EGFR‐mutated metastatic lung adenocarcinoma patients and their response to tyrosine kinase inhibitors.A relationship between the EGFR signaling pathway expression in skin and the use of targeted cancer therapies has been previously demonstrated. Consistent evidence to support the use of skin biopsies as a surrogate for therapeutic evaluation is needed. The purpose of this study was to establish the relationship between the expression of EGFR signaling pathway markers in skin samples from EGFR‐mutated lung adenocarcinoma. Immunohistochemistry was performed: EGFR, p27, Ki67, STAT3 and MAPK, as well as H&E histopathological analysis, in order to determine their treatment response to tyrosine kinase inhibitors.This was a prospective single blind analysis of 35 skin biopsies from 31 patients with confirmed advanced P = 0.025, 0.015, 0.017, 0.041, 0.039 respectively). EGFR, p27 and number of layers of the stratum corneum were related to a better median progression‐free survival (P = 0.025 and P = 0.030).EGFR, Ki67, STAT3, stratum corneum thickness (number of layers and millimeters) from skin samples had a statistical correlation with an adequate treatment response ; adequate response to treatment (12 patients: (34.3%); and no response to treatment (11 patients: 31.4%). We found that EGFR expression, STAT3, and the thickness of the stratum corneum (both in millimeters and in number of skin layers) had a statistically significant correlation with an adequate response to treatment with EGFR inhibitors in patients with stage IV lung adenocarcinoma. EGFR, p27 and the number of stratum corneum layers were related to better progression‐free survival. The relationship between EGFR inhibition in skin and in the tumor explains the parallel biological effect. Lung cancer represents the number one cause of cancer‐related mortality worldwide, with an estimated two million new cases reported in 2018 alone, representing 11.6% of the total cancer incidence burden.EGFR) mutation is one of the most common genetic disorders in non‐small cell lung adenocarcinoma, which is the most common lung cancer subtype worldwide, resulting in an ideal therapeutic target.Epidermal growth factor receptor , Janus kinase (JAK) and RAS.A decrease in the proliferation of basal keratinocytes has been demonstrated by the reduced expression of markers such as Ki67 and an increase of p27 and other negative growth markers. Additionally, it has been observed that as EGFR inhibition increases, transcription factor, STAT3, levels also increase at the basal layer of the epidermis.The inhibition of the aforementioned signaling pathways in patients treated with EGFR inhibitors produce documented histopathological skin changes, with the most frequent being: basket weave alteration of the stratum corneum (33%), spongiosis (79%–90%), perifollicular inflammation (80%–100%), infiltration by lymphocytes (100%), histiocytes (100%) and neutrophils (90%), sebaceous adenitis (70%), granuloma formation (60%), infiltrated dermis (59%), follicle inflammation (51%), perivascular infiltration (82%) vascular changes (100%) and vascular dilatation (82%).EGFR‐mutated lung adenocarcinoma patients allow us to establish a relationship between EGFR expression or its signaling pathway derivatives, and gefitinib treatment response, potentially acting as a tool to avoid tumor biopsy related risks.The hypothesis is that skin sample studies from Receiver operating characteristic (ROC) curve analysis was applied to set cutoff value parameters for EGFR, P27, Ki67, STAT3, and MAPK expression. Finally, clinicopathological and prognostic significance of biomarker's expression in skin tissue samples were analyzed.EGFR‐mutated lung adenocarcinoma, treated with gefitinib and ECOG ≤2 were included. Exclusion criteria were: patients with cutaneous disease at the time of the study, concomitant treatment with systemic steroids , previous treatment with TKIs, concomitant use with other antineoplastic treatments, patients undergoing anticoagulant treatment, impaired healing, or patients with immunological diseases, pregnancy, lactation, and nonevaluable treatment responses. A signed inform consent form was obtained from each patient included in the study. The immunohistochemical and financial related issues were absorbed by the investigation group from the study.A prospective single blind study was performed at the Thoracic Oncology and Pathological Anatomy departments at the “Instituto Nacional de Enfermedades Respiratorias, Ismael Cosio Villegas” (INER), in Mexico City. The study design was approved by INERs Institutional Ethics Board in accordance with the Declaration of Helsinki, Fortaleza Brazil 2013 . Patients older than 18 years old with a histopathological diagnosis of EGFR‐mutated lung adenocarcinoma, taken by 4 mm punch in the scapular region, which was subsequently formalin‐fixed and paraffin‐embedded. Patient samples were classified into three groups: (i) Pretreatment; (ii) adequate treatment response ; and (iii) no treatment response (stable disease and progression) according to RECIST 1.1 criteria assessed by our group's radiologist with a computed tomography (CT) scan. One patient was included in three groups , and two patients were included in two groups (adequate response to treatment and no response to treatment); these biopsies were taken at different times during the patients’ evaluation according to RECIST 1.1 criteria.An expert dermatologist performed 35 skin biopsies from 31 patients with a confirmed diagnosis of stage IV Histopathological analysis with hematoxylin and eosin staining was performed to confirm stratum corneum configuration, stratum corneum thickness measured in micrometers and number of layers, parakeratosis, spongiosis and infiltrate . The measurement mentioned above was taken from the top of the stratum corneum to the top of the granular cell layer at several sites Fig .Immunohistochemical analysis measured the percentage of expression of EGFR , p27 , STAT3 , MAPK  and Ki67 . The number of cells in the basal layer of the epidermis were also counted  was used for statistical analysis. A ROC curve analysis was used to determine the cutoff value for each biomarker, using the shortest distance from the curve to the point with the maximum sensitivity and specificity  dichotomizing the response to treatment as mentioned in the tissue samples section (adequate response or no response), to compare changes between the two groups Fig .17 CorFrom the 35 biopsies obtained, 21 (60%) of the patients were women and 14 (40%) men; mean age of participants was 60.6 ± 11.7 years. From the three groups previously described in the methods section, 12 (34.3%) were in the pretreatment group, 12 (34.3%) had an adequate response to treatment and 11 (31.4%) were in the no response to treatment group. The mean duration of treatment was 8.6 months with a median progression‐free survival of nine months.The ROC curve analysis defined the next cutoff values to consider a positive change between the pretreatment patients and post‐treatment groups: EGFR 72.5%, p27 45%, MAPK 45%, Ki67 61 keratinocytes, STAT3 0.5 keratinocytes, thickness of the stratum corneum 0.025 μM and 3.5 layers from the stratum corneum, these values represent a change from the basal state of 5.83%, 3.75%, 18.75%, 10 keratinocytes, 1.5 keratinocytes, 0.0083 μM and 0.6 layers, respectively. These cutoff points were pre‐established by using baseline means and performing a preanalytical ROC curve to set the change parameters.P = 0.025), Ki67 (P = 0.015), STAT3 (P = 0.017) stratum corneum thickness (P = 0.039) and number of layers in the stratum corneum (P = 0.041). A tendency to statistical significance was found with the use of MAPK (P = 0.059).The next biomarkers were significantly related to an adequate response to treatment by using a bivariate correlation test: EGFR (P = 0.025) and number of layers in the stratum corneum ; however, for p27 a better median progression‐free survival was shown in those with a value below the cutoff mentioned previously   than those with disease progression or pretreatment. By facing disease progression, the tumor is able to gain treatment resistance and find genetic escape routes which allow tumor proliferation. To date, the amplification of EGFR, has not been identified as one of those escape routes, which is why we have not found differences in cutaneous EGFR expression at the time of disease progression.These findings are related to the systemic inhibition of EGFR, which has effects on the skin by reducing keratinocyte proliferation and migration to upper layers in the epidermis. In addition, this is also probably due to the fact that the EGFR pathway has not been effectively blocked in patients who present with disease progression, and they come close to the basal levels observed in pretreatment patients. Considering that these lung cancer patients receive a tyrosine kinase inhibitor aimed at their specific et al.18 our study shows that epidermal cell proliferation decreases after exposure to tyrosine kinase inhibitors against EGFR. We found more Ki67 positive keratinocytes in pretreatment than post‐treatment patients. Patients with an adequate treatment response had lower Ki67 