfoliation because of the weakening of Van der Waals bonding .Most of the graphene synthesis through electrochemical exfoliation has used acidic electrolytes typified by HIn electrochemical exfoliation with alkaline electrolyte, both OH ions (have a size of ~0.958Å) and the hydrated OH ions (have a size of ~2.503 Å) are smaller than the graphitic interlayer spacing of 3.35 Å. The electrolyte with hydrated OH- ions (size ~2.503 Å) behave as polar, and due to the applied electric field, they enter the graphitic layers. These ions will polarize the graphitic layers located above and below, and the hydrated OH- ions will get coupled with the graphitic layers with electrostatic interaction. Under optimum electric force, the hydrated ions will pull the graphitic layers above and below out of the graphitic stack. The graphene-hydrated OH- configuration forming bilayer Graphene will thus get formed and float in the alkaline electrolytic solution [166]. The three main advantages of an electrochemical method of graphene production are: Firstly, it is used to obtain high-quality graphene with no use of hazardous oxidizing materials. Secondly, the method is relatively simple such that a scale-up to industrial production levels could be comparatively straightforward. Moreover, thirdly, hazardous chemicals and the lack of necessity for temperature control and makes the electrochemical production of GO more environmentally friendly . Table 1 summarizes the previous and recent representative studies on the synthesis of graphene by exfoliation from different carbon sources and reacting media.This paper reviewed the most common graphene fabrication methods. It involves the concept and technical development and modification in each process since its first inception in 1898. This review focuses on graphite exfoliation and its progress because it is considered the most popular method for graphene production with high yield and different properties. Most of the scientific works are interested in the mechanism of exfoliation, targeting how to get more yield, low O content, fewer layers, and large lateral size, best properties, low cost, more eco-friendly, and with fewer steps. Many excellent reviews have been published on the production and processing of graphene, including Cai et al. [13], Bonaccorso et al. [119], and Chung et al. [104].Cai et al. [13] presented an excellent general review on graphite exfoliation methods and discussed the bulk production of graphene via exfoliation, focusing on the exfoliation techniques and yields. Different precursors are used for exfoliation such as graphite oxide, pristine graphite, graphite intercalation compounds, and expanded graphite, but the yield of monolayer graphene is still relatively low. Furthermore, another drawback to the exfoliation of graphene sheets in solvents is the aggregation of these sheets due to Van der Waals attraction, which compromises the exfoliation efforts. Chung et al. [104] gave an excellent general review on exfoliated graphite (EG), which is graphite that has a degree of separation of a substantial portion of the carbon layers in the graphite. Graphite nanoplatelet (GNP) is commonly prepared by mechanical agitation of EG. Bonaccorso et al. [119] reviewed graphene as a new class of 2D materials derived from layered bulk crystals. They emphasize the requirement of graphene application and the development of industrial-scale, reliable, inexpensive production processes.


The middle cerebral artery (MCA) dot sign is an important radiological sign in patients presenting with acute ischemic stroke (AIS). If identified and intervened early, a good clinical outcome may be achieved The middle cerebral artery (MCA) dot sign is an important radiological sign in patients presenting with acute ischemic stroke (AIS). If identified and intervened early, a good clinical outcome may be achieved. On examination, left‐sided hemiplegia was noted. The sensation on the same side was markedly reduced, with brisk reflexes of both the ipsilateral upper limb  and lower limb , as well as hypertonia. A nonenhanced cranial computed tomography (CT) showed a right middle cerebral artery (MCA) dot sign Figure , with an3The MCA dot sign is a radiological sign representing a thromboembolus within a segmental branch of the MCA located within the Sylvian fissure (M2 or M3 segment). One study of 100 patients noted a higher detection rate of the MCA dot sign (16%) when compared to the more familiar, hyperdense MCA sign (5%), in patients with AIS presenting within 3 hours of symptom onset.None declared.MSF: is responsible for the care of the patient, literature review, and preparation of this manuscript.The author's institution does not require ethical approval for the publication of single case studies.Informed consent obtained from patient.


