However, the precise atomic structure, the key to understand the origin of the improved catalytic activity, is yet to be clarified. Here, we present a computational study to investigate the structure of platinum adsorbed on graphene with special emphasis on the edges of graphene nanoribbons. By means of density functional theory based thermodynamics, we find that single platinum atoms preferentially adsorb on the substitutional carbon sites at the hydrogen terminated graphene edge. The structures are further corroborated by the core level shift calculations. Large positive core level shifts indicate the strong interaction between single Pt atoms and graphene. The atomistic insight obtained in this study will be a basis for further investigation of the activity of single-atom catalysts based on platinum and graphene related materials.Single-atom catalysis, which utilizes single atoms as active sites, is one of the most promising ways to enhance the catalytic activity and to reduce the amount of precious metals used. Platinum atoms deposited on graphene are reported to show enhanced catalytic activity for some chemical reactions,  Single-atom catalysis, which utilizes single atoms as active sites, is one of promising ways to enhance the catalytic activity and to reduce the amount of precious metals used. Here by means of density functional theory based thermodynamics we show that the single platinum atoms preferentially adsorb on the substitutional carbon sites at the hydrogen terminated graphene edge. Tremendous efforts have been devoted to achieve these goals, including the use of non-precious metals for Pt–metal alloys,3–9 or the use of noble metal-free catalysts.10–13 Particularly interesting are Pt clusters supported by graphitic materials, such as carbon black, carbon nanotubes, and graphene, which have been extensively studied both experimentally,14–20 and theoretically.21–34 A better catalytic activity of small Pt clusters supported by graphene sheets has been demonstrated experimentally.16,17 These results lead to the hypothesis that downsizing the Pt clusters to single atoms can enhance the catalytic activity. Recently, Sun et al. have successfully deposited single Pt atoms on graphene nanosheets using atomic layer deposition and then demonstrated a significant improvement of the catalytic activity for the methanol oxidation reaction.35 Cheng et al. also showed that a Pt single-atom catalyst deposited on nitrogen doped graphene nanosheets exhibits enhanced catalytic activity for the hydrogen evolution reaction.36 The vacancies usually formed during the preparation are expected to have strong interactions with the Pt atoms as demonstrated in several theoretical studies.23,31,32 Back et al. have predicted the great potential of a single atom catalyst supported on defective graphene for CO2 electroreduction applications.37 However, the dispersion of the single Pt atoms on graphene is limited to the number of point defects. On the other hand, the graphene edge might offer more space for depositing single Pt atoms. Kong et al. have theoretically investigated Pt single-atom adsorption at the edges of graphite nanofibers and found that the atoms are tightly bound to the edges due to the existence of active dangling bonds.38 By employing transmission electron microscopy, the structure and dynamics of Au,39 Fe,40 Cu,41 and Pt41,42 atoms at the edges have been studied. Some experimental studies also observed that Pt nano-clusters at the graphene edges are stable at high-temperatures.43–45 However, the adsorption state and the catalytic activities of single Pt atoms are not yet fully understood.High-performance electrocatalysts are highly desired for electrochemical energy conversion devices, such as photovoltaic cells and fuel cells. Platinum (Pt) is widely used as an electrocatalyst, as it exhibits high catalytic activity not only for hydrogen oxidation but also for oxygen reduction at low temperatures.via DFT based thermodynamics.46 We find that single Pt atoms adsorb preferentially at the edge rather than on graphene, and the substitutional carbon site is the most stable one under the conditions relevant to experiments. Furthermore, the core level shift (CLS) of Pt atoms is calculated for each structure, which is used to validate the predicted structure against the experiment.In this work, we investigate Pt single-atom adsorption on graphene by means of density functional theory (DFT) that includes van der Waals forces. We perform systematic calculations to determine the adsorption state of single Pt atoms on graphene, including defective graphene structures and graphene edges. Special emphasis is devoted to the graphene edges, as they are abundant under realistic conditions. We consider both zigzag and armchair edges, including the dependence on hydrogen termination. We examine their stability by taking into account the environmental effects, 247,48 with ultrasoft pseudopotentials.49 A plane-wave basis set is used to expand wave functions and charge density with cutoff energies of 36 Ry and 400 Ry, respectively. We use rev-vdW-DF2 54 functional and the use of PBE pseudopotentials in rev-vdW-DF2 calculations is validated in V1), and di-carbon vacancy (V2) graphene structures. We introduce the 7575 membered rings of graphene as a boundary between zigzag and armchair as observed in Γ-centered 4 × 4 k-point mesh for pristine, mono-carbon vacancy, and di-carbon vacancy graphenes; 2 × 3 k-point mesh for GB-GR; and the Γ-point for DisGR. To investigate the edge effect in the Pt adsorption, we employ graphene nanoribbons (GNRs) with zigzag (zGNR) and armchair (aGNR) edges with different terminations, including non-hydrogenated, mono-hydrogenated, and di-hydrogenated ones. We follow the convention used in i.e., zn to denote zGNR with n hydrogen atoms at the edge carbon (C) site, and an for aGNR with n hydrogen (H) atoms. Spin polarization is taken into account for all systems. zGNR has the localized spin with ferromagnetic order along the edge and antiparallel orientation between the edges as reported in aGNR. GNRs are modeled using a periodic supercell along the edges, having the same configurations on both edges. Following the conventional notation, the width of GNR is specified by the number of zigzag chains and dimer lines for zGNR and aGNR, respectively, and we use 5-zGNR and 10-aGNR in this work. We use supercells containing 4 hexagons for both zGNR and aGNR, and the resulting supercells contain 40 carbon atoms. Supercells in the graphene plane directions correspond to  and  supercells of graphene for zGNR and aGNR, respectively, and graphene planes are separated by a vacuum of ∼15 Å thickness. Vacuum thicknesses between edges are 16.48 Å and 18.70 Å for zGNR and aGNR, respectively. Brillouin zone integration is performed using the Γ-centered 6 × 2 k-point set for both zGNR and aGNR. All the graphene based structures considered in this work are constructed using the lattice constant of graphene obtained using rev-vdW-DF2 (2.46 Å),61 which is in good agreement with the experimental value for graphite (2.4589 ± 0.0005 Å).62 The structures are fully relaxed until the forces acting on the atoms become smaller than 5.14 × 10−2 eV Å−1 (1 × 10−3 Hartree/Bohr). The Pt 4f CLS including the final state screening is calculated as the difference between the core level binding energy of Pt adsorbed GNR and bulk Pt.63,64All the DFT calculations are performed by using STATE code-vdW-DF2  exchange33.1EPt/G, EG, and μPt are the total energy of the adsorption system, the total energy of the substrate, and the chemical potential of Pt, respectively. We use the total energy of an isolated Pt atom for μPt. In 54 functional). The Pt single-atom adsorption on graphene with vacancy is significantly stable, because the Pt atom terminates dangling bonds associated with the C vacancy. However, the energies necessary to create the C vacancies (formation energies for C vacancy with respect to the C atom in graphene) are 7.77 eV and 7.86 eV for V1 and V2, respectively, and the effective binding energies are +0.07 eV and +0.29 eV, respectively. Thus we conclude that the Pt single-atom adsorption at the C vacancy site is thermodynamically less favorable. The binding energies of a single Pt atom for GB-GR and DisGR are apparently much larger than that for pristine graphene. However, this is because GB-GR and DisGR are less stable and thus more reactive than the pristine one. They are less stable than pristine graphene by 0.17 and 0.39 eV per C atom, respectively.We first consider pristine and defective graphene structures and adsorption of single Pt atoms on them as shown in 3.1.1EGNR, EGR, and μ2H are the total energy of GNR, total energy of C atoms in bulk graphene , and chemical potential of H2 molecule, respectively; NC (NH) is the number of C (H) atoms in GNR; and L is the length of the unit cell along the edge. 2 chemical potential. It is found that at high H2 chemical potential, aGNR (a2) is stable, whereas zGNRs are more stable at low H2 chemical potential, and non-hydrogenated GNR is unstable in a wide range of H2 chemical potential, in good agreement with the previous study.58 The positions of the intersections differ from those reported in We then investigate the most stable GNR by calculating the formation energy defined byL in NC). At μ2H = E2H, the formation energy per C atom of z0, z1, z2, a0, a1, and a2 are 0.61, 0.06, 0.10, 0.44, 0.03, and ∼0.00 eV, respectively. Thus, although most GNRs considered here are less stable than pristine graphene (i.e. most GNRs have positive Eform at μ2H = E2H), hydrogenated GNRs are more stable than defective graphene structures.To compare the stability of the GNRs with the defective graphene structures, we calculate the formation energy per C atom (replace 23.2zGNR (z0). We systematically construct the adsorption configurations with and without defects, and label them according to the Kröger–Vink notation,65 as shown in z0, denoted as PtT@z0 0, and a Pt atom adsorbed at the long-bridge (LB) site of the SW defect, denoted as PtLB@z(57)0  denoted as PtαC@z0 (βC@z0 (VC), and obtained the Pt substituted with two VC's (Pt(Cα)@z0, VC 0 . We intr PtCα@z0  and PtCβ0 . μPt is chosen to be the total energy of an isolated Pt atom (EPt) and μC varies around the chemical potential of graphene. μC = EGR (the system is in equilibrium with graphene) are summarized in μC = EGR, suggesting that Pt single-atom