levels than pretreatment patients. Patients with adequate response also showed a tendency to express lower Ki67 levels than patients without response, and subsequently, patients without response showed lower Ki67 levels than pretreatment patients.Ki67 is a nuclear protein present in cell cycle growth phases  used as a proliferation marker.et al.EGFR mutations.CDKN1B or p27 is a tumor suppressor protein that regulates cell cycle progression from G0 to S phase; induced by tyrosine kinase inhibitors to halt tumor cell proliferation.In contrast to other studies, the expression of Ki67, STAT3 and MAPK in skin did not show a relationship with adequate treatment response with EGFR inhibitors related to progression‐free survival in patients with stage IV lung adenocarcinoma. However, this may be related to the skin biopsy collection times, since our skin biopsies were taken at different times and there may be escape routes for the keratinocytes to adapt to EGFR inhibition.et al.18 reported that changes observed in the epidermis were related to treatment response, since patients with an adequate response to treatment are those who present with major changes at the stratum corneum (configuration and thickness), even when compared with patients without treatment response. The inhibition of EGFR in skin was related to a better median progression‐free survival, which explains the strong relationship between EGFR expression in skin and the tumor response.We also found that the EGFR inhibition of the tumor was related to skin alterations, such as stratum corneum thickness. The systemic inhibition of EGFR has been previously reported to have effects on the skin by decreasing the proliferation, migration and survival of keratinocytes towards the upper layers of the epidermis.Similarly, a multicentre, phase 2 study in which non‐small cell lung cancer (NSCLC) patients were given increasing doses of erlotinib, showed altered differentiation of epithelial structures in biopsies collected during treatment when compared with pretreatment biopsies.The potential limitations in this study are related to the relatively small number of patients who participated in our analysis. Additionally, since our research center is a highly specialized referral institution, generalizing these results to patient populations at other medical institutions need to be analyzed further. Finally, even when the overall genetic effect of EGFR blockade was observed, the gene expression itself was not evaluated.In conclusion, the parallel biological effects of systemic EGFR pathway inhibition is observed in both tumor response and skin biopsies in patients with advanced lung adenocarcinoma. In this study, patients treated with tyrosine kinase inhibitors showed reduced EGFR and Ki67 expression, especially evident in those who presented with an adequate treatment response. Alterations in the configuration of the stratum corneum and decrease in its number of layers were also found to be related with treatment response and the start of treatment with tyrosine kinase inhibitors. No relationship between p27 and response to oncological treatment was found.We found a relationship between EGFR, stratum corneum and number of layers in the stratum corneum, in patients with treatment response. We also found that there was a better progression‐free survival for patients with high expression EGFR, decreased number of layers in the stratum corneum and low p27 expression. The relationship between EGFR pathway inhibition in the skin and the oncological outcomes obtained, explains the parallel biological effects of these agents.This study presents strong evidence that EGFR lowered expression in skin samples from stage IV lung adenocarcinoma patients treated with tyrosine kinase inhibitors could potentially be used as a surrogate to predict treatment response and avoid tumor biopsy‐related risks in this population. We hope that our work incites future research to help validate and assess the use of these markers as potential prognostic and predictive factors.Dr Jeronimo Rafael Rodríguez‐Cid has educational, investigational and advice relations with MSD, Bristol Myers, Roche, Takeda, Amgen, Abvie, Aztra Zeneca, Boehringer Ingelheim, Pfizer, Celgen, Novartis and Bayer; Dr Jorge Arturo Alatorre‐Alexander has educational, investigational and advice relations with MSD, Bristol Myers, Roche, Takeda, Aztra Zeneca, Boehringer Ingelheim and Pfizer. The other authors have no conflicts of interest to declare.


BMV). 68/78 BMV teams and 23/71 AAM teams adhered to the ventilation recommendations (p < 0.0001). BMV teams had lower compression fractions than AAM teams  resulting entirely from higher no-flow times for ventilation . Compared to BMV, AAM leads to significant hyperventilation and lower adherence to ventilation recommendations but favourable compression fractions. The cumulative effect of deviations from ventilation recommendations has the potential to blur findings in clinical trials.The role of advanced airway management (AAM) in cardiopulmonary resuscitation (CPR) is currently debated as observational studies reported better outcomes after bag-mask ventilation (BMV), and the only prospective randomized trial was inconclusive. Adherence to CPR guidelines ventilation recommendations is unknown and difficult to assess in clinical trials. This study compared AAM and BMV with regard to adherence to ventilation recommendations and chest compression fractions in simulated cardiac arrests. A total of 154 teams of 3–4 physicians were randomized to perform CPR with resuscitation equipment restricting airway management to BMV only or equipment allowing for all forms of AAM. BMV teams ventilated 6 ± 6/min and AAM teams 19 ± 8/min (range 3–42/min;  Guidelines of cardiopulmonary resuscitation (CPR) recommend that advanced cardiac life support providers should give artificial ventilation as soon as possible . Means oThere are no data from randomized controlled trials supporting or refuting the routine use of any specific approach to ventilation and the timing thereof during CPR ,5,6,7,8.Adherence to CPR algorithms is not always optimal and may vary between different components of the algorithm ,23,24,25The Working Group on Intensive Care Medicine, Arnsberg, Germany, regularly provides educational courses for physicians from all over Germany and German-speaking countries working in intensive and emergency care. Most participants are residents in their 2nd to 3rd year of postgraduate medical education in internal medicine, anaesthesia or surgery that are regularly confronted as first responders with CPR and have been exposed to medical simulations during their previous medical education to a variable extent. All participants of our educational courses were offered the opportunity to take part in voluntary simulator-based CPR workshops and informed that they would work in teams, simulations would be videotaped for scientific reasons, and video-recordings would be analysed in a strictly anonymous way. Physicians refusing to be filmed were offered identical CPR workshops without videotaping and were not included in the present study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Ärztekammer Westfalen-Lippe (2014-657-f-N) that waived the obligation to obtain consent. The study is registered at the German Clinical Trial Registry (DRKS00022132) and reported herein according to the extensions to the CONSORT statements of the Reporting Guidelines for Health Care Simulation Research .In this prospective randomized single-blind trial participants from single workshops were randomly assigned to teams of three to four. Teams were then randomly allocated to perform CPR under two different conditions: (1) equipment for airway management available (AAM group) or, (2) only bag and mask available (BMV group). Medical equipment provided to all teams consisted of a manual defibrillator and an aluminium case (Ulmer Case) used for transport of emergency and resuscitation material by many German emergency systems. For AAM teams, the usual content of the case remained unchanged and included, among others, a facemask, a self-expandable bag with reservoir, supraglottic airways, endotracheal tubes, and a laryngoscope. For BMV teams, all equipment allowing advanced airway management was removed from the case so that only a facemask and a self-expandable bag with reservoir were available. Prior to the start of the scenario, BMV teams were informed that their case lacked equipment for advanced airway management due to partial restocking in the aftermath of multiple resuscitations. Except for this study-specific difference in the content of the emergency case, both versions of the simulation were identical.