Epstein Barr virus (EBV) is one of the most successful pathogens of humans, persistently colonizing more than 95% of the adult human population. At the same time EBV encodes oncogenes that can readily transform human B cells in culture and threaten healthy virus carriers with lymphomagenesis. Cytotoxic lymphocytes have been identified in experimental models and by primary immunodeficiencies as the main protective immune compartments controlling EBV. EBV has reached a stalemate with these cytotoxic T and innate lymphocytes to ensure persistence in most infected humans. Recent evidence suggests that the non-coding RNAs of the virus contribute to viral immune escape to prevent immune eradication. This knowledge might be used in the future to attenuate EBV for vaccine development against this human tumor virus that was discovered more than 55 years ago. The associated B cell proliferation and rescue from apoptosis is thought to drive B cells into differentiation in vivo with successive down-regulation of viral transcripts (in vivo (in vivo (Epstein Barr virus (EBV) that is also called human herpesvirus 4 (HHV4) is a common human γ-herpesvirus establishing persistent infection in more than 95% of the human adult population . Charactnscripts . In germ co-infection . In factin situ hybridization  in EBV positive gastric carcinoma cell lines . This enin vitro and in a xenograft model in mice. In addition, ATM/ATR dependent DNA repair is augmented by BART miRNAs  and rpL22 in the nucleus  the roleAlthough most of these pro-tumorigenic functions have been assigned to BART miRNAs of EBV, recombinant EBV that is deficient in BHRF1 miRNAs was found to infect mice with reconstituted human immune system components with a slower kinetic and attenuated systemic dissemination compared to wild-type virus . This se+ T cell help leading to a less functional CD8+ T cell phenotype  cells, NKT cells, and γδ T cells, controlling lytic and various stages of latent EBV infection, respectively  recognition receptors (PRRs) that detect viral RNAs, including toll-like receptor 3 (TLR3) and 8 (TLR8), RIG-I  and PKR + T, and CD8+ T cell responses. This realization offers the opportunity to explore miRNA deficient viruses, probably with additional safety mutations as vaccine candidates. Their superior ability to be recognized by both CD4+ and CD8+ T cell responses should be harnessed to induce both systemic and tissue resident T cell memory to protect from symptomatic acute EBV infection, namely infectious mononucleosis, and the associated risk for EBV positive malignancies and the autoimmune disease multiple sclerosis (MS)  .in vitro and in vivo systems of EBV infection and immune control to reveal the function of EBER1 and 2, possibly with more physiological migration behaviors of EBV infected cells from mucosal sites to secondary lymphoid organs (In contrast, EBERs remain an enigma of EBV biology. Their high expression in all EBV infection programs mocks us, having not identified a non-redundant function for these non-translated RNAs. It might require more sophisticated d organs . EBERs aCM wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KRAS mutations drive a wide variety of cancers. Drugs targeting the protein product of KRASG12C mutations are currently being evaluated show preliminary efficacy in clinical trials. A clinical trial of VS-6766, a dual RAF–MEK inhibitor, has reported early single agent activity in non-G12C mutated KRAS driven cancers. KRAS mutations are commonly associated with a range of solid tumours and haematological malignancies.1 Initial attempts to drug KRAS proved challenging due to the high affinity of KRAS to GTP and the relative abundance of GDP/GTP in human cellular tissue.2 This led a plethora of efforts to target downstream effectors such as RAF, MEK, PI3K, AKT, mTOR and the combination of these signalling nodes,3 but these approaches have not led to registration of a drug or drug combinations in the setting of KRAS mutant (KRASM) cancers. There has however been significant progress in developing drugs directly targeting protein products of specific subsets of KRAS mutations i.e. KRASG12C. These inhibitors (sotorasib and adagrasib) have shown promising activity in non-small cell lung cancer (NSCLC) with KRASG12C mutations5 and will no doubt be further explored in registration enabling studies.KRAS mutations.6 There are several interesting aspects to this study.Guo et al. reported a Phase 1b dose-escalation, basket expansion study of a dual RAF–MEK inhibitor, VS-6766 (previously known as CH5126766 and RO5126766), which showed promising anti-tumour activity in patients with solid tumours and multiple myeloma harbouring non-G12C-7 leading to reduction of phosphorylation of both MEK and ERK as demonstrated in pre- and post-treatment biopsies. This is distinct to a reduction of phosphorylation of ERK but not MEK caused by currently licensed MEK inhibitors. The inhibition of signalling in two distinct nodes in the MEK–ERK signalling network by a single drug may explain the preliminary single agent efficacy of VS-6766 reported.Firstly, the drug has interesting pharmacological properties. It is a MEK inhibitor but also blocks in-complex (CRAF–MEK) phosphorylation of MEK,8 This reflects previous preclinical studies by the group where they had studied the importance of maximal inhibition of MEK signalling in KRASM models.9 The intermittent schedule has significantly improved the therapeutic index of the drug compared with previous studies that explored continuous dosing schedules.10Secondly, the authors have exploited an unusually long half-life of the drug of approximately 50 hours (which is significantly longer than other RAF or MEK inhibitors) to run pharmacokinetic simulations to predict drug concentrations and design a twice a week dosing schedule. The highly intermittent twice a week schedule established in the trial enables patients to tread the fine line between efficacy and toxicity. Further, a dose modification strategy to drop to three-weeks on-one week off rather than reducing the dose of the drug is different from dose modification strategies commonly used in combination studies of targeted agents.KRASM cancers in a cohort of heavily pre-treated patients. Of note, they have demonstrated partial responses in 3/10 patients with