adsorption at the edges of z0 is thermodynamically unstable. Among the Pt adsorption structures on z0, PtαC@z0 is the most favorable configuration. However, the calculated CLS for PtαC@z0 is too small compared with the experimental value of (+2.0 ± 0.4) eV,42 suggesting that this adsorption configuration is unlikely. On the other hand, PtβC@z0 and PtβCγC@z0 show a relatively large CLS of +1.44 and +1.67 eV, respectively. However, their Gibbs free energies are significantly large, ruling out these configurations. Thus, we conclude that Pt single-atom adsorption on the non-hydrogenated GNR is unlikely under the equilibrium conditions.For the optimized structures with a single Pt atom at the edge of zGNRs (z1 and z2). The structures are similar to those adopted for non-hydrogenated zGNRs as shown in z1 and z2, respectively. We optimized all the structures and calculated the Gibbs free energy given byEPt@GNR (Eref) is the total energy of the adsorbed system (reference system), μPt = EPt, ΔNC, and ΔNH are the difference of numbers of C and H atoms from the reference system, respectively, and μC and μ2H are chemical potentials of C atom and H2 molecule, respectively. As in the case of z0, pristine graphene is chosen as the reference. We calculate ΔΩ for z0, z1, and z2 as a function of μ2H and μC and generate the phase diagram as shown in Ω at μC = EGR and μ2H = E2H for the structures considered, where E2H is the total energy of an isolated H2 molecule. The results are summarized in zGNR is unstable and does not appear in the phase diagram. We find that PtαC@z1 only exhibits negative ΔΩ at μC = EGR and μ2H = E2H, suggesting that this structure is thermodynamically stable under these conditions. Furthermore, the calculated CLS for this structure is in reasonable agreement with the experiment. Among the hydrogenated GNR structures, calculated CLSs for PtβC@z1 and PtαC@z2 are also reasonable. However, they show positive ΔΩ's, implying that these structures are less likely than PtαC@z1.We then investigate the stability of Pt single-atom adsorption on mono- and di-hydrogenated 3.3aGNRs. We do not consider non-hydrogenated aGNRs as the non-hydrogenated structures are unstable as observed in the case of zGNR. We consider the following structures: Pt adsorbed at short-bridge (SB) and long-bridge (LB) sites  sites ; substitB) sites . We conses  and Eref is the total energy of the reference system, where we use perfect z1 and perfect z1 on graphene (z1/GR) as references for the binding energies for a Pt single-atom at z1 and at z1/GR, respectively. Calculated binding energies and corresponding CLSs are summarized in βC@z1. The most stable adsorption structure (PtαC@z1) is unchanged and the binding energy difference is 0.16 eV upon inclusion of the substrate. The substrate does have an insignificant impact on the calculated core level shift, especially for the most stable PtαC@z1. The change is −0.19 eV and the conclusion is unaltered.Here we examine the effect of the substrate, as in the experiment,4zGNR and aGNR on the same footing, and investigate the favorable adsorption site when both edges are exposed. For this purpose, we define the binding energy of a single Pt atom asEz(a)GNRPt@ and Ez(a)GNR are total energies of Pt adsorbed zGNR (aGNR) and pristine zGNR (aGNR), respectively. We note that the total energy of the most stable form of GNR at a given μ2H is used as a reference (Ez(a)GNR, see also Eb is an indicator of the strength of Pt single-atom adsorption and can be used to discuss the stability of Pt when both zGNR and aGNR are present. Calculated Eb's as a function of μC and μ2H (phase diagram) are shown in μC = EGR and μ2H = E2H are summarized in aGNRs and Pt adsorbed aGNRs are thermodynamically more stable than zGNRs in terms of the Gibbs free energy 2@a2 appears as a stable structure among PtαCβC@z1. This narrow a2 region corresponds to the region where intersections of formation energies are found 2@a2 emerges as a stable adsorption structure. To determine the precise stability (boundary) when the different phases compete, however, more accurate and precise calculation of total energy is required. Nevertheless, the stability at around μC = EGR and μ2H = E2H is unaffected, and we pursue this in future work. Very recently, Yamazaki et al. proposed the threefold-coordinated Pt atom at the edges of graphene flakes based on X-ray photoelectron spectroscopy and DFT calculations,42 which contradicts our finding that the thermodynamically most stable configuration (PtCa@z1) has a twofold-coordinated Pt atom. On the other hand, the metastable PtβC@z1 in our study, which has a threefold-coordinated Pt atom, gives relatively large binding energy and reasonable CLS, and we do not rule out the possibility of the threefold-coordinated Pt atom at the edge at this point. To resolve the discrepancy, we may need to employ the state-of-the-art theoretical method to calculate the absolute binding energy,67 and to investigate the models for the graphene edge, because we and Yamazaki et al. use different models  for the graphene edge. Nevertheless, we can conclude that the Pt single-atom adsorption takes place dominantly at the edge of graphene, and determination of a more precise position and CLS will be done in the near future.Experimentally, both zigzag and armchair edges coexist depending on the environment. Thus, it is desirable to compare the stability of single Pt atoms adsorbed at the edges of ergy see  and 2, Pre found  and the αC@z1 (Fig. S3 in the ESIxy state hybridizes strongly with the C sp state and forms a fully unoccupied antibonding state, resulting in the formal oxidation state of 2+. We also performed the Bader charge analysis (Table S2 in the ESId states with GNR.Here, let us discuss the oxidation state of a single Pt atom, as it is crucial to the understanding of its catalytic activity. We calculated the density of states projected on the atomic orbitals of Pt for Pt5aGNR is thermodynamically more stable than zGNR under a wide range of hydrogen pressure, single Pt atoms preferably adsorb at the edge of hydrogenated zGNR. The calculated core level shifts for the stable structures are in reasonable agreement with the experiment, supporting our findings. Our study will serve as a basis for further investigation of the catalytic activity of single-atom catalysts based on single Pt atoms and graphene based nanostructures.We present a systematic density functional theory-based thermodynamics study of Pt single-atom adsorption on graphene. We find that single Pt atoms adsorb more preferably at the graphene edge than on the bulk. Although pristine There are no conflicts to declare.NA-001-C8NA00236C-s001


Cisplatin (CDDP), carboplatin (CP), and oxaliplatin (OXP) are three platinating agents clinically approved worldwide for use against a variety of cancers. They are canonically known as DNA damage inducers; however, that is only one of their mechanisms of cytotoxicity. CDDP mediates its effects through DNA damage-induced transcription inhibition and apoptotic signalling. In addition, CDDP targets the endoplasmic reticulum (ER) to induce ER stress, the mitochondria via mitochondrial DNA damage leading to ROS production, and the plasma membrane and cytoskeletal components. CP acts in a similar fashion to CDDP by inducing DNA damage, mitochondrial damage, and ER stress. Additionally, CP is also able to upregulate micro-RNA activity, enhancing intrinsic apoptosis. OXP, on the other hand, at first induces damage to all the same targets as CDDP and CP, yet it is also capable of inducing immunogenic cell death via ER stress and can decrease ribosome biogenesis through its nucleolar effects. In this comprehensive review, we provide detailed mechanisms of action for the three platinating agents, going beyond their nuclear effects to include their cytoplasmic impact within cancer cells. In addition, we cover their current clinical use and limitations, including side effects and mechanisms of resistance. During the first world war, the field of cancer treatment shifted from a surgical and radiology focus thanks to the use of the mustard gas . A paperSince the approval of cisplatin in the late 1970s, Pt-based drugs have remained as prominent anticancer drugs, and are the dominant treatment for many solid tumours, including testicular, ovarian, bladder and colorectal cancers. Cisplatin, carboplatin and oxaliplatin are all considered to canonically act via the generation of DNA adducts which lead to subsequent DNA damage; however, the scope of their complete cytotoxicity within the cell is much broader and not limited only to the nucleus ,7,8. In Cisplatin, also referred to as cis-diamminedichloroplatinum (II) (CDDP) , is a wiEscherichia coli (E. coli), but inadvertently rediscovered new properties of CDDP. Using Pt electrodes, Rosenberg and his team applied a current to a chamber containing a solution with E. coli; they found that the bacteria stopped dividing but continued to grow up to 300 times their normal size  platinum(II) , is anotBoth CDDP and CP are structurally similar; however, their leaving groups differ. Where CDDP has two chloride ligands, CP has a bidentate cyclobutane dicarboxylate ligand . The difCP is used in a wide variety of cancers including ovarian cancer, lung cancer, and certain head-and-neck cancers . In partOnce the drug has been activated and enters the nucleus, it can then bind to the DNA and form Pt-DNA adducts . CP can Regardless of which adduct CP forms, the resultant crosslinks create deformations in the DNA helix that initiate a similar cell death processes as CDDP; DNA replication and repair can become inhibited leading to transcription errors and inhibition of cell function ,242 FigA. As a rIn 2015, Soori et al. demonstrated through spectroscopy that CP had a weaker binding affinity to chromatin-bound DNA compared to free DNA . FurtherOnce DNA is transcribed into mRNA, it can undergo posttranscriptional regulation by microRNAs (miRNA); short, single-stranded RNA, is endogenous to the cell and non-coding, and acts on the mRNA by targeting the 3′ untranslated region . In partAs CDDP is a well-known ER stress inducer, research into assessing the ability of CP to cause ER stress is prudent ,245. Bro2+, increased membrane potential, and mitochondrial depolarization  into the cytoplasm, which lead to the activation of caspase-9, triggering apoptosis platinum(II) , was syn in 2002 ,259. OXP in 2002 . In