® Wireless Mega Code Trainer  was used. All participants received a standardized introduction to the workshop and were made familiar with the manikin. Team members were then informed that their role during the following scenario was that of an in-hospital resuscitation team summoned to an unwitnessed cardiac arrest in their hospital’s cafeteria. The victim of the arrest (simulator) was pulseless, apnoeic, and did not react to verbal commands or painful stimuli. When attached, ventricular fibrillation could be diagnosed on the display of the defibrillator. The study period started with the first touch of the patient by a team member and ended with the return of spontaneous circulation (ROSC) upon the third defibrillation after the third cycle. Trained tutors who were instructed to refrain from any intervention until the end of the study period operated the manikins. After the simulation, tutors gave educational feedback to the teams.The full body manikin Ambu ManTeam performance was analysed using video recordings obtained during simulations by one of the authors (L.V.). Any uncertainties in ratings were solved by jointly watching the videos with a further author (T.S. or S.M.) The first touch of the patient was defined to be the starting point for the timing of all events. Advanced airway management was defined as insertion of an ETI or a SAD. The 2015 American Heart Association (AHA) and European Resuscitation Council (ERC) guidelines for basic and advanced cardiac life support recommend for BMV the delivery of two breaths during pauses in chest compressions with an inspiratory inflation time of 1 s for each breath ,29,30. TThe 2015 AHA and ERC guidelines recommend that after insertion of an advanced airway continuous chest compressions should be performed while ventilation rates should be 10/min  and “appFor each second the presence or absence of chest compressions was noted and coded if absent chest compressions  were related or unrelated to ventilation and/or airway management. Chest compression fraction was defined as the sum of all time intervals with chest compressions expressed as percentage of the total time interval available for chest compressions.p < 0.05 was considered to represent statistical significance.Primary endpoints were chest compression fraction and adherence to guidelines with regard to ventilation recommendations. A difference of ≥5% between the groups for the primary outcome was considered to be of clinical significance. A power analysis, based on data of pilot experiments, revealed that a minimum of 75 teams had to be studied in each group to detect this difference with significance levels of 0.05 and 80% power. For organisational reasons, the number of available videotapes of sufficient quality could be assessed only after completion of each educational course. Secondary outcomes included request, timing, time consumption, and interruption of chest compressions for AAM. All data were analysed on an intention-to-treat basis. Data are medians (interquartile range (IQR)) unless otherwise stated. Statistical analysis was performed using IBM SPSS Statistics for Windows, version 22 . Student’s t-test, Mann–Whitney test, and Fisher’s exact test were applied as appropriate. A All BMV teams performed bag-mask ventilation only. By the end of the 3rd cycle, 71/76 (93%) AAM teams had inserted an advanced airway and therefrom ETI in 19/76 and a SAD in 52/76 teams. 5/76 AAM teams had not inserted an advanced airway by the end of the 3rd cycle  in two). 17/76 AAM teams performed AAM right from the start of CPR  while the remaining 59 AAM teams initially performed bag-valve-ventilation (58) or mouth-to-mouth ventilation (1).p < 0.001 vs. BMV; p < 0.001 vs. ventilation after insertion of an advanced airway; p = 0.6 vs. BMV teams).Over the first three cycles BMV teams ventilated at a median respiratory rate of 5/min (IQR 4–6) (range 3–24), whereas AAM teams ventilated at a respiratory rate of 17/min  for delivering two rescue breaths. 66/70, 67/70, and 69/70 teams interrupted chest compressions for ≤4 s, ≤5 s, and ≤6 s respectively. Thus, from a total of 78 BMV teams 85%, 86%, and 88% adhered to the target of interrupting chest compressions for applying rescue breaths and limiting interruptions to ≤4 s, to this target within a range of 20%, and to this target within a range of 50% respectively.p < 0.0001 vs. adherence of corresponding values in BMV teams).2/71 AAM teams erroneously continued ventilating in a 30:2 mode with corresponding interruptions of chest compressions after insertion of an advanced airway. In 4 of the remaining 69 AAM teams, ventilation rates were between 9 and 11/min, while in 6 and 23 teams ventilation rates were between 8–12/min and between 5–15/min, respectively. Thus, from a total of 71 AAM teams actually performing AAM, 6%, 8%, and 32% adhered to the target of uninterrupted chest compressions and a ventilation rate of approximately 10/min, to this target within a range of 20%, and to this target within a range of 50%, respectively (p < 0.001) chest compression fractions than AAM teams: 79% (IQR 75–82) vs. 88% (IQR 83–90). This was entirely due to the no-flow times related to ventilation and/or airway management (9% (IQR 8–12) vs. 3% (IQR 1–6); p < 0.001) while there was no difference in no-flow times unrelated to ventilation and/or airway management (12% (IQR 9–14) vs. 10% (IQR 8–14); p = 0.16).BMV teams had lower  and completed the insertion after a further 49 s (IQR 35–94) resulting in the advanced airway being in place after 116 s (IQR 74–170) .p = 0.04) between endotracheal intubation (18 s (IQR 13–24)) and supraglottic airway (11 s (IQR 7–23)). Teams interrupted chest compressions for placing an advanced airway for 0 s  with no difference (p = 0.22) between endotracheal tube  and supraglottic airway (0 s (IQR 0–0) range 0 to 8; mean 1 ± 2 s). 56/71 AAM teams did not interrupt chest compression at all for inserting an advanced airway while 64/71 AAM teams adhered to the recommendation of a pause in chest compressions of ≤5 s, if any, for inserting an advanced airway [The time interval required for the placement of an advanced airway was 13 s  with a significant difference  ,7,35 andAAM was associated with an immediate tripling of ventilation rates independently of the airway chosen. Hyperventilation is associated with persistently high airway pressures in humans  and decrBMV teams demonstrated a high adherence to the recommendation of limiting interrupting chest compressions for applying rescue breaths: The median interruption time of 2 s of our physician BMV providers compared favourably to a median time of 7 s of lay rescuers in a previous trial . HoweverIn keeping with a previous simulator-based  and a laStrengths of this randomized trial include the large sample size, participants currently acting as first responders in real life, and identical conditions for all teams. Importantly, we were able to capture and record airway management occurring early in the course of CPR, which is extremely difficult in real cases. Moreover, restricting first responders’ airway equipment for a scientific purpose only would be difficult, if not impossible, to justify ethically and medically in patients. Limitations of simulator-based studies include the absence of real patients. However, when performance markers like chest compression fraction and technical quality of airway management are assessed, findings in simulator-based studies show a high agreement with findings in real cases ,23,24,25The present study adds the following insights to the ongoing debate on optimal airway management: First, advanced airway management is associated at the same time with both potentially beneficial (less no-flow time) and potentially harmful (hyperventilation) features, which may partly explain conflicting data in clinical studies. Second, hyperventilation occurs consistently and starts immediately after insertion of an advanced airway regardless of the type used. Third, the incidence and degree of further deviations from ventilation recommendations in both BMV and AMV has the potential to blur true differences in clinical trials. Fourth, prolonged no-flow times are a very unlikely explanation for worse outcomes, if any, associated with AAM in general or differences in outcomes between different methods of AAM. Fifth, provided the equipment necessary is available, advanced airway management is carried out in the vast majority of cases in the very early phase of resuscitation (20% right from the start and over 90% within the first two minutes).Given the potentially detrimental effects of hyperventilation, training and teaching of CPR should include an additional focus on adherence to ventilation recommendations. Such teaching should be easily implementable in current teaching frameworks. Potential first responders and leaders of CPR teams should be made aware of the frequent occurrence of hyperventilation after insertion of an advanced airway and other deviations from ventilation recommendations and of their duty to avoid, prevent, or correct these events. Interestingly, a favourable effect of a ventilation feedback device on the quality on manual ventilation has been recently reported in a simulator-based trial . This simulator-based randomized trial of CPR demonstrates that AAM is associated with higher chest compression rates than BMV but more deviation from ventilation recommendations, and especially hyperventilation. The cumulative effect of deviations from ventilation recommendations in both BMV and AAM has the potential to blur findings in clinical trials. Training and teaching of CPR should include awareness of hyperventilation associated with AAM and measures to prevent it.