KRASM NSCLC as a single agent and interestingly two of the three patient who responded had KRASG12V mutations. Given there are no KRAS targeted therapies for KRASG12V mutations, VS-6766 could be explored further in this disease space both as a single agent or in combination. Of note, 3/5 patients with RAS/RAF mutations with gynaecological cancers responded to treatment. There is considerable excitement and activity of MEK inhibitors (trametinib and binimetinib) in low grade serous ovarian cancer (LGSOC).12 The combination study of VS-6766 with defactinib have shown promising activity in Phase 1 studies with expansions in LGSOC13 and randomised a Phase 2 trial is exploring the activity of the combination in LGSOC is ongoing (NCT04625270). Further, an interesting response was seen in patient with KRASG12V driven multiple myeloma. While anecdotal responses of MEK inhibitors have been noted in myeloma,14 this further confirms response of this agent in an independent cancer type driven by KRAS mutations.Finally, the authors have demonstrated single agent activity in a variety of 15  and defactinib (NCT03875820). Randomised studies of the combination of VS-6766 and defactinib to explore efficacy of the combination in KRASM LGSOC (NCT04625270) and NSCLC (NCT04620330) are currently ongoing.Attention to detail of pharmacokinetics, pharmacodynamics, toxicity and predictive biomarkers of efficacy are part of the pharmacological audit trail and remain critical to successful development of targeted therapy


In conclusion, childhood maltreatment may mediate epigenetic mechanisms through DNA methylation, thereby affecting physiological responses and conferring a predisposition to an increased risk for psychopathology and forensic repercussions. Similar evidence for somatic illnesses is not yet available.Epigenetic mechanisms are potential mediators of the physiological response to abuse by altering the genetic predisposition of the cellular response to the environment, leading to changes in the regulation of multiple organ systems. This study was established to review the epigenetic mechanisms associated with childhood abuse as well as the long-term determinants that these epigenetic changes may have on future illness. We retrospectively analysed the effect of exposure to adverse childhood experiences  between the ages of 0 and 16 years on the human epigenome, as well as possible clinical associations. After meeting inclusion and exclusion criteria, 36 articles were included in this systematic review. Eight of these studies did not find a relationship between childhood maltreatment and DNA methylation. Of the remaining 28 studies, nine were genome-wide association studies, whereas the rest were candidate gene studies, mainly studying effects on neuroendocrine, serotoninergic and immunoregulatory systems. Meta-analysis of correlation coefficients from candidate gene studies estimated an association of childhood adversity and DNA methylation variation at Adverse childhood experiences are associated with increased mortality partly explained by acquired epigenetic changesThere is a positive correlation between childhood abuse and DNA methylation at specific gene sitesThe cumulative effect of different types of childhood abuse and neglect may lead to changes in DNA methylationEpigenome changes associated with childhood abuse appear to be involved in the development of psychiatric illness in adulthoodStudying epigenetic changes may have important public health and forensic applications in the future The adverse childhood experiences (ACEs) study is a seminal work that laid the foundation for research into the long-term effects of child abuse and household dysfunction, in particular by increasing the risk of mortality due to ischaemic heart disease, cancer, chronic lung disease, liver disease and fractures . A recenA commonly accepted explanation for the association between childhood adversity and increased disease risk relies on the use of coping behaviours such as smoking, illicit drug use and risky sexual behaviours , which aEpigenetic mechanisms are potential mediators of the physiological response to abuse by altering the genetic predisposition of the cellular response to the environment, eventually leading to changes in the regulation of multiple organ systems at the most basic level , 16. DNAIn the literature, there are attempts to summarize the impact of ACEs, socioeconomic position and early environmental stressors on the human genome and future risk of disease , in someThis study thus aims to summarize the epigenetic mechanisms associated with childhood abuse as well as the long-term effects that these epigenetic changes may have on future illness. More specifically, this study was established to perform a meta-analysis of epigenome-wide association studies (EWASs) to quantify the effect that these ACEs may have on the epigenome, as well as a separate meta-analysis to study whether differentially methylated genes could be used as potential biomarkers for childhood maltreatment, with clinical and forensic implications.Appendix I. In this search query, terms relating to childhood trauma, childhood adversity and familial adversity were used to determine exposure to ACEs. Terms including the acronym ACE but not focussing on child abuse and victimology were excluded from the search. Articles analysing the effects of prenatal or familial stress, low socioeconomic status and institutionalization alone were excluded since those studies exclusively focused on genetic polymorphisms. It was assumed that DNA methylation levels would be equivalent irrespective of the tissue sample used. Therefore, studies were included if they: i) were performed on adult human subjects (over the age of 16), ii) used a validated method for ascertaining exposure to childhood trauma, and iii) included a relevant control group .The systematic review and meta-analysis were carried out in accordance with the Cochrane Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . Five daP-sample size-based fixed-effects model with genomic control correction [P-value threshold set at P < 0.0001 for this study. In addition, pathway analysis of significant CpGs was undertaken using ConsensusPathDB [Articles were screened at the title/abstract level to filter out unoriginal study designs and irrelevant