factThe structure of OXP differs from the other Pt-drugs based on its carrier ligand, 1,2-diaminocyclohexane (DACH) and an oxalate leaving group ,7. The DOXP was specifically developed to overcome the resistance to CDDP or CP, and in the NCI 60-cell human tumour panel, OXP showed a different pattern of sensitivity than CDDP, indicating a differing array of cancers OXP could treat. Reports have also shown that OXP has promise in cancers for which CDDP is weak against . FurtherOXP has been explicitly approved for the treatment of adjunctive and/or metastatic colorectal cancer by the US FDA and other drug administration bodies globally ,267,268 Beyond colorectal cancer, there are off-label uses of OXP in the treatment of other digestive system cancers such as esophageal and gastric cancers. Advanced biliary adenocarcinoma, chronic lymphocytic leukemia, advanced pancreatic cancer, and advanced ovarian cancer all list OXP as an indication ,267,268.OXP, like other Pt-based agents, is a DNA-damage inducer via the formation of Pt-DNA adducts, causing growth arrest and triggering apoptosis ,270 FigA. OXP haThe adducts formed by OXP, predominantly 1,3-d(GpNpG) and Pt–dG or Pt-dA monoadducts ,271, oftOXP can enter the cell via passive diffusion and is only known to use the main Pt-influx transporter, CTR1, at lower concentrations, whereas higher concentrations of OXP tends to enter the cell independently of CTR1 ,272,273 2], where the oxalate was replaced with chloride leaving groups, like that of CDDP, showed higher rates of adduct formation at equimolar concentrations of OXP [The chemical structure of OXP also hinders its ability to form DNA adducts. While the DACH ligand allows for a strong deformation of the DNA strand, the slow dissociation of the oxalate ligand results in lower rates of Pt accumulation. An OXP analogue, [Pt(DACH)Cls of OXP . Due to s of OXP ,276,277.s of OXP ,279.As mentioned with respect to CDDP and CP, OXP interacts predominantly with chromatin rather than free DNA . Soori eIn addition to the inhibition of DNA replication, OXP was shown to also be effective in inhibiting transcription as demonstrated by a study which utilized immobilized DNA templates to create site specific 1,2- and 1,3-crosslinks for CDDP and OXP . Yuan etBruno et al. assessed RNA interference (RNAi) signatures, comparing CDDP, CP, and OXP, to predict the mechanism of their cytotoxic drug action . The autRibosome biogenesis is often elevated in cancer and inhibition of the process is currently being studied as a treatment mechanism ,282. TheStudies looking at OXP-treated lymphoma and lung adenocarcinoma cells showed levels of pre-RNA that fluctuate in a similar pattern to that of a known ribosome biogenesis stressor, actinomycin D ,282. In As ribosome biogenesis occurs within the nucleolus, nucleolar morphology can be assessed for tumour severity, and it is therefore of interest with OXP treatment ,283. RibPol I inhibition can occur from ribosomal DNA damage; however, direct DNA damage can also show an increase in Pol I inhibition as with CDDP-treated cells. On the other hand, clinically relevant doses of OXP also increased Pol I inhibition, but DNA damage was not found abundantly surrounding the nucleoli, suggesting that the mechanism of Pol I inhibition was separate from DNA damage and likely correlated with the nucleolar perturbation (NPM1 redistribution)  Figure C. The reBeyond the nucleosome, there is also the potential for OXP affecting ribosome biogenesis by forming canonical DNA crosslinks such as CDDP and CP . For insAlthough OXP is able to induce cell death through DNA damage, it also plays a larger role outside of DNA. For instance, Gourdier et al. demonstrated that OXP was able to induce mitochondrial apoptosis via activation of pro-apoptotic factors Bax and/or Bak in a p53-independent manner in HCT116 colon carcinoma OXP-sensitive cells; a defect in the Bcl-2 family of proteins was shown to be a critical mechanism for OXP-resistance ,279,286 A unique mechanism of OXP in treating cancerous cells is the induction of immunogenic cell death (ICD) ,287. ICDER stress, from drug treatment, results in caspase-8 activation that prompts the translocation of CRT and ERp57 from the ER to the plasma membrane surface, where CRT acts as an “eat-me” or engulfment signal to the nearby immune cells. The translocation of CRT occurs early on in apoptosis, prior to any morphological changes or phosphatidylserine exposure ,288,289.Colorectal cancer is heavily influenced by the immune system, with the type, density, and location of lymphoid infiltrates present in the tumour bed affecting the development and progression of the disease; thus, cytotoxic T cells and T-helper cells can be beneficial in treating this cancer . As OXP An important point to acknowledge is the requirement of a functioning immune response for OXP efficacy. Progression-free and overall survival is lowered in colorectal cancer patients receiving OXP treatment in those who have a loss-of-function TLR4 mutation, indicating that even with a release of HMGB1 and translocation of CRT, without an active immune system an anti-cancer vaccine environment cannot be formed .In a study looking at colorectal cancer cell lines (HCT116 and HT29CC) and hepatocellular carcinoma (HCC) cell lines , OXP was shown to induce necrosis and a stronger inflammatory state in the HCC cells compared to the colorectal cell lines which more often underwent apoptosis . In this+ T-cells and reduced Foxp3+ T-reg cells, which are a strong indication of survival [In a 2020 study, murine HCC (H22 and Hepa-16) and human HCC (HepG2 and SMMC7721) cells were exposed to multiple doses of CDDP and OXP each; a single maximally effective dose of 20 µM was chosen to avoid higher dose-side effects . Similarsurvival . The presurvival . The stusurvival . These r2. A suggested mechanism for chronic OIPN is through mitochondrial damage and death of sensory neurons (nerve cell necrosis) [As with CDDP and CP, OXP has limitations to its use, either because of side effects, hypersensitivity, or chemoresistance. The major dose-limiting side effect of OXP is peripheral neurotoxicity, occurring acutely or chronically ,296,297 ecrosis) ,268. Symecrosis) ,297. WhiOther major side effects from OXP treatment include gastrointestinal reactions, myelosuppression, and hypersensitivity reactions ,296,297 Gastrointestinal problems tend to be of minor concern, limited to mild-to-moderate nausea, vomiting and diarrhea; generally, there are no impacts on the liver or kidney unless previously impaired ,268. HowWith respect to myelosuppression and myelotoxicity, symptoms tend to be moderate: a greater amount of grade 3–4 neutropenia compared to anemia or thrombocytopenia; fever only tends to present in less than 4% of patients ,268 Tab.Although OXP was designed to overcome hypersensitivity induced by CP or CDDP, patients can still develop hypersensitivity to OXP as the cumulative dosage increases . NotablyAs with all drugs, resistance can be innately present or acquired over time and use. The mechanisms that increase resistance to OXP include cellular influx/efflux and detoxification, DNA adduct repair, and cell death mechanisms ,268,270.+K+-ATPase β1 subunit was found to be correlated with OXP resistance in an Na+K+-ATPase-pump independent manner; this result suggested that exogenous introduction of the β1 subunit was able to increase uptake and sensitization of the cell to OXP, whereas resistance was maintained with a loss of this subunit [Even though OXP is not known to be solely reliant on CTR1 for its predominant influx into cells, OXP is still reliant on transporters. The levels and function of transporters such as soluble carrier transporter family (OCT 1–3) are critical for OXP influx into the cell . Therefo subunit ,300.Another method of resistance to OXP is detoxification, namely by the glutathione system, mediated by the ABCC transporter subfamily . The aqumodels of preclinical intrinsically low levels of ERCC1 have been correlated with OXP sensitivity, while elevated levels have been associated with resistance [With respect to DNA repair, OXP is unhindered by a deficiency in MMR due to the bulky DACH ligand. OXP-treated cells are therefore able to undergo apoptosis irrespective of MMR pathway functionality ,264,266.sistance ,304,305.sistance ,306. In sistance . OXP is sistance . In colosistance ,308,309.Once OXP has caused its damage and the repair mechanisms have failed to protect the cell, the cell inevitably must undergo cell death; however, there are resistance mechanisms that at this stage too, can interfere with OXP. Alterations to levels of certain pro-apoptotic and anti-apoptotic factors, such as the Bcl-2 family, Bax, inhibitors of apoptosis proteins (IAP) including survivin and BIRC, and p53 have all been correlated with resistance to the intrinsic apoptotic pathway ,310,311.The introduction of CDDP, CP, and OXP into cancer chemotherapeutics has resulted in dramatic improvements in survival and quality of life for cancer patients. However, in the literature, their nuclear effects have been the predominant focus, while their non-nuclear targets have often been neglected in assessing cytotoxicity. More research needs to be conducted on their cytoplasmic mechanisms of action, as these effects can provide information on the cellular responses and toxicity that may be missed by solely evaluating nuclear damage. Furthermore, elucidating additional cellular targets of these drugs can provide insight into potential methods of reducing side effects and resistance, while enhancing cytotoxicity. A conclusion to be taken from this review is the need for further research to differentiate the actions of CP from those of CDDP. CP is often considered to be equivalent to CDDP and conclusions reached from research on CDDP cytotoxicity is often applied to CP. However, as demonstrated in this review, these drugs do not show identical cytotoxicity towards cancer cells, and while certain mechanisms are common between the two, current research inadequately covers the scope of effects induced by CP. In addition, OXP requires further comparison within CDDP-effective cancers to determine whether it could be used as a low-side effect substitution. Overall, further research needs to be conducted on CP and OXP so as to provide a more well-rounded conclusion of their mechanisms of action, resistance, and what cancers they could be used to treat.