Schizophrenia is a neuropsychiatric disorder characterized by multifactorial etiology involving complex interactions among genetic and environmental factors. “Multiple-hit” models of the disorder can explain its variable incidence and prevalence in related individuals. Hence, there is a dire need to understand these interactions in the emergence of schizophrenia. To test these factors in the emergence of schizophrenia-like behaviors, we employed a genetic mouse model of the disorder (harboring the DISC1 mutation) along with various environmental insults, such as early life stress  and/or pharmacological interventions (ketamine injections). When assessed on a battery of behavioral tests, we found that environmental interventions affect the severity of behavioral phenotypes in terms of increased negative behavior, as shown by reduced mobility in the forced swim and tail suspension tests, and changes to positive and cognitive symptoms, such as increased locomotion and disrupted PPI along with reduced working memory, respectively. Among the various interventions, the genetic mutation had the most profound effect on behavioral aberrations, followed by an environmental intervention by ketamine injections and ketamine-injected animals that were maternally separated during early postnatal days. We conclude that although environmental factors increased the prevalence of aberrant behavioral phenotypes, genetic background is still the predominant influence on phenotypic alterations in these mouse models of schizophrenia. Schizophrenia is a neuropsychiatric disorder whose etiology encompasses the interaction of several genetic and environmental factors. Heritability of the disorder is as high as 80%  gene confers a 2% risk of schizophrenia in carriers  and single nucleotide polymorphisms (SNPs) studies  compared with controls. We found that although environmental variables increased the number of animals exhibiting aberrant behaviors, the genetic composition of the animals was still the major driver in the expression of schizophrenia-related phenotypes.Despite the advances in understanding the genetic and environmental factors involved in the etiology of schizophrenia and a plethora of molecular interactions of DISC1 protein at presynaptic, synaptic and/or postsynaptic sites  of the Louisiana State University School of Veterinary Medicine.A total of 12 animals were used in each group: 129SvEv (129S:ΔDISC1) a DISC1 mutation model and C57BL/6J (control), and the intervention groups described below. Mice were obtained from the Jackson Laboratory . These animals were assessed on a battery of behavioral tests when 8 weeks old, with the least stressful tests performed first . In adulthood, they were injected with ketamine (i.p. 30 mg/kg), as described above. These animals were then tested under the behavioral test battery as follows.All behavioral experiments were performed by the same investigator during the late morning. The behavioral procedures have been described in detail in our previous work  patterns can provide an indicator of the affective state of the animal . Percent entries into the correct arm were calculated using the formula described previously .ANOVA followed by Tukey’s p ≤ 0.05; see DISC1 mutation showed a variable degree of time at the periphery differing from control animals . Different interventions on the control background did not affect this behavior, except in the control+MS+ketamine group . These results support the interpretation that all the environmental interventions affect animal behavior differently depending on the genetic background and their specific interactions.While in the open field, the total distance covered by all groups was assessed. Control animals traveled a significantly greater distance as compared to other groups, i.e., DISC1 animals alone and with interventions . However, environmental interventions on a DISC1 mutation background did not change the sociability behavior of these animals significantly.Control animals exhibited significantly higher time socializing compared with other groups, with the exception of maternally separated (MS) controls (where different groups differed at p ≤ 0.05), maternally separated DISC1 (p ≤ 0.01), MS+DISC1 injected with ketamine at adulthood (p ≤ 0.01), and the DISC1+ ketamine injection animals exhibited an increased social novelty vs. DISC1 animals alone (p ≤ 0.01), suggesting a complex interplay in DISC1 animals with pharmacologically induced NMDAR hypofunction  exhibited a different outcome , with cop ≤ 0.001) and other groups  as compared to DISC1  can be used as a measure of the degree of despair in animal models of behavioral disorders  was measured to determine sensorimotor gating and pre-attentive deficits. As previously described, the first test block measured habituation , DISC1 (p ≤ 0.05) and DISC1+ketamine  and control+MS+ketamine [p ≤ 0.01; at 30 (ISI)_85 (Prepulse intensity); p ≤ 0.05 at 100 ms (ISI)_75 dB (Prepulse Intensity); p ≤ 0.001 at 100 ms (ISI)_85 dB (Prepulse Intensity) combinations; p ≤ 0.01 at 100 ms (ISI)_85 dB  and pre-pulse intensity combinations. We found that control animals showed inhibition to all trial (ISI-PP intensity) combinations see , signifiDISC1 gene) on a behavioral test battery , environmental factors , and interactions of these factors see . Among tIn many of the behavioral tasks, environmental interventions on the DISC1 background did not increase the severity of behavioral phenotypes. We suggest that the severity of symptoms in many of the behavioral task have a lower/upper limit. However, an interesting finding from our data is that the animals harboring the DISC1 mutation often exhibit a bimodal distribution in the expression of behavioral phenotypes, which is not found following environmental interventions. Thus, we propose that the effects of the environmental interventions may not necessarily be on the magnitude of the behavioral effects, but rather the probability that these animals may develop schizophrenia-related behavioral phenotypes.As models to study schizophrenia, these interventions are argued to have faced, construct and/or predictive validity  and negative symptoms  of the Louisiana State University School of Veterinary Medicine.CL designed the study, edited and finalized the manuscript. RS designed, executed the experiments, collected data, statistically analyzed and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Meanwhile, the proposed thermal emitter possesses near unity emissivity at the wavelength of 6.3 μm to increase radiation heat dissipation, maintaining the thermal stability of the system. The underlying mechanism relies on fundamental magnetic resonance and the interaction between the high-order magnetic resonance and anti-reflection resonance. When the environmental background is blackbody, the tunable emitter maintains signal reduction rates greater than 80% in middle-IR and longer-IR regions from 450 K to 800 K and from room temperature to 800 K, respectively. The dependences of thermal camouflage on crystallization fraction of GST, incident angles and polarization angles have been investigated in detail. In addition, the thermal emitter can continuously realize thermal camouflage for various background temperatures and environmental background in atmospheric window in the range of 3–5 μm.Thermal radiation control has attracted increasing attention in a wide range of field, including infrared detection, radiative cooling, thermal management, and thermal camouflage. Previously reported thermal emitters for thermal camouflage presented disadvantages of lacking either tunability or thermal stability. In this paper, we propose a tunable thermal emitter consisting of metal-insulator-metal (MIM) plasmonic metamaterial based on phase-change material Ge GST is the past ,33,34.Thermal camouflage is important in military tactics, which is usually utilized by warships and submarines to make them invisible for IR detectors and thermal cameras. With the fast development of IR detection technology, dynamical control of thermal radiation is a critical issue in thermal camouflage of military objects and critical targets as external circumstances changes. To date, several thermal camouflage devices have been practically presented and experimentally demonstrated in aspects of thermal camouflage performance. By electrically tuning charge density of graphene, the emissivity of a thermal emitter consisting of a multilayer-graphene electrode on a porous polyethylene (PE) membrane and a back gold-electrode can be dynamically tailored in a broadband wavelength range from 5 μm to 25 μm, achieving adaptive thermal camouflage . Using aIn this paper, a tunable thermal emitter, consisting of MIM metamaterial based on phase-change material GST, is theoretically investigated to realize tunable control of thermal radiation and thermal camouflage in both atmospheric windows. Meanwhile, the tunable thermal emitter has near unity emissivity at wavelength of 6.3 μm to improve radiation heat dissipation, maintaining the thermal stability of the system. When the environmental background is blackbody, the tunable emitter maintains signal reduction rates greater than 80% in middle-IR and long-IR regions from 450 K to 800 K and from room temperature to 800 K, respectively. Subsequently, the dependence of thermal camouflage on crystallization fraction of GST, incident and polarization angles have been also investigated. In addition, the thermal emitter can continuously realize thermal camouflage for varies background temperatures and environmental background in atmospheric window ranges from 3 μm to 5 μm. Our proposed thermal emitter will pave the way towards the tunable thermal radiation control, thermal management and thermal camouflage in both civilian and military applications.t3 = 100 nm, t2 = 240 nm and t1 = 100 nm, respectively. The array periodicity is p = 3 μm, and the diameter of the nanodisks is d = 1.5 μm. The deposited GST is in an amorphous phase . An annealing process above phase-change temperature on a hot plate is applied to obtain GST in crystalline phase (termed as cGST). It should be noted that the interlayer diffusion between GST and gold disks exists in the annealing process [Thermal camouflage is achieving low observability of vital targets by decreasing the IR radiation intensity of object itself. As shown in  process . Herein, process  and Ref.eff (λ) of different intermediate GST phases can be estimated through effective-medium theories [c (λ) and εa (λ) are the permittivities of GST in the crystalline and amorphous phase, respectively.The GST layer in the intermediate stage is composed of amorphous and crystalline molecules in different proportions. In terms of numerical simulation, the effective permittivity εtheories  based ontheories ,43,44: is directly proportional to the refractive index and thickness of dielectric layer. Consequently, the wavelength of anti-reflection resonance has a red-shift with increasing the refractive index and thickness of GST layer with fixing other geometrical parameters. In conclusion, all these facts are necessary for designing thermal emitter achieving tunable thermal camouflage and radiation heat dissipation. In addition, the spectral emissivity of the MIM thermal emitter based on GST in intermediate phase, has also been investigated theoretically by varying the crystallization fractions of GST from 0% to 100%, showing a tunable and continuous manipulations of the thermal radiation. Therefore, the proposed MIM thermal emitter possessing continuously tunable emissivity shows the ability of adaptive thermal camouflage when the external environment changes. In the following, we will demonstrate that the MIM thermal emitter based on cGST can realize the performance of thermal camouflage in both 3–5 μm and 8–14 μm and maintain thermal stability owing to high emissivity in 5–8 μm.To unveil the underlying physical mechanism of different emission peaks, the enhancements of magnetic field component |Hy| in x-z plane have been investigated and presented in The magnetic field at a wavelength of 3.8 μm is confined to the vicinity of interface between the bottom gold layer and the GST layer, which can be generally regarded as anti-reflection resonance  βT=1−lated by :(3)Ebλ,T follows :(5)δT=∫λIn the following, thermal camouflage performance of the MIM thermal emitter based on cGST will be investigated, with the blackbody background temperature varying from 300 K to 800 K. Considering the radiant intensity from the normal direction, However, low emissivity in the wavelength ranges of 3–5 μm and 8–14 μm will lead to a decrease in radiation heat dissipation and influence its own temperature. To compensate the decrease of radiation heat dissipation, high emissivity with FWHM of 2.7 μm has been achieved in the 5–8 μm ranges a, and thThe dependence of the thermal camouflage performance on the different crystallization fraction is further studied at temperature 500 K. Multiple spectral radiation peaks of MIM thermal emitter gradually shift toward middle wavelength IR ranges and finally form single spectral radiation peak in wavelength ranges 5–8 μm when the crystallization fraction of GST from 0% to 100%, as shown in The dependence of the thermal camouflage performance on the incident angles is also studied. When the incident angle increases from 0° to 25°, the spectral emissivity changes little in the wavelength range of 5–8 μm, but has a bit of enhancement in the wavelength range of 3–5 μm and 8–14 μm, as shown in As mentioned above, our proposed MIM thermal emitter based on GST is investigated in the blackbody background with temperature equaling to emitter temperature to evaluate the performance of the thermal camouflage device. However, most nonmetallic or metallic environments, such as vegetation (emissivity ε = 0.97), desert (emissivity ε = 0.9), iron (emissivity ε = 0.21), and aluminum (emissivity ε = 0.09) have different emissivities from a blackbody. Furthermore, the temperature of the ambient background is constantly changing, while the temperature of the object remains constant under most circumstances. Herein, our proposed MIM thermal emitter based-on the phase-change material of GST is theoretically demonstrated to continuously hide objects at varying background temperatures and different ambient backgrounds.P (maxP − minP) is the difference in the radiant intensity between the aGST-Emitter and cGST-Emitter. When the radiant intensity of the background is in the range from minP to maxP, continuous thermal camouflage can be achieved by controlling the GST crystallization fraction, to meet the requirement of radiant intensity of the MIM thermal emitter equal to radiant intensity of the background. When the radiant intensity of the background is maxP, the upper limit of the background temperature is maxT. Similarly, the lower limit of the background temperature corresponding to minP is minT. Therefore, the temperature range of background camouflage ΔT is the difference between maxT and minT. In addition, the object can be camouflaged over a wide range of background temperatures by only increasing the object temperature.The radiant intensities of different background, aGST-Emitter, and cGST-Emitter are shown in As shown in Simulated IR images demonstrate that our proposed tunable thermal emitters can continuously achieve thermal camouflage at different background temperatures and ambient background. When the vegetation background temperature is 20 °C, the radiation temperature of both cGST-Emitter and vegetation background is approximately 19 °C, and therefore the cGST-Emitter is difficult to distinguish from background in the IR image, as shown in In summary, a tunable thermal emitter, consisting of MIM plasmonic metamaterial based on GST, is proposed to realize tunable manipulations of thermal radiation and thermal camouflage, owing to low emissivity in both atmospheric windows. Meanwhile, the tunable thermal emitter possesses near unity emissivity in 6.3 μm to compensate for decreasing radiation heat dissipation, which can maintain the thermal stability of the system. The underlying mechanism relies on fundamental magnetic resonance and coupling between the high-order magnetic resonance and anti-reflection resonance. When the environmental background is blackbody, the tunable emitter maintains middle-IR signal reduction rates greater than 80% from 450 K to 800 K, and longer-IR signal reduction rates greater than 80% from room temperature to 800 K. In addition, our proposed thermal camouflage GST device theoretically demonstrates the ability to continuously modulating camouflaging objects at varying background temperatures and differently environmental background. This ultrathin plasmonic metamaterial thermal emitter based on phase-change material of GST provides a scheme of worthy reference for thermal emission control, radiative cooling, infrared detection, thermophotovoltaics and tunable thermal camouflage.


Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced. With the growing prevalence of obesity and its associated hepatic disorder, non-alcoholic fatty liver disease (NAFLD), obesity has become the main cause of chronic liver injury in developed countries . HepaticThe underlying molecular mechanisms that induce the progression from steatosis to steatohepatitis have not been fully elucidated. According to the “multiple-hit hypothesis”, hepatic lipid accumulation represents one of multiple concurrent insults, such as insulin resistance, mitochondrial dysfunction, and oxidative and endoplasmic reticulum (ER) stress, eventually leading to cellular damage and apoptosis ,6.Reactive oxygen species (ROS) are byproducts of mitochondrial oxidative processes, such as free fatty acid (FFA) β-oxidation. In a physiological state, ROS are detoxified by antioxidants, including glutathione (GSH) . In steaExperimental studies have shown that ER stress is another component involved in the development of steatohepatitis and liver fibrosis ,15. It hThis study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in primary human hepatocytes (PHHs) upon exposure to palmitate and oleate. The levels of oxidative stress were measured directly and indirectly, and the protein expression profiles of the three major UPR signals  were analyzed. Our results suggest that, although both oxidative and ER stress are induced in steatotic hepatocytes, a leading role in the progression to inflammation and apoptosis can be ascribed to the translation-inhibiting properties of the UPR.We isolated PHHs from six human liver tissue samples A.The seeded PHHs were treated with 0.6 mM FFA every 24 h for five days to induce steatosis in vitro. Microscopic observation of PHH cultures from donors 1–6 continuously treated with FFA showed a time-dependent accumulation of neutral lipids C. DuringQuantification of the lipid accumulation in the PHH cultures of cells from donors 1–6 showed different dynamics and capacities for lipid storage in the LLCH and HLCH B. PHHs iContinuous lipid accumulation in vivo leads to fatty liver, whereas excessive lipid storage is linked to pathological tissue transformation and hepatocyte loss. Therefore, we sought to determine whether prolonged treatment of PHHs with FFA increases cell death rates. Pathological effects were evaluated by the measurement of cell activity using XTT -2H-tetrazolium-5-carboxanilid) assays, and membrane integrity was evaluated by measurements of LDH (lactate dehydrogenase) and AST (aspartate transaminase) release .In general, cell activity levels were lower in the HCLH than in the LLCH group A. In theThe initially high LDH and AST values showing substantial deviation among donors reflected the stress of PHH isolation. LDH B and ASTAltogether, we did not observed signs of lipotoxicity as a result of the continuous in vitro lipid accumulation for five days.High hepatic lipid content is associated with increased oxidative damage linked to increased ROS formation generated by FFA-stimulated mitochondrial ß-oxidation. Therefore, we investigated whether continuous lipid accumulation leads to an increase in ROS formation. ROS were measured directly using DCF (dichlorodihydrofluorescein) assays and indirectly by investigation of the level of lipid peroxidation and availability of intracellular antioxidants. Lipid peroxidation leads to the formation of electrophilic 4-hydroxynonenal (4-HNE), which can be quantified as 4-HNE-protein adducts by ELISA. The influence on the intracellular antioxidant status was quantified by GSH availability and formation of oxidized GSH, that is, the GSSG level. The LLCH and HLCH showed significant time-dependent decreases in ROS with tendentially lower ROS values after FFA treatment than were shown in the control group A. These GSH is a strong endogenous antioxidative compound that detoxifies ROS by oxidizing GSH to generate GSSG. GSH and GSSG were quantified using Ellman’s assay . The iniAltogether, we were not able to detect elevated ROS levels in steatotic hepatocytes as described in many in vivo studies. However, the initial GSSG concentration was higher in the HLCH than it was in the LLCH, which suggests increased oxidative stress in steatotic PHHs in vivo. The additional in vitro lipid accumulation was not accompanied by elevated ROS levels. The results suggest that a certain lipid load protects against ROS damage.To test the hypothesis that steatotic hepatocytes are more robust to oxidative damage, we induced oxidative stress in cultures of steatotic hepatocytes using menadione. Menadione induces the generation of ROS via redox cycling, leading to