articles. Full-text screening was performed for the remaining 90 articles, with 36 articles being included after discussion among the authors. The Newcastle–Ottawa Scale for assessing the quality of nonrandomized studies  was applrrection . Probes usPathDB .metafor package in R was used. The effect size used was Pearson’s r correlation coefficient measuring the association between DNA methylation of certain loci and childhood trauma, with the modulus of r (|r|) used to represent the strength of differential methylation variation. A random-effects model was applied for all analyses. When no data were reported for a nonsignificant association, the authors assigned z = 0.00 as a conservative estimate.To study whether candidate genes could be used as biomarkers for child abuse, the A flow chart of the selection process is presented in There are nine genome-wide studies among the 36 articles included. DNA methylation of the central nervous system at the genome-wide level was only examined in one study. Labonté et al.  used hipIn another study , gene exCecil et al.  studied KITLG, which mediated the relationship in 32% of Caucasians.Houtepen et al.  performeFinally, only one genome-wide association study of the nine studies included in this review found no changes relating DNA methylation and child abuse, regardless of whether a genome-wide or site-specific level was considered .P-sample size-based approach, leading to 44 CpGs being considered significant for this meta-analysis, 97.7% of which were hypo-methylated. The full sample size included 209 individuals, with moderate heterogeneity (mean I2 = 58%). A QQ-plot for the FDR-corrected values showed an approximately normal distribution  to analyse DNA methylation across the genome, which made them eligible for inclusion in a meta-analysis of EWASs , 35–37. ribution . Two CpGNR3C1 [NR3C1 exon 1 F in a subgroup of 33 depressed patients who reported early life adversity.Nine studies explored the consequences of childhood maltreatment on the glucocorticoid receptor. Labonté et al.  examinedNR3C1 . In a caNR3C1  found a NR3C1 promoter methylation at CpG1 and CpG3 in response to adversity. In contrast, childhood adversity was associated with decreased NR3C1 promoter methylation in a separate group of 340 adults [NR3C1 [Two cross-sectional studies designed by the same group of researchers found contrasting results. In the first study, Tyrka et al.  recruite0 adults . Three os [NR3C1 , 45, 46.FKBP5 gene and its relationship with PTSD and major depressive disorder (MDD). Two studies did not find any relationship between FKBP5 methylation and adversity or between depressed patients and controls [FKBP5 in patients with MDD carrying a high-risk allele (rs1360780), which also correlated with reduced grey matter concentration in the inferior frontal orbital gyrus. Decreased activation in this region was also found to be related to the severity of depression [FKBP5 region [Four studies focused on the controls , 47. In pression . Another5 region .SLC6A4 gene. In a group of 26 women reporting a history of child sexual abuse, symptoms of antisocial personality disorder, depression and substance abuse were more prevalent and methylation of the CpG island surrounding exon 1 of SLC6A4 was increased [SLC6A4 methylation [Three studies examined the effect of childhood maltreatment on methylation of the ncreased . Two casncreased  found sehylation .BDNF gene. Perroud et al. [BDNF methylation was associated with increased childhood trauma. A separate analysis found that women with bulimia nervosa who suffered physical abuse in childhood had a greater percentage of DNA methylation across all CpGs than women without eating disorders, whereas childhood sexual abuse in bulimic patients was associated with differential methylation of specific BDNF sites [Two studies examined the effect of childhood maltreatment on the d et al.  found hiNF sites .DRD2 gene was studied in a group of Canadian women, most of who suffered from bulimia spectrum disorder, a condition that when compounded with childhood sexual abuse was associated with higher methylation levels [IL6 promoter. The group exhibiting IL6 hypo-methylation had a reduced cortisol response and prolonged IL6 elevation after stressful stimuli [MAOA gene. Physical abuse was particularly associated with hyper-methylation at CpG7/8, whereas sexual abuse was particularly associated with hyper-methylation of the MAOA first exon as well as an increased risk of current depression.Several studies looked at other candidate genes to describe any epigenetic changes associated with childhood maltreatment, specifically in regions governing pain sensitivity, immune response and neurotransmission. For example, the n levels . In addi stimuli . Moreove stimuli  designedOPRK1) using brain tissue of suicide victims with and without a history of child sexual and physical abuse and neglect compared with a group of psychiatrically healthy victims who had died of accidental causes. Methylation of intron 2 in the Kappa variant 1 region was significantly lower in child abuse suicide cases, which was related to decreased kappa expression in the anterior insula.Lutz et al.  studied OXTR methylation was studied in a group of 393 African-American subjects. This study described an interaction between child abuse and CpG methylation, which was predictive of both depression and anxiety [In addition,  anxiety .SKA2 methylation in a group of 85 healthy controls. However, pre-deployment SKA2 methylation levels and childhood trauma exposure did significantly predict post-deployment PTSD symptoms in a cohort of 94 military servicemen.Boks et al.  did not SSTR4 methylation, despite 21.6% of this population displaying increased SSTR4 promoter methylation compared with 2.3% of controls [Moreover, a case–control study on 176 Polish individuals with ethanol dependence and 127 healthy volunteers found that the former group reported significantly higher numbers of childhood adversities. However, childhood trauma was not found to be a predictor of controls .MT-DN6 (NADH dehydrogenase 6) methylation levels in a group of 42 individuals with an ACE score ≥4, an epigenetic change also associated with higher baseline cortisol levels and blunted cortisol response to stress [A further study considered epigenetic changes in