This reversible conversion might be a novel and facile approach to make graphene nanoscrolls and to store the graphene. Also, it may bring new sight to the conversion research between two-dimension and one-dimension materials.In this paper, the reversible conversion between pristine graphene nanosheets and pristine graphene nanoscrolls at room temperature was reported. The graphene nanosheets were rolled up into the graphene nanoscrolls by silver nitrate in ethanol solution, and the fabricated graphene nanoscrolls were unfolded back to the graphene nanosheets in ethanol solution by adding ammonium hydroxide. The dynamic conversion state of the process was confirmed by the morphology of the intermediate samples captured using an optical microscope and scanning electron microscope. Also, AFM, TEM and Raman spectroscopy displayed that the graphene transformed from its nanoscrolls remained the structure and morphology of the started graphene. The reason for the formation of the nanoscrolls was assigned to the silver cyanide particles generated on the edge of the graphene. The freshly formed silver cyanide caused the unbalanced energy of the graphene surface by changing the pi electron distribution and triggered off the graphene to roll up. The unfolding of the graphene nanoscrolls back to the graphene nanosheets was attributed to the removal of the silver cyanide by the ammonia  The reversible conversion between the pristine graphene nanosheets exfoliated from graphite and the pristine graphene nanoscrolls was achieved at room temperature. It was first reported by Bacon in 1960 , which was formed in a dcarc under a pressure of 92 atmospheres of argon and at 3900 K.8 Graphene nanoscrolls not only inherit graphene's superior properties but also have new excellent features. They can be useful candidates for microcircuit interconnects due to their high current density being sustained up to 5 × 107 A cm−2;9 applied to supercapacitors, batteries, hydrogen storage, and catalyst supports due to their considerable surface area and highly porous structure;10–12 weaved as high-strength structural materials because they have a high degree of flexibility, tensile strengths up to 2000 kg mm2 and Young's modulus of more than 7 × 1012 dyne cm−2.8 Also, graphene nanoscrolls can be used as ion channels, controllable nano filters, tunable water channels and molecular sensors.13–18 Therefore, the preparation of graphene nanoscrolls has attracted increasing attention. Viculis et al. prepared graphene nanoscrolls11 through exfoliating graphite-intercalated with alkali metals at 200 °C under sonication with the high-energy of 500 W. Li J. L. et al. made graphene scrolls by high energy ball milling of graphite.19 Savoskin et al. obtained graphene nanoscrolls by using graphite-intercalation compounds and sonication.20 Xie reported a controlled way to prepare graphene nanoscrolls via rolling the monolayer graphene by evaporation of isopropyl alcohol solution.9 Quintana et al. rolled the exfoliated graphite sheets into graphene nanoscroll in dimethylformamide by using ferrocene aldehyde.21 Zheng J et al. reported the production of high-quality graphene nanoscrolls with microwave spark assistance in liquid nitrogen.22 Carotenuto G. et al. obtained nanoscrolls from graphite nanoplatelets by using a mechanical technique.23 Nevertheless, these methods can only produce a tiny amount of the graphene nanoscrolls, and the fabrication conditions are either harsh or difficult to control too. Therefore, the real practical application of the graphene nanoscrolls is sparsely reported. Notably, no articles on the reversible conversion between graphene nanosheets and graphene nanoscrolls have been published up to date.Graphene has attracted considerable attention since it was discovered because it has potential applications in numerous fields owing to its excellent electronic, mechanical, optical and thermal properties.In this letter, we reported a novel and facile approach to realize the mutual transformation of graphene nanosheets and graphene nanoscrolls by using silver salt and ammonia for the first time. The graphene nanoscrolls can be produced mainly from the graphene exfoliated from graphite and converted back to the graphene. SEM, TEM, AFM, XRD, and Raman spectroscopy were applied to characterize the morphology and structure of the graphene, the graphene nanoscrolls and the intermediate state of the conversion. The reversible conversion mechanism is also explored.2 (99.5%) were obtained from the Shanghai Jiao Tong University chemical stores. Ammonium hydroxide (25–28%) and silver nitrate (99.8%) were purchased from Sinopharm Chemical Reagent Co., Ltd.Graphite powder was purchased from Sinopharm Chemical Reagent Co., Ltd. Absolute ethanol (99.5%) and CO24 Then, a certain amount of the as-prepared graphene was dispersed in ethanol solution into which a certain amount of silver nitrate was added forming the concentration of 0.01 mg ml−1. After stirring by a magnetic mixer for 30 min, the black sediment was formed and separated from the solution via vacuum filtration. The collected black sediment (named as graphene nanoscrolls) was dried in a vacuum oven for 24 h.The graphene was firstly prepared by shear exfoliation in supercritical carbon dioxide based on our previously published article.A certain amount of the as-prepared graphene nanoscrolls were firstly dispersed in 95% aqueous ethanol solution. Then, the ammonium solution (25–28%) was added dropwise into the ethanol solutions of the graphene nanoscrolls from which a small amount of solution was taken out at regular interval for characterization.−1. Raman spectroscopy was measured on Renishaw inVia Reflex Raman System equipped with a 532 nm laser source.The morphology and the size of the samples were characterized by scanning electron microscopy  on silicon substrates and were analyzed by transmission electron microscopy  on standard copper grids. The lateral size and thickness of samples were examined by atomic force microscopy  on mica substrates. X-ray diffraction (XRD) patterns were carried out on a model of D/max-2200/PC X-ray diffractometer . The samples were irradiated at 10–70° with a scan rate of 5° min25,26 Their thickness and the lateral size can be identified from the AFM images. The average lateral dimension of the graphene nanosheets was around 1–5 μm as shown in In order to compare the graphene made from the graphite (named as GMG) with the ones converted back from the graphene nanoscrolls (designated as GNS), we characterize the morphology and structure of the GMG using SEM, TEM, and AFM firstly. An SEM image  displaysθ = 26.38° is attributed to the graphite crystallinity (002). The diffraction peaks at 2θ values of 24.03°, 29.74°, and 38.35° are assigned to the AgCN (101), AgCN (110) and AgCN (012) planes, respectively. Therefore, the nanoparticles formed on the surface of the GCN are identified to be AgCN. Also, the formed AgCN has been demonstrated using other methods.27 The role of the forming AgCN in the conversion of the GMG into the GNS will be explained in later.Then, a certain amount of the as-prepared graphene was scrolled by a certain amount of silver nitrate in ethanol solution. We investigated the conversion of GMG into the GNS by applying SEM, TEM, and XRD, respectively. We can find an intermediate state of the isolated GNS from the SEM image , which h−1). It was observed by an optical microscope at an interval of five minutes until the GNS converted into the GCG completely. We found an interesting phenomenon when aqueous ammonium was added to the GNS ethanol solution to remove the AgCN: the GNS was converted back to the graphene sheets (named as GCG). To investigate this exciting phenomenon, we designed a series of experiments for the reaction of the GNS with the aqueous ammonium. The morphology and structure of the resultant samples obtained at a regular interval period were characterized. The images of the optical micrograph shown in −1) for 10 min. Also, we captured the process of the samples during the conversion of the GNS into the GCG using SEM to observe the micro-process of the translation. To confirm the point aforementioned, we applied TEM, AFM, TEM and Raman spectra to characterize the morphology and quality of the GCG. TEM images (Fig. S1A and Bvia the intermediate state (27 triggers the curling of the graphene, which overcomes the stain energy barrier when the graphene starts to bend. Upon the overlap of the graphene sheet occurs, the van der Waals force between the graphene sheets is more significant than the bending force of the graphene, which resulting in the curling process to continue until reaching the stable state of the nanoscrolls. The silver cyanide nanoparticles might be generated much more at one edge of the graphene but scarce in the other sides because the reaction was at random. Then, the unbalanced energy of the graphene surface was generated by the formed silver cyanide nanoparticles through changing the pi electron distribution of the graphene sheet because of silver ion attraction like the isopropyl alcohol causing a surface strain in graphene by immersion.29 This unbalanced surface strain caused the opposite edge of the graphene sheet to start to bend. Upon the surface strain is big enough to overcome the energy barrier of the graphene bending, the graphene sheet would begin to roll until reaching the stable state, a graphene nanoscroll, by van der Waals forces. This speculation is proved by the fact that the silver cyanide nanoparticles were on the surface of the scrolls but not trapped in the scroll structure as shown in via reacting with the ammonium ion when the aqueous ammonium was dropwise into the solution. With the dissolving of the silver cyanide nanoparticles, the spreading force of the graphene nanoscrolls was big enough to make itself loosen gradually (28 Sharifi et al. claimed that the scrolling of the nitrogen-doped reduced graphene oxide sheets into the nanoscrolls was initiated by the strong adsorption of the maghemite nanoparticles at nitrogen defects in the graphene lattice and their mutual magnetic interaction, and the nanoscrolls could be returned to half-open structures and open sheets fully upon removal of the maghemite nanoparticles by acid treatment in different period. Therefore, we think the mechanism of the scrolling and unwinding in our work were similar to the Sharifi's ones regarding the rolling and unrolling caused by forming nanoparticles and removing nanoparticles. However, the role of the silver cyanide nanoparticles in initiating curling in our work might be different because the silver cyanide does not have magnetic properties, which suggests that the nanoparticles without magnetic properties can cause scrolling too if the conditions are suitable. Also, the system in the published paper28 was complicated, while our scrolling system is straightforward.The interconversion of the pristine graphene and the pristine graphene nanoscrolls is reported for the first time. We proposed a mechanism to explain this process. As shown in te state . The comradually  and finaradually . In the A novel and facile approach has been developed to achieve the reversible conversion between graphene nanosheets and graphene nanoscrolls. We have demonstrated the products of the mutual transformation by using an optical microscope, SEM, TEM, and AFM and Raman spectra, respectively. The graphene converted from the graphene nanoscrolls remained its initial structure and morphology. The silver cyanide at the edge of the graphene played essential roles in the transformation of the graphene into the graphene nanoscrolls and the reverse. The approach is expected to be useful in making graphene nanoscrolls and storing graphene.There are no conflicts to declare.RA-008-C8RA00475G-s001