GSH depletion and cell death . PHHs weControl and FFA-treated PHHs showed comparable ROS levels when incubated with the vehicle control and 1 and 5 µM menadione, while the 10 µM menadione treatment led to a significant decrease in the ROS levels in both groups A. MeasurTaken together, the data show that the incubation of PHHs cultured for 72 h without and with the addition of FFA and with 10 µM menadione led to oxidative stress in the PHHs. The oxidative damage was more pronounced in the control PHHs, while the steatotic PHHs showed less sensitivity towards ROS.ER stress results in the activation of the UPR mediated by the signaling pathways of IRE1a, PERK, and ATF6a. These pathways and their downstream targets inhibit protein translation (PERK) and induce autophagy and protein turnover (IRE1a) and lipid metabolism (ATF6a) . When thIn general, we observed a slight time-dependent increase in ATF6a and PERK expression in the control LLCH and HLCH at early time points. These values decreased over the cultivation time, suggesting that ER stress was related to the initial lipid load and decreased as a result of time-dependent lipid degradation in the control PHHs. In particular, PERK expression was strongly correlated with the lipid levels in the PHHs. In contrast, the time-dependent course of IRE1a expression was stable in the control LLCH and decreased in a time-dependent manner in the HLCH .In the LLCH, the lipid accumulation through 48 h did not lead to changes in UPR signaling proteins. Additional lipid storage led to a significant time- and lipid level-dependent increase in IRE1a, ATF6a, and PERK expression. The data indicate that prolonged lipid accumulation in the LLCH led to activation of autophagy (IRE1a) and lipid metabolism (ATF6a), but progression to apoptosis (PERK) was not clearly detected .In contrast, we observed an immediate increase in IRE1a and ATF6a in the FFA-incubated HLCH after 24 h, which remained at this level through 72 h independent of further lipid accumulation. In this time span, the lipid accumulation was tolerated, as PERK expression was not increased in the FFA-treated HLCH compared to the level in the control HLCH. Further 24 h lipid storage (96 h) led to a significant peak in IRE1a and ATF6a expression accompanied by a significant increase in PERK expression. Furthermore, at 96 h, all these protein levels were significantly elevated in the FFA-treated PHHs compared to those in the control group. The IRE1a and ATF6a levels decreased after 120 h of lipid accumulation, while the PERK levels remained constant .Taken together, the data suggest that lipid accumulation for 72 h in the HLCH led to ongoing activation of autophagy, protein metabolism (IRE1a), and lipid metabolism (ATF6a). Further lipid accumulation exceeded the threshold favoring the inhibition of translation and induction of apoptosis (PERK) over the induction of repair mechanisms , leading to a persistent high PERK level and reduction of IRF1a and ATF6a levels.As PERK activation leads to the inhibition of protein translation, we used SRB staining to investigate the total expression of membrane proteins dependent on the lipid load. In general, we found an approximately 2-fold higher level in the initial amount of surface protein on the LLCH compared to the amount on the HLCH . In LLCHTaken together, the data suggest that the first 48 h of primary cell culture was dominated by regenerative processes that restored surface protein expression levels after proteolytic damage incurred during the cell isolation process. Then, this protein amount remained constant (LLCH control), while a higher lipid load (LLCH FFA) resulted in a decrease in the surface protein level or the inhibition of the initial regeneration of these proteins (HLCH). The decrease in the lipid level by a specific amount in the HLCH control cells enabled the restoration of their surface proteins. Therefore, the lipid load negatively influences the ability of the hepatocytes to undergo regenerative processes.Advanced steatosis in vivo is known to lead to severe lipid accumulation that is linked to oxidative and ER stress. The activation of both stress pathways leads to increased cellular regenerative capacities that reduce the cause of cell stress. Therefore, oxidative stress leads to the induction of antioxidative activity, resulting in an enhanced availability of the endogenous antioxidative peptide GSH. However, ER stress also activates the unfolded protein response (UPR), leading to higher protein and lipid turnover to eliminate and recycle misfolded proteins and lipids. If both pathways fail to reduce the stress level, then apoptotic pathways are activated. The persistent stress of cells results in cell death, as observed in the progression from steatosis to steatohepatitis.Therefore, this study aimed to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis in humans.Persistent high blood concentrations of free fatty acids (FFAs) increase lipid accumulation in the form of triacylglycerides (TAGs) in hepatocytes . The invThe different dynamics of in vitro lipid accumulation are associated with the physiology of the liver before hepatocyte isolation. Since the HLCH reflects the NAFLD condition, adapted expression of lipid transporters in hepatocyte membranes may be the basis of the higher lipid uptake rate. CD36 was shown to be abnormally increased under the NAFLD condition , while uHepatic TAG accumulation is considered a nontoxic mechanism of lipid storage in the liver . Our expOxidative stress is a general feature of NAFLD in vivo . IndepenThe lower amount of 4-HNE in the FFA-treated compared to the control PPHs indicated reduced lipid peroxidation and suggests an increased cellular protection towards oxidative stress in dependence of the lipid accumulation as well. This effect may be explained by the regulatory function of the nuclear factor erythroid 2-related factor 2 (NRF2) signaling, which is known to limit the progression from NAFLD to NASH by activating genes that promote the elimination of ROS and electrophiles derived from lipid peroxidation and mitochondrial dysfunction . The addTo test the sensitivity of the PHHs towards oxidative stress, we treated steatotic hepatocytes with the redox cycling compound menadione. In fact, 10 µM of menadione showed a clear hepatotoxic effect by decreasing membrane integrity and cellular activity. However, menadione incubation did not alter ROS levels significantly. Additionally, PHHs showed a menadione concentration-dependent increase in 4-HNE-protein adducts, which was more pronounced in the control group. Both suggests that additional ROS from redox cycling can remain in balance and can be detoxified and confirms an increased protective capacity towards oxidative stress by steatotic hepatocytes. This finding was confirmed by the GSH concentration, which was increased at 5 µM and decreased at 10 µM menadione treatment in control PHH, indicating an adaptive antioxidant effect at lower oxidative stress levels. These data confirmed the results from Sentellas and coworkers, who showed that a 24 h treatment with 10 µM menadione efficiently reduced intracellular GSH and increased GSSG in rat and human hepatocytes . HoweverIt is known that progression from NAFLD to NASH is accompanied by increasing ER stress in hepatocytes . The ER The induction of lipid stress within the ER is accompanied by an increase in PERK activation. In our experimental setting, the protein amount of PERK showed a general increase with culture time independent of the FFA treatment, probably due to an increasing stress reaction from the artificial 2D culture. However, the change in PERK levels during this culture time was more pronounced in the HLCH cultures suggesting a relation to absolute lipid load. In contrast to steatotic hepatocytes, PERK levels decreased in the control cells in correlation with the decrease in the lipid load. Consequently, PERK peaked in FFA-treated hepatocytes after 72 h in the LLCH and after 96 h in the HLCH, similar to ATF6 activation. Persistent PERK activity leads to the inhibition of protein translation and eventually to apoptosis. However, our data did not show a clear decrease in cell viability or signs of lipotoxicity. Additionally, PERK-activation may also lead to NRF2 nuclear translocation and increased transcription of antioxidant response elements . Our datThe activation of the PERK pathway is linked to protein translation inhibition. Therefore, we investigated the impact of lipid accumulation on protein synthesis. It is striking that the HLCH generally showed lower surface protein concentrations than did the LLCH. We previously showed that most surface proteins on PHHs are damaged due to the cell isolation process . The surIn surgery, remaining hepatocytes are likely to proliferate at high levels depending on the protein biosynthesis realized after liver resection. In this process, liver cells carry out increased protein biosynthesis, which in turn causes an increase in protein folding at the ER. This process leads to a rapid increase in ER stress-associated genes such as PERK, pIF2a, and CHOP within the first hours after liver resection . In thisOur study is limited by the numbers of donors we investigated. Additionally, the usage of PHHs is accompanied by high individual variances depending on age, sex, disease, lifestyle, and medication. Our data analysis revealed that the initial lipid content has a high impact on the results and that the samples from the six donors had to be separated into PHH groups based on high and low lipid content. However, the analysis of the effects on various interacting pathways confirmed our results with different read-outs, leading to a consistent analysis and revealing a plausible picture. Further investigations should address the activation of single ER stress pathways in dependence of prolonged lipid accumulation, because the phosphorylation patterns of ER stress proteins are complex as shown for IRE1a . HoweverIn summary, our data revealed that hepatocytes are capable of storing enormous amounts of TAG. Lipid accumulation alone did not seem to induce lipotoxicity over the investigated time span. However, FFA-treated PHHs showed signs of different kinds of cell stresses. Although prolonged lipid accumulation did not disrupt redox homeostasis or antioxidative capacity, we observed a lipid load-dependent increase in ER stress. The FFA-dependent increase in ER stress showed thresholds for the activation of pathways associated with lipid stress (ATF6) and inhibition of protein translation and apoptosis (PERK). We concluded that when a certain threshold of lipid-induced ER stress is passed, cellular functions, including regeneration, are impaired. Additionally, persistent PERK activation can induce the activation of apoptosis. In our study, the