mitochondrial DNA, finding significantly higher average o stress .LINE-1 and BAGE were also studied in 48 patients presenting with first-episode schizophrenia and 48 healthy controls. Schizophrenic patients with a history of child abuse had significantly lower LINE-1 methylation, which was predicted by higher emotional abuse and total trauma scores [Repeating DNA sequences a scores .rRNA promoter methylation was studied in cerebral samples from 13 individuals with a history of child abuse who had committed suicide and 11 sudden death controls. The former group had greater rRNA promoter methylation in hippocampal tissue.Finally, in an earlier study by McGowan et al. , rRNA prr correlation coefficients was undertaken for SLC6A4, NR3C1, FKBP5, IL6 and OXTR. Correlation coefficients for four studies [z = 0.00 [r = 0.291 (P < 0.0001), although examination of the forest plot (2 = 85.23%). Egger’s regression test indicated the presence of publication bias  , while analysis for NR3C1 gave a less significant result .A random-effects meta-analysis of studies reporting  studies , 48, 55 z = 0.00 , 58. Risest plot  suggeste 0.0004) . SubgrouThis review strongly suggests that epigenetic changes throughout the genome have a positive, long-lasting relationship with childhood abuse and adversity, with possible clinical and forensic repercussions. These changes tend to be both gene-specific and specific to the type of childhood trauma experienced, with the strongest association for total trauma scores, followed by sexual and physical abuse in childhood . It is aInduced network module analysis  of the gNR3C1 gene encodes the glucocorticoid receptor involved in inflammatory responses and cellular proliferation and differentiation in targeted tissues through the HPA axis, a major neuroendocrine system responsible for regulating the stress response through numerous biological feedback mechanisms [NR3C1 gene at specific loci). FKBP5 was only found to be hyper-methylated in individuals with a high-risk T allele, underlining the importance of genotyping when certain phenotypes have known genetic risk profiles. This gene encodes an immunophilin protein, which participates in immunoregulation as well as protein folding and trafficking within the cell [SLC6A4 encodes a membrane protein responsible for the transport of serotonin across the synaptic space [SLC6A4, providing a basis for a gene environment interaction between trauma and depression [BDNF, a gene encoding a protein involved in nerve growth [The chanisms . Intereschanisms . This rethe cell . SLC6A4 ic space . A systepression . BDNF, ae growth , has beee growth .DRD2, IL6, MAOA and Kappa genes showed differential methylation profiles, some of which were associated with bulimia nervosa and depression [Although less evident, other candidate genes are also promising regions for further study as these genes are often related to disease phenotypes. Indeed, pression , 56, 57.2 = 85%), which was accounted for in a random-effects model, recognizing the increased probability of sampling and effect variability in the populations studied.It is also becoming evident that a number of research groups are interested in using these differentially methylated genes as clinically meaningful prognostic biomarkers , such asThe evidence base of the studies included in this analysis is moderate in quality, considering the parameters outlined in the Newcastle-Ottawa Scale . This maFKBP5 high-risk T allele rs1360780 [A major difficulty in interpreting candidate gene studies is the lack of consistency in nomenclature of the genetic regions under study, especially when discussing differentially methylated loci. Comparing findings is also difficult when the studied regions vary significantly among studies focussing on the same gene, with some studies looking at the whole promoter region, while others focus on specific CpG islands. The genetic regions chosen should be selected based on previously studied regions, while researchers who wish to study novel sites should include loci overlapping with those from previous research to aid comparison. It should be noted that disease associations may be specific to a certain genotype, as seen for the s1360780 , 48. ResAlthough increased DNA methylation of a gene is generally associated with decreased expression, it is of fundamental importance that future studies both measure gene expression and quantify associated levels of hormones or biomarkers. These functional results are essential to understand whether certain loci are sensitive to a determined exposure or pathology and may help to explain results that appear contradictory. Research should also include CpG sites located in transcription factor binding sites, as it is through inhibition of these sites that DNA methylation silences genetic expression .The tissues used in these studies may be responsible for the variation in the obtained results regarding epigenetic changes. As changes after childhood trauma are generally associated with neural pathways, studies utilizing brain or neuronal tissue should be favoured over those using peripheral measures. The use of peripheral blood may bring further challenges, aside from possibly not being representative of central tissues . A possA clear limitation of this meta-analysis is that the authors are not part of a consortium, as is usual for meta-analyses of genome-wide association studies, allowing the results to be pooled across multiple cohorts. Although an attempt was made to contact the authors of five articles considered for an EWAS meta-analysis, only two authors made their data available for use in this study. Another limitation is the existence of publication bias in the candidate gene meta-analysis, most likely due to the small-study effect.SLC6A4 gene. Future studies should consider the differing effects that certain types of adversity may have on the epigenome, as well as on the presentation of disease, and should aim to characterize each of these ACEs separately. Another major gap in research is apparent. Despite ACEs being associated with changes in several genes with effects on various physiological processes, studies identified using the search query outlined in this review only examined psychiatric illness. Even using search parameters specifying somatic diseases such as “neoplasms”, “autoimmune diseases” or “cardiovascular diseases” (illnesses strongly associated with childhood trauma), no study definitively relating epigenetic changes after childhood adversity with any of these pathologies in humans was identified, even though correlations with somatic illnesses may be inferred through known epigenetic modifications of the HPA axis and immune system.Despite these limitations, different ACEs appear to have different effects on the epigenome, especially in the Acknowledging the importance of prevention in reducing the nefarious effects of childhood abuse at both the individual and societal levels, studying these epigenetic effects and clinical outcomes may lead to more effective public health policies, interventions and better forensic decisions based on trauma-informed care models , 19. On