The experimental data shows the hysteresis effect of the silica gel in the region between 30% and 80% relative humidity and its dependence on the relative humidity earlier in the process. It also shows there are multiple equilibrium water contents at a relative humidity of 40%, depending on the previous relative humidity. The relative humidity barely influences the fitted reaction rate constant during adsorption. However, during the desorption process, not only the current relative humidity but also the history of relative humidity have an influence. A higher relative humidity in the previous step can slow down the desorption rate in the following step.Mathematical models can provide estimates for the shelf life of water-sensitive products like food or pharmaceuticals. This study presents a simple kinetic model using two first-order reactions for the evaporation and condensation of water. Furthermore, the model can be simplified to contain only one free parameter, the reaction rate constant  Moisture content in the product and the headspace composition strongly influence the shelf life and quality of a wide variety of products, like food, pharmaceuticals, or even artworks ,2,3. DepIn summary, many different isotherm models already exist and are successfully used within their individual range of applications and boundaries. Therefore, this paper does not want to propose a new isotherm model. Instead, the experimentally generated data is used to calculate individual points on that isotherm for the different relative humidity and interpolates between this grid points. However, the isotherm only describes the equilibrium but not the kinetics, which are necessary for the integration into a complete shelf-life model, with varying storage conditions and the coupling of additional processes, like permeation through the packaging. Such kinetic behaviour is also important for the design of active packaging systems, like oxygen scavengers, antimicrobials and moisture absorbers, because multiple concurring processes occur simultaneously. In this study, a simple kinetic model for the sorption process is presented. Silica gel is chosen as a well-controllable system for general model development here instead of an actual food. Its main benefit is that it is chemically inert and stable during the experiment, which lasts for around three weeks. Actual food could decay during this time and distort the results. While silica may not be a quantitative proxy for a food system, the results on silica itself may still be quantitatively useful as silica gel is the most commonly used desiccant for preventing excess water in food packages  and is u2/g. The pore volume of silica gel is around 0.4 cm3/g.Silica gel beads with a grain size of 2–5 mm were obtained by Carl Roth, Karlsruhe Germany. Moreover, silica gel is a chemically inert, nontoxic material with a high internal surface area of around 750 mThe water vapour sorption measurements were carried out using the dynamic vapour sorption (DVS) system . Silica gel beads with a weight of approximately 0.05 g are placed inside a sealed chamber. During the experiment, the samples are exposed to a predefined relative humidity and temperature profile and are weighed every minute. Equilibrium is assumed, whenever the change in the measured weight over five consecutive data points is lower than a defined threshold. If all samples are in an equilibrium state, the experiment continues with the next step of the humidity profile.In this study, the relative humidity profile shown in A pseudo-first order reaction (PFO) is a commonly used kinetic model to describe the adsorption kinetics for a wide variety of substances .(1)dwdtk is the reaction rate constant, which describes how fast the equilibrium will be reached. This parameter also depends on the current relative humidity and again can be affected by the history of relative humidity, because there are different sorption processes, like surface sorption, pore sorption and pore condensation occurring for different levels of relative humidity.In (1) I of the experiment as:However, using the assumption that the equilibrium and the reaction rate are constant as long as the current relative humidity stays constant, the experiment shown in i-th step of the experiment. It has to be emphasized that the calculated reaction rate constant Hereby i of the humidity profile of the sorption experiment  can be solved analytically using the initial condition (3):i-th step. The analytically solution for the water content is:Hereby It has to be emphasized that the pseudo-first-order approach for the description of the adsorption and desorption is not based on a physical explanation. Instead it uses a simplifying assumption commonly applied in literature because of its good agreement with experimental data for both the adsorption as well as the desorption .lsqnonlin resulting in 18 reaction rate constants and isothermal water contents. Lsqnonlin uses the trust-region-reflective algorithm based on the Newton method described in [N is the number of experimental observations per step i. Because each step has the same duration of 24 h and there is one measurement point per minute N has the same value for every step and can be calculated as 1440. The index j describes the 1440 experimental data points per step i. The quality of the fit is described using a relative root mean square error (6):The model parameters ribed in . The solribed in .(5)SSQip. Since the temperature during the experiment is kept constant, the surface tension, and the molar volume of the water are also constant. If the pore size distribution of a substance is known and it is assumed that the diameter of a pore stays constants over the complete pore length this would allow the calculation of a sorption isotherm. However, for the same relative humidity, Equation (8) always leads to the same critical pore diameter, and therefore the same water content, regardless of adsorption or desorption, and therefore cannot explain the hysteresis. Nevertheless, the pore network of the silica gel can explain this behaviour, because in reality, the pores of the silica gel are non-uniform and there are parts of the pore with a larger diameter, the cavities and parts with a narrower diameter, the bottlenecks. According to [The maximum pore radius the gas :(7)lnppsrding to , when thAn essential factor for shelf-life modelling is the water content of the product. Its behaviour is influenced by the sorption isotherm of the product and the sorption kinetics. Therefore, this study researched the influence of relative humidity on the sorption kinetics of silica gel as a model substance.i,final,messw, which is the last measurement point of each subset is also included in The measured data is divided into 18 subsets for further analysis, one for each new relative humidity condition. The 18 subsets are then regrouped into 6 parts, each containing 3 subsets. Only the relative humidity of the first subset of each group is different and varies between 10% and 80% relative humidity. The second subset of each group has always a relative humidity of 40%, the third subset of each group has always a relative humidity of 30%. The fitted parameters i,isow and i,final.messw indicates that equilibrium was reached in this experiment for every step. The RRMSE calculates the average relative deviation between the experimental and predicted model values. For every subset, the deviation is less than two percent and demonstrates a good fit of the proposed model for the experimental data. As an example, The reaction rate constantsThe calculated equilibrium water content for every subset is shown in The measurement of the standard isotherm shows that the hysteresis effects of silica gel are located in the relative humidity range between 30% and 80%. It can be seen that there is a linear correlation of the water content and the relative humidity for a relative humidity up to 30%. As seen in This finding also indicates that there are not only two states for the isotherm, one for the adsorption and one for the desorption. Instead, there are multiple states with a partial hysteresis depending on the history of the sample.k of silica for all 18 subsets.k of the relative humidity suggests that the kinetics of the pore condensation is independent of the pore diameter. This finding also validates the ink-bottle theory, which suggests only influences on the desorption process, not on the adsorption process [The data for the kinetic of the adsorption is not quantitatively fully conclusive. However, the apparent trends can be physically interpreted. For low relative humidity below 60%, the reaction rate k seems independent from the relative humidity and the scatter may be attributed to the model simplifications and slight experimental errors. The main factor of water uptake in silica gel between 30% relative humidity and 80% relative humidity is due to pore condensation. Condensation in pores with higher diameter is possible with higher relative humidity and therefore higher water pressure in the gaseous phase. Thus, the independence of the calculated reaction rate constant  process . Only fohistory two steps before, which is varied between 10% and 80%.The different values at a relative humidity of 30% are noteworthy, suggesting the values of the reaction rate constant history of 10% and 20% in k. It is also shown that this exponential behaviour can be derived mathematically using two first order reactions for the evaporation and condensation of water onto the silica gel. These results can also help by the design of more efficient packaging concepts for preservation of food or artwork. It shows that more silica gel is needed to protect against drying compared to moisturizing of the product because of the slower desorption kinetics of silica gel. The knowledge of the partial hysteresis of silica gel also allows the more detailed calculation of the water uptake or release capacity of silica gel under varying outside relative humidity, also helping by the design of the active packaging. Additionally, these experimental results can be used as a proof-of-concept for this measurement setup with a varying relative humidity profile, and can be used on perishable food in the future.It could be shown that the desorption kinetic of water on silica gel depends not only on the current relative humidity, but also on the relative humidity earlier in the process, because of hysteresis effects. A closer look at scatter on the fitted reaction constants shows a difference between adsorption and desorption. While both were not constant for individually fitted parts of the experimental data, the scatter was lower for the absorption process. In both cases, the experimental data can be described using an exponential expression containing only one free parameter, the reaction rate constant