lipid load was still in a tolerated range, and PERK activation did not lead to cell death.Our study clearly showed that steatosis per se is a physiological condition and that even severe lipid accumulation can be balanced by cellular defense and regenerative mechanisms. However, fatty liver is also a stressful condition limiting the ability of tissues to deal with additional stress and tissue damage. Tissue damage and regeneration, as it occurs in pathologies, hepatotoxicity, and surgical interventions, intensify the cellular stress that progresses to additional cell loss. Additionally, the regenerative capacities of steatotic hepatocytes are reduced, leading to an increased risk for liver failure. One of the biggest risk factors for liver failure after surgical liver resection is the presence of NAFLD, the incidence of which has dramatically increased in recent years in the Western population. Therefore, this study can help to understand the link between advanced steatosis and impaired cellular regeneration.Phosphate-buffered saline solution supplemented with calcium and magnesium (PBS) was purchased from Gibco and Trypan Blue was provided by Biochrom . All other chemicals were purchased from Sigma-Aldrich , if not stated otherwise. Rat tail collagen was prepared in our laboratory according to the protocol established by Rajan et al. .Liver tissue samples were obtained from macroscopically healthy tissue retained after resected human liver in patients with primary or secondary liver tumors or benign local liver diseases . All sub®, Merck, Darmstadt, Germany).PHHs were isolated from liver tissue samples by a two-step EDTA/collagenase perfusion technique as described previously ,48. The In vitro induction of steatosis was achieved on the basis of the model of Gomez-Lechón et al. . After oOil Red O is a reddishly colored azoic dye used to detect intracellular neutral lipids. Cells were washed once with PBS and fixed with 4% buffered formaldehyde  for 5 min. For staining, cells were covered in Oil Red O working solution (0.2% of 8.6 M Oil Red O in isopropanol) for 15 min. Afterwards, unbound dye was removed by intensive washing with tap water, followed by a drying phase of at least 20 min. Oil Red O dye was completely dissolved in 100% 2-propanol  by gentle rocking. The absorbance was measured at 492 nm using a microplate reader . To calculate relative lipid content the Oil Red O measurement was normalized by the SRB values of the respective sample.The protein dye SRB binds basic amino acid residues. After the Oil Red O assay, cells were washed twice with PBS and covered with SRB solution for 30 min at room temperature protected from light. Unbound SRB was removed from cells by washing the cultures four times with 1% acetic acid solution. For quantification, SRB was resolved in 10 mM TRIS solution for 10 min. The absorbance was measured at 565 nm using a microplate reader.In order to evaluate the cellular and mitochondrial activity as well as changes in energy metabolism, the cell activity was determined using the XTT assay . The assay is based on the reduction of the yellow tetrazolium salt XTT -2H-tetrazolium-5-carboxanilide) to a highly colored formazan dye by dehydrogenase enzymes in metabolically active cells. The assay was performed according to the manufacturer’s protocol. After an incubation with XTT reagent of 24 h or 1.5 h for induction of in vitro steatosis or menadione incubation, respectively, the absorbance was measured at a wavelength of 492 nm using a microplate reader.® reaction kits . The assay was performed according to the manufacturers’ instructions. The absorbance was measured at a wavelength of 340 nm at 37 °C using a microplate reader.To evaluate cellular damage of PHHs, the cell membrane integrity was quantified by measuring the enzymatic activity of LDH and AST in the supernatants using the Fluitest2 in a humidified incubator. Subsequently, the supernatants were aspirated, and the cells were incubated with RPMI medium for 1 h. Fluorescence was measured in a microplate reader at an excitation wavelength of 492 nm and an emission wavelength of 520 nm.The formation of intracellular ROS was measured by using the fluorogenic substance dichlorodihydrofluorescein diacetate (DCF-DA) according to Kegel et al. . In brieGSH, a ubiquitous tripeptide, and its oxidized form, glutathione disulfide (GSSG) form a redox couple, preventing cellular structures from oxidative damage by neutralizing ROS and are commonly used markers of oxidative stress .2PO4 0.1 M in distilled water and 84% K2HPO4 0.1 M in distilled water, adjusted to pH 7.5 or pH 6.1 for GSH or GSSG measurement, respectively, and then adding 8.7 mM EDTA. For quantification, samples were replenished with 220 µL cold extraction buffer (23.6 mM of [0.1% Triton X-100 and 0.6% 5-SSA] in 0.1 M KPE) and lysed by sonification and freeze-thaw cycles.Experimental setup followed the protocol of Rahman et al. . BrieflyAfter centrifugation at 3000 g, 4 °C for 4 min, 100 µL of the supernatant was immediately frozen for later GSH measurements. For GSSG measurement, the pH of another 100 µL of the supernatant was adjusted to 6.1–6.4 and 2 µL of 10% 2-vinylpyridin  in KPE solution was added. After 1 h incubation on ice, cold 16.6% triethanolamine solution was added until a neutral pH was reached. A calibration curve was prepared with different GSH and GSSG concentrations (0.78–50.00 µM). Then, 20 µL of each sample were transferred to a 96-well plate and 100 µL of a solution of 1.33 U/mL glutathione reductase  and 0.84 mM DTNB ) in KPE was added. After 30 s reaction time for the conversion of GSSG to GSH, 50 µL NADPH solution were added, followed by the immediate measurement at 405 nm. The previously frozen GSH samples were thawed. Then, 20 µL of each sample were transferred to a 96-well plate, 0.84 mM DTNB ) in KPE was added, and immediately measured at 405 nm.4-HNE is an α,β-unsaturated hydroxyalkenal that is a product of lipid peroxidation. 4-HNE forms covalent protein adducts, which were quantified by ELISA using the Lipid Peroxidation (4-HNE) Assay Kit  according to the manufacturers’ instructions.Menadione, a quinone and potent redox cycler, is a commonly used agent for the study of oxidant-induced liver injury ,53. Mena3VO4, 1 M glycerol phosphate, 4.35% proteinase inhibitor dissolved in  in PBS). Protein content was quantified with the bicinchoninic acid  assay according to manufacturer’s protocol. For calibration, a standard series with concentrations from 1000 µg/mL to 62.5 µg/mL was established from a serially diluted solution of 20 mg bovine serum albumin (BSA) in 10 mL distilled water. Samples were adjusted to a protein quantity of 1 µg/µL with water and 5-fold sample buffer and incubated at 96 °C for 5 min prior analysis. For each sample, 20 µg total protein were separated using SDS-PAGE containing 10% acrylamide . Protein extracts of MCF-7 cells served as positive control. The electrophoresis was performed at a continuous voltage of 80 V.For protein analysis, cells were harvested with a cell scraper after 10 min incubation with lysing buffer  at a continuous voltage of 30 V at 4 °C for 15–20 h. Primary and secondary antibody solutions were prepared as listed in The proteins were transferred to a polyvinylidene difluoride  membrane with a tank blotting system  was considered significant, and further significance levels were not differentiated in the figures. All statistical comparisons and significance levels are listed in Statistical analyses and chart design were performed using GraphPad Prism 7 . Data are plotted as the mean values of biological replicates ± standard deviation. Each data point was measured at least in triplicate. For statistical analysis of single values, an unpaired t-test was performed. To test differences among multiple groups, a two-way ANOVA  was employed, followed by a Dunnett  or Bonferroni (comparison of different treatments) post hoc analysis. A


We trained and validated risk prediction models for the three major types of skin cancer— basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma—on a cross-sectional and longitudinal dataset of 210,000 consented research participants who responded to an online survey covering personal and family history of skin cancer, skin susceptibility, and UV exposure. We developed a primary disease risk score (DRS) that combined all 32 identified genetic and non-genetic risk factors. Top percentile DRS was associated with an up to 13-fold increase (odds ratio per standard deviation increase >2.5) in the risk of developing skin cancer relative to the middle DRS percentile. To derive lifetime risk trajectories for the three skin cancers, we developed a second and age independent disease score, called DRSA. Using incident cases, we demonstrated that DRSA could be used in early detection programs for identifying high risk asymptotic individuals, and predicting when they are likely to develop skin cancer. High DRSA scores were not only associated with earlier disease diagnosis (by up to 14 years), but also with more severe and recurrent forms of skin cancer. Predicting who will develop skin cancer is difficult. Here, the authors from 23andMe developed a polygenic risk score for skin cancer based on a questionnaire and genetic data from more than 210,000 individuals and suggest that the score could be used in early screening programmes. It has been estimated that 50–60% of cancers could be prevented or successfully treated by efficient cancer prevention and early detection programs3. Skin cancer is the most commonly diagnosed cancer, and it is also among the more preventable forms of cancer. In the United States, one in five Americans are likely to develop skin cancer during their lifetime4. Even melanoma, the more aggressive form among the three main skin cancers, which include basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), has a 5-year survival rate of 98%, if diagnosed at an early stage5. Despite being preventable, more than 15,000 Americans die every year from skin cancer6.Cancer screening and early diagnosis are recognized as key public health strategies for reducing cancer burden. Each year, more than 14 million people worldwide are diagnosed with cancer, and 8.8 million will die from cancer, representing one in six deaths globally7. For early skin cancer detection, medical organizations have recommended full body skin examination, a quick, inexpensive, and noninvasive method. Unfortunately, there is no convincing evidence that visual skin examination is an effective method to detect skin cancer in the general population8. Furthermore, epidemiological studies have identified many additional risk factors, including skin pigmentation, skin susceptibility to sun exposure, family history of skin cancer, and genetics9. There is thus a