Lipoprotein(a) (Lp(a)) is a highly proatherogenic lipid fraction that is a clinically significant risk modifier. Patients wanting to learn more about Lp(a) are likely to use online patient educational materials (OPEMs). However, the readability of OPEMs may exceed the health literacy of the public.This study aims to assess the readability of OPEMs related to Lp(a). We hypothesized that the readability of these online materials would exceed the sixth grade level recommended by the American Medical Association.Using an online search engine, we queried the top 20 search results from 10 commonly used Lp(a)-related search terms to identify a total of 200 websites. We excluded duplicate websites, advertised results, research journal articles, or non–patient-directed materials, such as those intended only for health professionals or researchers. Grade level readability was calculated using 5 standard readability metrics  to produce robust point (mean) and interval (CI) estimates of readability. Generalized estimating equations were used to model grade level readability by each search term, with the 5 readability scores nested within each OPEM.A total of 27 unique websites were identified for analysis. The average readability score for the aggregated results was a 12.2 (95% CI 10.9798-13.3978) grade level. OPEMs were grouped into 6 categories by primary source: industry, lay press, research foundation and nonprofit organizations, university or government, clinic, and other. The most readable category was OPEMs published by universities or government agencies . The least readable OPEMs on average were the ones published by the lay press . All categories exceeded the sixth grade reading level recommended by the American Medical Association.Lack of access to readable OPEMs may disproportionately affect patients with low health literacy. Ensuring that online content is understandable by broad audiences is a necessary component of increasing the impact of novel therapeutics and recommendations regarding Lp(a). Lipoprotein(a) (Lp(a)) is a highly proatherogenic lipid fraction that is increasingly recognized as a clinically significant risk modifier. The 2018 American College of Cardiology and the American Heart Association guidelines recommend using Lp(a) as a risk-enhancing factor favoring initiation of statin therapy in persons with intermediate atherosclerotic cardiovascular disease (ASCVD) risk . StatinsPatients wanting to learn more about Lp(a) are likely to use online patient educational materials (OPEMs) . HoweverWe used Google, the largest online search engine, to query the first 20 results for each of the following 10 search terms: “lipoprotein(a),” “lipoprotein(a) cardiovascular risk,” “lipoprotein(a) elevated,” “lipoprotein(a) high,” “lipoprotein(a) levels,” “lipoprotein(a) screening,” “lipoprotein(a) test,” “Lp little a,” “Lp(a),” and “Lp(a) screening” . LocatioThis research is exempt from human participant institutional review board approval as no human participant data were used, and only publicly available OPEMs were included in the analysis.Industry OPEMs were published by for-profit companies or offered a proprietary test or service related to Lp(a). Lay press OPEMs were published by news organizations and health care reporters that do not have a specific research or scientific focus. Research foundations and nonprofit OPEMs were published by health-related organizations that have a specific research or scientific focus. University or government OPEMs were published by academic institutions or national, state, or local government agencies. Clinic OPEMs were published by Williams Integracare clinic, an independently owned family medicine, chiropractic, and physical therapy clinic in St. Cloud, MN. Other includes Wikipedia.org, a crowdsourced online free encyclopedia, which published an article on Lp(a).OPEMs were grouped into 6 categories by primary source. Websites meeting the criteria for OPEMs were converted into plain text in separate Word (Microsoft Corporation) documents and prepared in accordance with recommendations from the Centers for Medicare and Medicaid Services prior to scoring readability . Using mSimilar to prior readability studies, grade-level readability was calculated using 5 standard readability metrics  to produce robust point (mean) and interval (CI) estimates of readability. Averaging across multiple readability metrics has been demonstrated to yield more reliable results than relying on a single readability metric . This waAmong the 27 OPEMs, the largest category of were university and government sources, which included 30% (n=8) of unique search results. The next largest categories were research and nonprofit foundations and industry publications, which each comprised 22% (n=6) of the results. The third largest category of OPEMs were articles in the lay press, which comprised 19% (n=5/27) of results. The smallest categories were clinic publications and other, both of which had 1 (3%) website .The average readability score across unique websites was at a 12.2 (95% CI 10.9798-13.3978) grade level. The most readable category was OPEMs published by universities or government agencies . The least readable OPEMs on average were the ones published by lay press . Research and nonprofit foundations  and industry  had intermediate readability. Clinic (10.2) and other publications (14) had 1 site per category and therefore are not averaged figures. Of the 27 unique search results, only 1 site, the University