Testicular involvement is a rarely encountered complication of sarcoidosis. There are unique challenges involved when sarcoidosis affects the genitourinary system. These include differentiating the findings from testicular cancer, which often results in invasive procedures such as orchiectomies. It can also be a cause of secondary infertility in men. Additionally, it may also be underdiagnosed. Here, we describe a case where a patient showed a constellation of findings suggestive of sarcoidosis, along with testicular involvement at initial presentation. In this case, the diagnosis was made clinically with supporting laboratory, pathology, and ultrasound findings. The testicular findings were not biopsied, as the patient had easily accessible skin findings to confirm sarcoidosis. His testicular findings are continued to be monitored via ultrasound. Sarcoidosis is a systemic disease prevalent globally that forms noncaseating granulomas, affecting mainly young adults . It can In the United States, African Americans have a 2.4% lifetime risk of sarcoidosis, and it preferentially also afflicts patients who are Scandinavian, Irish, German, and West Indians [The pathogenesis involves the activation of T-cells after an interaction between antigen-presenting cells and major histocompatibility complex-II. This sets off a cascade that results in granuloma production, which is non-necrotizing in most cases . SarcoidHowever, at times, sarcoidosis can affect other organs too, including the genitourinary (GU) system . This isWe are reporting a rare case of sarcoidosis with non-painful testicular involvement. The patient discovered multiple masses by self-exam and presented with a constellation of other symptoms and findings suggestive of sarcoidosis.A 40-year-old African American male with a past medical history of type II diabetes on insulin, gastroesophageal reflux disease (GERD), and a BMI of 41 presented with a concern to remove multiple raised, nontender plaques on his anterior shins. He had no surgical history, and his social history was positive for significant alcohol use, and daily cannabis use. He underwent corticosteroid injections of his skin findings, thought to be keloids at the time. Labs were subsequently drawn, which showed mild hypercalcemia of 10.8 (8.4-10.4 mg/dL). Due to chronic bilateral lower extremity edema, an echocardiogram was also ordered, which showed an ejection fraction of 60-65%, with no concerning findings.At follow-up, the patient had a satisfactory response to corticosteroid injections on his skin findings. He had not completed repeat labs for a follow-up on his mild hypercalcemia but was concerned about a non-painful scrotal mass. Ultrasound of the scrotum showed an enlarged epididymis bilaterally with numerous hypoechoic lesions measuring up to 6 mm, along with a left varicocele. Additionally, there were numerous hypoechoic testicular lesions measuring up to 2 mm. The differentials included sarcoidosis or lymphoma. Urology was contacted, and they recommended a biopsy of an easier accessible site rather than the patient’s testicular growths Figure .The patient followed up within a week, denying any fevers, chills, or night sweats. He had no lymphadenopathy on an exam. He was concerned about right eye erythema and pain. He was urgently sent to ophthalmology due to concern of uveitis. Additionally, blood work and a chest X-ray were obtained. The patient was also sent to dermatology for a skin biopsy, as it was thought that the plaques originally diagnosed and treated as keloids could potentially be skin manifestations of sarcoidosis.The patient’s angiotensin-converting enzyme (ACE) level was found to be 274 U/L (Reference range: 9-67 units/L), and his IL2R antibodies were 2335 pg/mL (127.3-858.2 pg/mL). Calcium remained elevated at 11.1 (8.4-10.4 mg/dL), 25-OH vitamin D was found to be 7.1 (30-80 ng/mL), and his intact parathyroid hormone (PTH) was 6.7 (14-97 pg/mL). 1,25-dihydroxycholecalciferol was found to be 48.7 pg/mL (19.9-79.3 pg/mL). A chest X-ray was subsequently performed which showed interstitial micronodular disease. The patient’s purified protein derivative (PPD) test was found to be negative. Additionally, ophthalmology diagnosed the patient with uveitis of his right eye, and he was started on appropriate therapy with Pred Forte and Cyclomydril. Sarcoidosis was placed above lymphoma in the differential.A high-resolution CT scan was performed, which showed a nonspecific finding of micronodules and ground-glass opacity, along with bronchiectasis, mediastinal lymphadenopathy, and probable hepatosplenomegaly Figure . BiopsieGenitourinary manifestations of sarcoidosis have been described in case reports in the past. Sixty cases of histologically proven sarcoidosis with GU manifestation had been described prior to 2004. In that small sample size, 48% of the patients were African American, 73% of patients had involvement in the epididymis, and 47% had involvement in the testes [When differentiating sarcoidosis and testicular cancer, it’s paramount to understand that Lactate dehydrogenase (LDH) is not reliable, as sarcoidosis may cause a raised LDH as well. However, it does not cause elevations in alpha-fetoprotein (AFP) or human chorionic gonadotropin (b-HCG) . ClinicaFurthermore, De Cinque et al. suggest using contrast-enhanced ultrasound to differentiate between sarcoidosis and testicular cancers. Although the most accurate method in differentiation remains a biopsy, orchiectomies are often undertaken as it can be misdiagnosed for a neoplasm . On ultrThere are other concerns with testicular sarcoidosis as well. Kovac et al. report a case of azoospermia from testicular sarcoidosis, resulting in secondary infertility. The case presented with normal follicle-stimulating hormone (FSH) levels and azoospermia after a new-onset difficulty conceiving. A diagnosis of sarcoidosis was made based on CT findings, testicular biopsy, and uveitis. After treatment with corticosteroids, the patient’s sperm concentrations and motility rapidly improved and the couple was able to conceive naturally again .Testicular sarcoidosis continues to pose a multitude of diagnostic challenges and is likely an underdiagnosed entity. To compare, sarcoidosis affects the testes in fewer than 0.2% of all clinically diagnosed cases, while pulmonary manifestations are diagnosed in 90% of cases ,10. ManaTo our knowledge, the amount of literature on testicular sarcoidosis is limited. The diagnosis of testicular sarcoidosis poses unique challenges as well, as it has to be differentiated from testicular cancer, which can result in invasive procedures, such as orchiectomies. This case demonstrates an alternative management strategy that involves a more conservative approach. However, due to the strong association between sarcoidosis and testicular cancer, the patient will have a long-term follow-up with scrotal ultrasounds to ensure that his testicular growths remain stable or resolved.


Patient-specific 3D models are currently being used preoperatively for trainee medical education in planning surgical approaches and intraoperatively to guide decision-making in several specialties. Our study group utilized a modified Delphi process to create a standardized assessment for trainees using patient-specific 3D models as a tool in medical education during pre-surgical planning.A literature review was conducted to identify survey questions administered to clinicians in published surgical planning studies regarding the use of patient-specific 3D models. A core study team reviewed these questions, removed duplicates, categorized them, mapped them to overarching themes, and, where applicable, modified individual questions into a form generalizable across surgical specialties. The core study panel included a physician, physician-scientist, social scientist, engineer/medical student, and 3D printing lab manager. A modified Delphi process was then used to solicit feedback on the clarity and relevance of the individual questions from an expert panel consisting of 12 physicians from specialties including anesthesiology, emergency medicine, radiology, urology, otolaryngology, and obstetrics/gynecology. When the Radiological Society of North America (RSNA)/American College of Radiology (ACR) 3D Printing Registry Data Dictionary was released, additional survey questions were reviewed. A final cross-disciplinary survey of the utility of 3D printed models in surgical planning medical education was developed.The literature review identified 100 questions previously published in surveys assessing patient-specific 3D models for surgical planning. Following the review, generalization, and mapping of survey questions from these studies, a list of 24 questions was generated for review by the expert study team. Five additional questions were identified in the RSNA/ACR 3D Printing Registry Data Dictionary and included for review. A final questionnaire consisting of 20 questions was developed.As 3D printed models become more common in medical education, the need for standardized assessment is increasingly essential. The standardized questionnaire developed in this study reflects the interests of a variety of stakeholders in patient-specific 3D models across disciplines.The online version contains supplementary material available at 10.1186/s12909-022-03581-7. The utilization of three-dimensional (3D) printing has grown dramatically in recent years, bringing new techniques to the healthcare industry. Unlike traditional manufacturing methods , this form of additive manufacturing offers a low-cost, rapid method to generate objects from a digital model or computer-aided design (CAD) file into physical shapes. Moreover, patient-specific data (CT/MRI imaging) can now be processed and printed into 3D form. Applications of 3D printed products in healthcare include tissue and organ fabrication and the creation of customized prosthetics and implants . In addip < 0.05) in post-interventional assessments for medical students [Within graduate medical education and residency training, 3D printing has been used to enhance knowledge and skill acquisition. The ability to rapidly produce detailed mimetic models can be beneficial for visualizing complex anatomy and structural relationships. 3D models have shown benefits to students and trainees in anatomy education and procedure simulation , 3. A mestudents . SmerlinThe use of patient-specific 3D models for pre-procedure planning has been reported in a variety of surgical and medical specialties. Within cardiology, 3D models have been utilized to visualize the complex, variable pathology of congenital heart diseases and to size implantable devices for left atrial appendage closure, among other uses , 7. UsesIn research reporting the uses of 3D models, various measures have been reported to assess the utility of 3D printed models for education and procedure simulation. In a review conducted in 2017 of 93 studies on the use of 3D models for surgical training and simulation, it was found that subjective and objective measures were used in 74 and 61% of studies, respectively . CommonlWe propose the use of a modified Delphi process to review prior measures across specialties to generate a concise, generalizable survey to serve as a gold standard for feedback on the use of 3D printing in surgical planning. The Delphi method is a structured and anonymous process used to collect opinions on a specific topic. It has previously been used in healthcare for a variety of purposes ranging from developing a curriculum for general surgery robotic education to creating a guideline for clinical trial protocol content , 24. TheAs the use of 3D models for surgical planning expands, there exists a need to more consistently assess the value of such models in trainee development and surgeon experience. Our aim was to provide a standardized survey tool for evaluating the use of 3D models in surgical planning across specialties.The core study team consisted of five individuals representing a diversity of professions with experience in the subject matter including a physician, physician-scientist, social scientist, engineer/medical student, and 3D printing lab manager/innovation director. A medical expert panel of 12 individuals was formed representing physicians in anesthesiology, emergency medicine, radiology, urology, otolaryngology, and obstetrics/gynecology. All members of the panel had previous experience working with patient-specific 3D printed models as part of clinical care at the study team’s institution. Participants were recruited through email.A literature search was conducted in 2019 using the electronic databases Pubmed, Ovid, and SCOPUS. The following keywords were searched in the title and abstract: model OR planning OR training OR education OR teaching OR assessment OR skills OR simulation AND “3D print” OR “3D printing” OR “3D Printed” OR “three-dimensional print” OR “three-dimensional printing” OR “three-dimensional printed”. Articles were screened by reviewing the title and abstract to determine if they met inclusion criteria. Inclusion criteria included 1) published in the English language, 2) primary research , 3) use of patient-specific 3D printed models for surgical planning, and 4) a questionnaire that assessed surgeon and/or trainee experience. Articles that initially passed inclusion criteria underwent full-text review. Reasons for exclusion after full-text review included: if the literature was a secondary report of the data, non-English, validation study of previously published survey, or solely discussed specific steps of a surgery.Articles that met the inclusion criteria were examined and data was extracted from them into a structured tabular form to ensure articles were reviewed consistently. Data extracted included study year, specialty, total survey questions administered, use of objective and subjective questions, type of survey response rating scale used, and each full-text