of Rochester Medical Center , had the sixth grade level recommended by the AMA within its 95% CI. The least readable OPEM was the HEART UK’s Lp(a) general information site . The readability of each unique website is shown in A list of the OPEM sources used for this study can be found in We found that the average reading grade level of OPEMs pertaining to Lp(a) generally exceeded AMA readability recommendations that OPEMs be written at or below a sixth grade reading level to be accessible to the public. Average grade-level readability of OPEMs on Lp(a) was a 12.2 (95% CI 10.9798-13.3978) grade level, exceeding the average reading level of US adults (eighth grade) [These findings have several important implications for how patients may make decisions about this important, proatherogenic lipid fraction that is receiving increased attention. Patients frequently use the internet to supplement health information from their clinicians . If presThe less readable OPEMs tend to cover topics in greater depth, including nuances around Lp(a) measurement and standardization, and the role of Lp(a) in thrombogenesis and wound healing. Thus, there appears to be a trade-off between readability and comprehensiveness in OPEMs.These findings align with results from prior studies across a broad range of health conditions that show OPEMs commonly exceed the recommended readability level. Ayyaswami et al  showed tThe percentage of adults with below basic health literacy is considerably higher for populations who identify as Black (24%), Hispanic 41%), American Indian/Alaska Native (25%), and Asian/Pacific Islander (13%) compared to non-Hispanic White (9%) . Similar%, AmericMany clinical preventive and screening services are underused by historically marginalized racial/ethnic communities and older adults due to inadequate health care access and low health literacy, but we suggest that providing more readable OPEMs may help bridge this gap in care. In the interim, our study findings remind clinicians to consider the readability of OPEMs and patient literacy when recommending Lp(a) evaluation. Shared decision-making requires adequate understanding of the risks and benefits of any diagnostic testing or risk stratification procedure. Actionability of OPEMs on Lp(a) is presently limited by the lack of approved therapies; however, with changes in emerging therapies and practice guidelines, understandability and actionability will be important parameters to assess systematically using the Patient Education Materials Assessment Tool in future studies.A strength of our study is that we incorporated readability results from five different standard readability metrics, which allows us to have a robust evaluation of readability regardless of the number of websites evaluated. All of the reading grade level estimates supported our hypothesis that Lp(a) OPEMs are written above the recommended sixth grade reading level. We also included a thorough review of possible patient queries by analyzing 200 search results for 10 commonly used search terms. Finally, we included examples of communication of a concept from both more readable and less readable OPEMs. This may serve as a real-world, practical guide for creators of OPEMs seeking to choose words and phrases that will be accessible to a broad audience.Our study should be interpreted in the context of certain limitations. We did not account for other search engines besides Google. This limitation is somewhat mitigated by the fact that 88% of global internet users use Google as their most frequent search engine . As withIn conclusion, we found that the grade level readability of OPEMs relating to Lp(a) generally substantially exceeded the sixth grade reading level recommended by the AMA. This gap in readability may disproportionately affect patients with low health literacy. Creators of OPEMs should be mindful of the readability of their content. Ensuring that online content is understandable by broad audiences is a necessary component of increasing the impact of novel therapeutics and recommendations regarding Lp(a).


The characterization of the immune component of the tumor microenvironment (TME) of human epidermal growth factor receptor 2 positive (HER2+) breast cancer has been limited. Molecular and spatial characterization of HER2+ TME of primary, recurrent, and metastatic breast tumors has the potential to identify immune mediated mechanisms and biomarker targets that could be used to guide selection of therapies.We examined 15 specimens from eight patients with HER2+ breast cancer: 10 primary breast tumors (PBT), two soft tissue, one lung, and two brain metastases (BM). Using molecular profiling by bulk gene expression TME signatures, including the Tumor Inflammation Signature (TIS) and PAM50 subtyping, as well as spatial characterization of immune hot, warm, and cold regions in the stroma and tumor epithelium using 64 protein targets on the GeoMx Digital Spatial Profiler.PBT had higher infiltration of immune cells relative to metastatic sites and higher protein and gene expression of immune activation markers when compared to metastatic sites. TIS scores were lower in metastases, particularly in BM. BM also had less immune infiltration overall, but in the stromal compartment with the highest density of immune infiltration had similar levels of T cells that were less activated than PBT stromal regions suggesting immune exclusion in the tumor epithelium.Our findings show stromal and tumor localized immune cells in the TME are more active in primary versus metastatic disease. This suggests patients with early HER2+ breast cancer could have more benefit from immune-targeting therapies than patients with advanced disease.The online version contains supplementary material available at 10.1186/s12967-021-03113-9. Human epidermal growth factor receptor 2 positive (HER2+) breast cancer accounts for 20–25% of all breast cancers , 2. ThesThe molecular subtypes and spatial interactions of immune, stroma, and tumor cells in the tumor microenvironment (TME) are not well defined in HER2+ breast cancer. HER2 is a natural antigen and the effect of some HER2-directed therapies could be potentiated with immune checkpoint inhibitors (ICI) , 11. A bImmunotherapy has revolutionized the treatment paradigm of several malignancies but its use has been limited in breast cancer and mostly studied in the TNBC subtype –17. Two In this study we utilized bulk tissue gene expression profiling  and digiSamples of patients with HER2+ breast cancer between 2001 and 2019 were analyzed by the Georgetown University Histopathology Tissue Shared Resource and 15 formalin fixed paraffin embedded (FFPE) samples were selected for this study. Deidentified archival samples were selected through Georgetown University Hospital Institutional Review Board (IRB) approved protocols .Samples included 15 specimens from eight patients. Histopathology of the specimens was obtained from the original pathology reports as were results of estrogen receptor (ER), progesterone receptor (PR), and HER2 status.RNA was extracted from one slide of unstained FFPE tissue and macro-dissection to remove non-tumor was performed based on a sequential hematoxylin and eosin (H&E) staining. Approximately 50-200 ng of RNA was run on NanoString’s nCounter PanCancer IO 360™ assay (IO360) containing 770 genes with an additional 28-gene PAM50 spike-in. Genes were normalized using a combination of geometric means of housekeeping (HK) gene expression and an IO360 panel standard run on the same cartridge. IO360 signatures were calculated as previously described , 23.CalcFFPE tissue from eight patients were profiled using GeoMx DSP as previously described . An H&E Differential gene expression (nCounter) is fit on a per gene or per signature basis using a linear mixed model for analyses with subject as a blocking factor to account for the temporal effects in the model. The statistical model uses the expression value or signature score as the dependent variable and fits a grouping variable as a fixed effect to test for differences in the levels of that grouping variable. The duplicate Correlation function within the limma  R packagP-values are adjusted within each analysis, gene or signature, and on the grouping variable level difference t-test using the Benjamini and Yekutieli False Discovery Rate (FDR) adjustment to account for correlations amongst the tests (FDR of 5%). All gene expression models are fit using the limma package in R .For the GeoMx DSP protein data, hierarchical clustering analyses were performed to look for broad patterns within and between subjects based on TIS status, PAM50 subtype, timepoint (primary or metastatic), ROI type , and AOI type . For hierarchical clustering, the HK protein-based normalized expression values were first log2 transformed and the resulting values were then centered and scaled. The Z-scores for each protein were then used as input to the hierarchical clustering in the R package pheatmap .i.e., 64 hypotheses), raw P-values were adjusted based on an FDR level of 0.05 using the Benjamini-Hochberg (BH) procedure [i.e., “least squared means”) in lmerTest. BH P-value adjustment for time series data was performed for a given pairwise contrast .Differential expression analyses for DSP data were used to look for specific protein differences between three different groupings: (1) Primary vs metastatic tumor, (2) Breast vs brain tumors, and (3) timepoint (three different samples of a single patient). To account for multiple observations  within a given patient, a linear mixed effects model was used in the R package lmerTest . Specifirocedure . This prBoxplots were used to better visualize the protein expression for a given patient across time using the R package ggplot2 . This waFifteen specimens from eight patients were analyzed, including 10 primary breast tumors, two soft tissue tumors, two brain metastases and one lung metastasis. Eight patients were receiving cancer-directed therapies  and PAM50 subtypes from bulk tumor tissue Fig. a.Fig. 1IMetastatic disease and primary samples clustered separately based on IO360 signatures, with 8/10 primary tumors having high TIS score status  in tumor rich (PanCK +) and stromal (PanCK−) compartments. A schematic of the DSP experimental design is shown in Fig. We then looked for differences between primary and metastatic lesions by spatially profiling ROIs of varying immune infiltrate  and six had low TIS (< 6.5). One patient (patient 2) had discordant TIS, meaning that the primary tumor and soft tissue recurrence had low TIS status and the residual disease had high TIS status Table . FinallyThree patients in our cohort had samples from two timepoints , PTEN, AKT (PanCK-) after therapy (Additional file Patients 1 and 2 had three timepoints. Figures DSP allows for a comprehensive analysis of protein expression by which we were able to assess immune markers that have been proposed as markers of response to ICI, such as PD-L1, and other potential therapeutic markers, such as B7H3 or LAG3. Larger studies are needed to determine if this technology can be used to guide treatment decisions in the future with the goal to provide individualized care to our patients.The breast cancer intrinsic subtypes have shown to provide prognostic  and predICIs have shown central nervous system penetration in lung cancer , 35 and Several studies have shown efficacy of the combination of ICI with HER2-targeted agents in PD-L1 positive HER2+ breast cancer , 19, 40.Limitations of this study include the small sample size, which restricts the correlation between the tumor immune characteristics, treatment, and outcomes. Even though our study is small the strength is the paired samples. Also, we will contribute our data to the GeoMx Breast Cancer Consortium for a meta-analysis with other investigators examining primary and metastatic breast cancer to increase the data set. Another limitation is that the ROI selection might not capture the heterogeneity of the entire tissue. We chose to look at DSP with proteins, other analyses can be done using in situ RNA examination of up to 18,000 genes using DSP, which gives spatially resolved information on signaling pathways as opposed to the bulk gene expression presented in this paper.This study underscores the importance of spatial resolution of the tumor or stroma within the TME to see distinct differences in immune activation markers. A better understanding of the TME and gene expression profiling of HER2+ breast cancer will allow us to tailor the treatment of patients to continue to improve outcomes.Additional file 1. Additional figures and tables.