survey question.To categorize the scope of questions found in the literature extraction, the full-text questions were assigned to one of six categories  based on the focus of the question, as determined by the core study team through consensus. Each question was further assigned a construct based on the question’s focus . Using the assigned categories and constructs, similar questions were then grouped. Duplicate questions were merged into a single question by consensus of at least two members of the study team. All questions, including both unique and merged questions, were modified into generalized forms that retained the given category and construct but removed references to specific diagnoses or procedures. An example of this categorization process is diagrammed in Table The core study team reviewed each resultant question from the categorization process. Questions that were too narrow in focus to be applied broadly across surgical specialties and cases as determined by the core study group consensus were removed. Questions too vague to allow consensus in interpretation were also removed. Dominant themes  were determined from the remaining questions, which were then grouped and mapped accordingly until all questions were exhausted Fig. . Each maA modified Delphi process was undertaken involving one round of expert feedback and one round of consensus from the core study group to evaluate the question list created by the study team from the literature extraction, categorization, and content mapping process. Questions were evaluated on a 5-point Likert scale for relevance to 3D printing use in presurgical planning and question clarity. Experts were also asked to select whether each question should be retained as-is, reviewed, or removed and to provide free-text comments to explain their recommendations. Individual questions were removed from the question list without further review if they received an average score below 4.0 for relevance . An example of this process is shown in Table Questions with an average score of 4.0 or above for relevance  underwent a second round of review and discussion by the core study team. Questions below 4.0 for clarity were reworded by two members of the study team and required consensus from the entire core study team to be included. The review included evaluating relevance and clarity through examination of the expert panel comments. Questions that were determined to be irrelevant or unclear through commentary feedback were removed. Questions where experts noted difficulty interpreting the meaning were reworded by a subgroup of the core study team and sent back to the remainder of the study team for final approval. Once 80% or greater consensus was reached on the new wording of the question, it was finalized.Concurrently with the development of our survey tool, the 3D Printing Data Dictionary (3DPDD) was released by the RSNA/ACR , 31. ThiAfter the final survey was developed, readability was measured using both the Flesch-Kincaid Grade Level and FORCAST grade level –34. The n = 9), whereas other specialties were represented singly or at maximum in duplicate  reported on both subjective and objective measures while the other half (n = 8) reported on subjective measures only. There were no studies that reported on objective measures only. Rating scales for survey responses varied among the 16 articles and included 10 point (n = 8), 6 point (n = 1), 5 point (n = 2), 5 point Likert (n = 2), and 4 point scales (n = 1).Of the research studies included in the dataset, none noted the completion of a validation process in the study methods to develop the survey questionnaire administered. Two studies utilized surveys that were previously published. The median number of questions per individual survey among the dataset was 5 (range 1–11). A total of 100 full-text questions were extracted from the articles. Half of the studies (Nine studies (56%) administered questions evaluating the anatomic accuracy of the 3D models. Five studies (31%) assessed whether the 3D model helped the surgeon communicate in general with patients, whereas two studies (12.5%) asked specifically whether the 3D models helped explain the procedure to patients. Of the included studies, nine (56%) asked users to rate whether the model was helpful for preoperative planning.The most commonly asked questions were whether the 3D model accurately reflected the region of interest (ROI) anatomy (56%) and whether the model was helpful in preoperative planning (56%). While each of these questions was asked by over half of the included studies, only six (37.5%) studies asked both questions.Of the 100 full-text survey questions extracted, 33 questions were categorized as pertaining to anatomy, 24 relating to surgery, 18 concerning planning, 12 assessing general experience, 11 exploring communication, and two representing diagnosis. Following the sorting and categorization of each question, 55 questions were noted to substantively duplicate at least one other question within its category. Duplicate questions were reviewed, merged, and re-written in generic form yielding 45 unique representative questions in the dataset  participated in a review of the 24 questions generated during the previous mapping session. Of the 10 initial anatomy questions, three questions received an average score below 4.0 for relevancy and were removed. Seven questions scored 4.0 or above for relevancy and underwent further review. Of these seven questions, four questions were mentioned to address similar concepts in feedback and were combined into two questions. The core study team added one question from the 3DPDD and another to reflect feedback from the Delphi process, yielding a total of seven anatomy questions in the final survey.A total of 12 questions related to the utility of patient-specific 3D models were reviewed by the experts and none scored below 4.0 for relevancy. After review, five questions were removed from the utility question pool, four for redundancy and one was moved for consideration in the experience section. One question was noted to be similar to a question in the 3DPDD and a modified version of the latter was used. After review of the expert feedback by the core study team, there were a total of seven utility questions.Three questions relating to the experience of using patient-specific 3D models were reviewed by the experts as all scored above 4.0 for relevancy. All three of these were kept. Additionally, there were three questions from the 3DPDD that were added to this section in a modified succinct format agreed upon by consensus of the core study team. The final survey is listed in Table After the final survey was developed, readability was measured. The Flesch-Kincaid Grade Level was found to be 8.3 and the FORCAST grade was 12.3. The length of the survey was calculated to be around 4 min. Preliminary data suggests a response rate of 74% across 38 cases in which 107 surveys were sent out. Cronbach’s alpha was calculated for each category within the survey and found to show good internal consistency . However, more data is necessary to fully validate the survey.We have described a multi-disciplinary approach to creating a survey that quantifies the experience of using a patient-specific 3D model for medical education in surgical planning. In the literature review performed by the study team, the majority of studies selected for full-text review were excluded for not administering a questionnaire –38. ThisOur approach builds on previous literature, employing both quantitative and qualitative methodology in the Delphi process. By utilizing the results of an extensive literature review, this method provides data already evaluated by those with experience in 3D printing. The use of an anonymous feedback technique to develop a survey to assess 3D printing has not been reported previously. The Delphi method has been used and validated in several fields, including healthcare , 39–41. Compared to other survey development methods, the Delphi process utilizes the feedback from an expert panel. An expert is generally considered someone with experience and knowledge of the field who is well-respected . Our stuTo create a more manageable survey for the modified Delphi process, the study team reviewed, categorized, and mapped the generalized survey questions found through the literature search that were used in previously published research of 3D printed models. Through the mapping process the number of potential questions decreased from 45 to 24 and created shorter, clearer questions. In keeping with the goal of the study to develop a generalizable survey tool to standardize 3D printing model feedback, questions found to be too specific, long, or vague were removed or reworded. Another reason this was done was to eliminate survey fatigue - a common problem in survey data collection. Survey length, along with question complexity and question type, are known to influence survey response . Given tThe release of the 3DPDD during the study period, after the Delphi feedback from our expert panel, influenced the final survey development. The Data Dictionary is composed of ten sections  with a total of 112 questions. The User Assessments section is directed towards clinical users of 3D models and consists of eight questions with Likert scale responses. In reviewing these questions, the core study team found that five were different from questions developed through the Delphi process, of which three related to the experience of using patient-specific 3D models. While these five questions did not undergo separate Delphi review by the expert panel, the core study team determined these questions were important to add by consensus, given that the 3DPDD and 3D Printing Registry were developed through expert agreement and leadership within these organizations. By including these questions in our survey, we hope it will promote the contribution of data into the 3D Printing Registry. Additionally, including the RSNA/ACR data in the final survey has improved the balance of emphasis between anatomy, utility, and experience.The 3DPDD is both comprehensive and generalizable, however, it is not designed as an off-the-shelf survey tool and does not include some questions that we found valuable for collecting information for the understanding of subjective claims of satisfaction and efficacy relating to both surgical planning and patient communication. Therefore, we found it important to continue generating this survey tool to be a readily deployable asset that augments the efforts of the RSNA/ACR and fills a need gap.In the initial review of the survey, a response rate of 74% was achieved across the 107 surveys sent out to a population of academic physicians indicating a positive majority response. Additionally, good reliability was noted across all categories of the survey with each category scoring above 0.700 for Cronbach’s alpha. Our future work will continue the collection of data to further validate the survey and report on data captured by the survey questions. This study illustrates how a literature review-based Delphi method can be used to produce a generalized survey to quantify the experience of using a patient-specific 3D model for surgical planning and trainee education. Through this anonymous feedback process, the study team engaged a variety of stakeholders across an institution to create a survey that can be used to track the use of 3D models. The strength of this survey tool is in that it allows for the comparison of 3D model features and utility in pre-surgical planning across institutions, practices, and specialties.The study team has generated a survey informed by quantitative and qualitative data collection that reflects the diverse needs and experiences of clinicians across healthcare specialties when utilizing patient-specific 3D models. As a valuable educational tool, 3D printed models are utilized and studied for surgical planning at an increasing rate. In the future, data from surveys will contribute evidence to the medical community in a standardized fashion to inform the design, utility, and experience of patient-specific 3D models in surgical planning.Additional file 1.


ERBB2 non-amplified. Endocrine therapy restrains tumor proliferation and is the mainstay of lobular BC treatment. Mutation of ERBB2 has been associated with recurrent ILC. However, it is unknown whether ERBB2 mutation impacts on the otherwise exquisite responsiveness of early ILC to endocrine therapy. We have recently profiled n = 622 HR-positive early BCs from the ADAPT trial for mutations in candidate genes involved in endocrine resistance, including ERBB2. All patients were treated with short-term preoperative endocrine therapy  before tumor resection. Tumor proliferation after endocrine therapy (post-pET Ki67 index) was determined prospectively by standardized central pathology assessment supported by computer-assisted image analysis. Sustained or suppressed proliferation were defined as post-pET Ki67 ≥10% or <10%. Here, we report a subgroup analysis pertaining to ILCs in this cohort. ILCs accounted for 179/622 (28.8%) cases. ILCs were enriched in mutations in CDH1  and ERBB2 , but showed fewer mutations in TP53  and GATA3 . Considering all BCs irrespective of subtypes, ERBB2 mutation was not associated with proliferation. In ILCs, however, ERBB2 mutations were 3.5-fold more common in cases with sustained post-pET proliferation compared to cases with suppressed post-pET proliferation . Moreover, ERBB2 mutation was associated with high Oncotype DX recurrence scores (P = 0.0087). In summary, our findings support that ERBB2 mutation influences endocrine responsiveness in early lobular BC.Invasive lobular breast cancer (ILC) is a special breast cancer (BC) subtype and is mostly hormone receptor (HR)-positive and  Lobular BC is typically hormone receptor (HR)-positive and HER2/ERBB2 non-amplified1. Lobular BC is driven by mutational inactivation of CDH1/E-cadherin and is a slow-growing tumor entity4. Endocrine therapy (ET) restrains ILC cell proliferation and is the mainstay of systemic treatment for lobular BC in the clinic6. Activating mutation of ERBB2 has been associated with recurrent and metastatic ILC and poor prognosis14. Estrogen receptor (ER)-positive human BC cell lines with isogenically incorporated ERBB2 mutation acquire resistance to growth inhibition by estrogen deprivation in vitro15. Accordingly, it is thought that ERBB2 mutation mediates endocrine resistance in vitro15.Invasive lobular breast cancer (ILC) is a special histological BC subtypeERBB2 mutation impacts on the otherwise exquisite clinical responsiveness of ILC cells to endocrine therapy, especially in early ILC. Assessment of the ERBB2 mutation status is not a standard diagnostic procedure in the clinic16. Furthermore, assessment of tumor cell proliferation in early BC pre-treated with endocrine therapy requires that anti-hormonal therapy is initiated a short time before tumor resection. This is not usually done in the clinic. Short-term preoperative endocrine therapy (pET) does not improve outcome, but it offers an empirical readout for endocrine responsiveness by measuring the post-pET Ki67 cell proliferation index19. So far, pET has only been implemented in a limited number of prospective clinical trials, including the POETIC and ADAPT trial22. Whether or not ERBB2 mutation impacts on tumor cell proliferation in early ILC treated with preoperative endocrine therapy has not yet been investigated in this context. Therefore, we extended our previous, retrospective exploratory analyses of BCs from the ADAPT trial (HR-positive/HER2-negative)23. Here we report on the relation between genetic alterations and tumor cell proliferation following pET, with a special reference to ILC and ERBB2 mutation.However, it is currently unknown whether or how much n = 622 HR-positive/HER2-negative early BCs from patients enrolled in the West German Study Group (WSG) ADAPT trial (NCT01779206). This cohort corresponds to a subset of patients enrolled in the ADAPT trial (run-in phase)23. Study design details were reported previously24. Short-term preoperative endocrine therapy  was administered for three weeks before tumor resection22. Oncotype DX recurrence score (RS) testing at baseline was performed at the laboratory of Genomic Health Inc . All tumors were subjected to prospective central pathology review  for histological subtyping and prospective assessment of tumor cell proliferation (documented in 2012–2016). Histologic BC subtypes were determined in accordance with the criteria of the “World Health Organization (WHO) classification of tumours of the breast”, 4th edition (2012)25. Histologic BC subtype calls were based on a consensus of three experts headed by H.K. and were aided by upfront E-cadherin immunohistochemistry (IHC), as described recently26. A comprehensive description of criteria for the diagnosis of ILC has been provided elsewhere2. From an initial set of n = 701 case, n = 79 cases were excluded from further molecular work-up (performed in 2018–2020) due to (i) missing Ki67 values, (ii) unavailable tissue blocks , (iii) divergent histological subtypes at baseline or post-pET or controversial classification as either lobular or non-lobular BC, including cases classifiable as BC with mixed ductal/lobular features (n = 12), (iv) triple-negative hormone receptor status, (v) insufficient DNA amount and/or quality23. The total number of cases for the final statistical analysis was n = 622 .Tumor tissues included rial NCT0779206. T23. BCs scored as HER2 2+ and 3+ were subjected to HER2/ERBB2 fluorescence in situ hybridization in accordance with ASCO/CAP guidelines16. Tumor cell proliferation before endocrine therapy (baseline) and after endocrine therapy (post-pET) was determined prospectively by standardized central pathology assessment of the Ki67 cell proliferation marker23. Immunohistochemical staining of Ki67 was performed with the anti-Ki67 antibody clone 30-9 28. Ki67 scoring was supported by computer-assisted image analysis 30. The Ki67 index was based on three independent evaluations 23. The semiquantitative Ki67 index that was nearest to the digital Ki67 index was accepted as the consensus Ki67 index23. Representative immunohistochemical stainings of BCs in each Ki67 category  are shown in the data supplement 26. E-cadherin IHC was scored as negative (complete loss) versus positive (any specific staining). All IHC stainings were performed on a Benchmark Ultra  automated stainer.Immunohistochemistry (IHC) for estrogen receptor (ER), progesterone receptor (PR) and HER2 was performed prospectively in the central pathology unit of the ADAPT trial as described previously23. In addition, mutational analysis of the CDH1 gene was carried out by next generation sequencing (NGS) using a customized NGS panel, which covered the complete protein-coding sequence including 10 bp of the flanking intron sequence of the CDH1 gene. Mean mapped reads per sample was 282,620 . Data processing and evaluation were performed as described previously23.Extraction of DNA and analysis of genetic alterations were performed (in 2018-2020) as described previouslyP ≤ 0.0500. Statistical analyses were performed with GraphPad Prism software Version 5.00 .For statistical evaluation of the association between genetic alterations and pathologic parameters, we focused on candidate genes with a mutation frequency of ≥2.5%. The two-sided Fisher’s exact test and the Chi square test for trends were used for contingency analyses. The Mann–Whitney test was used to determine statistical significance of different median Ki67 indices in BC subsets. Results were considered as statistically significant if n = 622 HR-positive early BCs from the ADAPT trial for genetic alterations in selected candidate genes involved in endocrine resistance or responsiveness23. All patients were treated with short-term pET (TAM or AI) for three weeks before tumor resection22. Here, we report a subgroup analysis pertaining to the lobular BCs included in this cohort. According to central pathology review, lobular BC accounted for 179/622 (28.8%) cases. ILC was associated with larger pT stage, lower histological grade, lower Oncotype DX recurrence scores, lower baseline Ki67 (before pET), and loss of E-cadherin , and CCND1, FGFR1 and PAK1 23. These genes were selected as candidate genes involved in endocrine tumor response based on the study of Razavi et al., which focused on metachronous BC recurrences after failure of (adjuvant) ET23. For the present subgroup analysis of ILCs we also performed mutational analyses of CDH1. As expected, ILCs were enriched in cases harboring mutations in CDH1   , but showed fewer mutations in TP53  and GATA3      resulting in single amino acid substitutions in the tyrosine kinase domain, such as p.L755S  were exon 20 in-frame insertions or duplications, such as p.Y772_A775dup, which are more common in non-small cell lung cancer (NSCLC) and induce a constitutively active ERBB2 protein conformation (Supplementary Table 33. One ILC (case 110290) harbored two different ERBB2 mutations (p.I767M and p.V777L) (Supplementary Table ERBB2-mutated BCs harbored a concomitant CDH1 mutation (P < 0.0001), confirming the strong association of these two alterations, as reported earlier  Fig. . CDH1 mu8%) Fig. . CDH1 miier Fig. 8. Overal21. Furthermore, post-pET tumor cell proliferation was prospectively determined by standardized central assessment of the Ki67 index in the framework of a controlled clinical trial (ADAPT)21. Moreover, central Ki67 scoring was supported by computer-assisted image analysis23. No such comprehensive information on post-pET proliferation is available for specimens in public BC mutation databases, such as METABRIC or TCGA4. Representative immunohistochemical stainings for Ki67 at baseline and post-pET are shown in Fig. n = 622) revealed that sustained tumor cell proliferation after preoperative endocrine therapy (defined as post-pET Ki67 ≥10%) was associated with TP53 mutation but not with ERBB2 mutation23. In fact, by considering all BCs irrespective of subtypes, ERBB2 mutation was not associated with any prognostic parameter or enhanced proliferation   . This association was also statistically significant in the subset of ILC patients that were treated with AI for pET (Supplementary Table ERBB2 mutations in ILCs were associated with the “high-high” dynamic Ki67 category (baseline and post-pET Ki67 both ≥10%), as defined recently in the POETIC trial (P = 0.0263)   (Supplementary Table ERBB2 mutation was associated with high risk Oncotype DX recurrence scores (P = 0.0087), but only in the subgroup of lobular BCs  Fig. . ConversBCs Fig. .Fig. 4ReERBB2 mutations may arise de novo during endocrine therapy10. In our BC cohort, ERBB2 mutations were concordantly detected in resections specimens (post-pET) and matched core needle biopsies  in 14/18 (77.8%) cases tested . Moreover, ERBB2 mutation was associated with high risk Oncotype DX recurrences scores, but only in the subgroup of lobular BC.In the present study we have extended our previous molecular analyses of BC specimens from the ADAPT (HR-positive/HER2-negative) trial, in which all patients were treated with pET before surgeryERBB2-mutated cases. However, compared with the situation in early HR-positive/HER2-positive BCs, there is a lack of prospective clinical trials designed specifically for early HR-positive and/or ERBB2-mutant ILC. Accordingly, retrospective exploratory subgroup analyses may represent a necessary intermediate step on the way towards new clinical trials for this distinct tumor type.Limitations of the present study include: (i) the retrospective approach of molecular analyses, (ii) the exploratory subgroup analysis for BCs with lobular histology, (iii) exclusion of a small minority of cases with controversial subtype calls  and (iv) the comparatively small absolute number of ERBB2 influences endocrine responsiveness in lobular BC. For the first time, this has now also been demonstrated by means of clinical tumor specimens obtained after pET. Even so, a proportion of ERBB2-mutant ILCs still showed optimal suppression of tumor cell proliferation by pET. Accordingly, it remains an open question whether ERBB2 mutation is a suitable prognostic factor that is independent from other parameters, such as the Onctotype DX recurrence score. Further analyses in larger cohorts treated with adjuvant endocrine therapy alone, or with adjuvant endocrine therapy plus adjuvant chemotherapy would be necessary to clarify this issue. The current retrospective, exploratory subgroup analysis of a proportion of patients enrolled in the ADAPT trial (run-in phase) was not sufficiently powered to address this problem. Second, this subgroup analysis exemplifies that biologically relevant associations may be masked and can remain unidentified, if HR-positive ILCs and non-lobular BCs are lumped together for statistical analyses in BC research. Accordingly, lobular BC deserves greater attention in clinical trials and translational research.In fact, the findings reported here are important for two reasons. First, this study further supports that mutant Supplemental Material