considerable interest in the development of new cost-effective screening programs that integrate all risk factors, environmental and genetics, and accurately identify asymptomatic individuals with high risk of developing skin cancer10. The ultimate goals of screening would be to identify individuals at high risk, construct personalized skin cancer monitoring plans for these individuals, detail how early and frequently skin cancer detection programs should be implemented, and to anticipate the type and intensity of potential treatments.Public health efforts for prevention have focused on education regarding the danger of sun exposure and increasing adoption of sun-protective habits11. Unfortunately, they have generally suffered from insufficient validation, an incomplete catalog of known risk factors, including genetics, and relied heavily on the current age of individuals, which limits their application for early detection. Building medically relevant individual risk scores is challenging, but the recent emergence of polygenic risk scores (PRS) has provided a potential blueprint for success13. The growth of interest in PRS has been fueled by several major innovations: the development of prediction models in large training datasets, the recognition that while risk scores can be weak predictors overall, their long tails permit the identification of individuals with high disease risk, and finally the ability to validate and characterize risk score distributions in large, independent, and preferably longitudinal cohorts. The open questions are whether non-genetic risk scores would also exhibit long tail behavior, and if they can be combined to improve individual risk predictions and their clinical utility14.Many skin cancer predictive models have been proposed over the years11. Over the course of approximately thirteen months from May 2016 to June 2017, more than 210,000 research participants responded to questions from an in-house designed cancer survey. , as well as risk factors identified after reviewing the recent skin cancer literatureThe demographic characteristics of the training and validation sets are described in Supplementary Table 15 from factors that are generally not included .We identified 32 risk factors that contributed to at least one type of skin cancer, following the procedure described in Fig. 17. As such, including associations from other publications could bias the assessment of our risk models in the validation set. An exception to this issue is provided by a recent large-scale PRS study of melanoma, which we were able to use to compare to our melanoma PRS18. The results are summarized in Supplementary Fig. P-value < 1.0e−3) in the training set .Genetic variants included in PRS were directly selected in the training set using a simple clumping and thresholding method (Supplementary Table r2 = 10%) but less so of melanoma (r2 = 3.3%). The presence of dysplastic moles increased the relative risk of melanoma by 6.7×, and explained 11.5% of the melanoma variance, but was less predictive for BCC and SCC, explaining 2.9% and 2.1% of the variance, respectively. In addition to dysplastic moles, the number and size of moles were generally more predictive of melanoma than BCC and SCC. On the other hand, a diagnosis with actinic keratosis before age 40 increased BCC and SCC risks by >3×. A family history risk score, computed as a simple sum of binary indicators of reported skin cancer in father, mother, siblings, and children, explained about 1% of the BCC and SCC variance, but only 0.3% of the melanoma variance. Pigmentation factors individually explained less than 1% of skin cancer variance, with consistent estimates between the three skin cancers. Among the exposure risk factors, sunbathing frequency before age 30 explained between 0.3% and 1% of the skin cancer variance. The typical sun exposure per week, which was measured by the current reported number of minutes per week spent in the sun, also increased the risk of skin cancer, and explained about 0.1% of variance. Tanning bed lifetime usage, which was reported by 41.6% of the participants, was associated with a moderate increase of skin cancer risk (maximum 1.3× in SCC for participants that reported using tanning beds more than 30 times). Finally, sex showed a moderate and consistent effect across the three skin cancers, with men having ~1.6x higher risk of developing skin cancer than women.Only two risk factors—current age and the presence of dysplastic moles—surpassed the explanatory performance of the PRS. Age was strongly predictive of BCC and SCC . The decreased risk with high BMI was large, with a 4× less risk of BCC cancer for participants with a BMI > 35, but it was only half of this effect in melanoma. It has been suggested that a direct protective link between obesity and skin cancer is unlikely, and may instead reflect other life-style factors22. Although the weight effects were more modest, with a 2.2 to 3.1× increase for participants reporting a current weight >300 lb relative to participants with a weight ranging between 100 and 200 lb, they could indicate direct effects of physiological and physical (increase of skin surface) changes in overweight participants23. A few other risk factors included in the final 32-factor models could also be surrogates of sun exposure behavior. For example, participants self-reporting a morning person chronotype showed a small increase of skin cancer risk (1.05 to 1.1×) relative to night person chronotype. Participants reporting as having engaged in intense physical activities also showed a slight (1.1×) increase of skin cancer risk. Curiously, a so-called ‘clean desk’ factor (referring to a participant’s self-reported preference for keeping their desk clean) was selected as the best representative variable from a cluster of correlated personality traits , and explained up to 0.3% of the varianceFor descriptive convenience, we grouped the non-genetic risk factors into six risk scores on the basis of physiological or behavioral similarity Fig. , 14.We evaluated the predictive performances of each risk score in the validation set. We computed the receiver operating characteristic (ROC) and the precision-recall (PR) curves, and estimated the AUC , 3.52 (3.42–3.63), and 2.58 (2.52–2.66), respectively. The upper tail of the DRS distribution showed substantial increase risk of developing skin cancer: the prevalence of skin cancer for participants in the top DRS percentile was 69.8%, 46,7%, and 31.8% for BCC, SCC, and melanoma, respectively. These correspond to a 5.2×, 8.1×, and 12.9× risk increase relative to participants with middle DRS percentile , a principal component analysis showed different contributions of the Susceptibility and Exposure risk scores that enable the identification of different high risk participants for BCC and SCC .In contrast to the DRS, the DRSA are not including the age effects. They however use the same weights for the remaining 31 factors than the ones used for the DRS calculations. These weights were, by definition, adjusted for age by the linear modeling , we investigated the ability of the DRS, DRSA, and PRS to predict incident cases of skin cancer, and their age of diagnosis. We defined incident cases as participants who did not report to have been diagnosed with skin cancer in the original baseline survey, but subsequently reported a diagnosed for skin cancer during the previous year in the follow up survey. We further defined cancer free participants as participants who reported to have been diagnosed with skin cancer in the original baseline survey, but who also reported not been treated for skin cancer during the previous year in the follow-up survey. Using these metrics, we showed that high baseline DRS, DRSA, and PRS were all associated with a higher rate of incident cases in the prospective data, and fewer cancer free participants overall. Consistent with the results from the baseline survey, we also showed that non-cancer participants with high baseline DRS, DRSA, and PRS developed new skin cancer earlier than participants with lower risk scores Table , 12. Fin26, we did not find them to significantly improve the prediction performance  and no evidence of dilution between variant effect estimates from the training set and the independent melanoma GWAS, suggesting a comparable level of misclassification in the 23andMe cohort and the independent melanoma cohort. Similar analyses for BCC and SCC are complicated for the fact that most recent large GWAS publications of these diseases have included 23andMe data, and the variant effect estimates are therefore not independent. However, for BCC, a study17 directly estimated self-reported misclassification in 23andMe cohort, with adjudicated medical records, and revealed a sensitivity and specificity of 93% and 99%, respectively.Our risk models were built using self-reported skin cancer diagnoses, and multiple studies have documented variable rates of self-reporting accuracy in skin cancersAge remains a key component of the skin cancer predictive models. It not only captures the detrimental effects of senescence processes, but it is also correlated with many of the non-genetic risk factors. Nevertheless, we demonstrated that DRSA, which combined all identified risk factors, except age, was an age independent predictor of skin cancer for 30 years or older individuals. This age independence allowed to tread the DRSA and age as two independent variables, and it permitted to draw the lifetime risk trajectories, stratified by DRSA scores, for the three skin cancers Fig. . In retu32. Additional work is also needed to understand the prediction performance in the different European sub-populations, and the portability to non-European ancestries. The current BCC and SCC models seem to perform equally well for individuals of Northern, Southern, or Eastern European ancestry, but the melanoma model shows signs of performance degradation . Over the course of approximately thirteen months from May 2016 to June 2017, more than 210,000 research participants  that accounts for switch errors and incorrect assignments, and gives probabilities for each reference population in each window. Finally, simulated admixed individuals are used to recalibrate the HMM probabilities so that the reported assignments are consistent with the simulated admixture proportions. The reference population data are derived from public datasets  and from 23andMe research participants who have reported having four grandparents from the same country.Individuals were only included if they had >97% European ancestry, as determined through an analysis of local ancestry37. Individuals were defined as related if they shared more than 700 cM IBD, including regions where the two individuals share either one or both genomic segments identical-by-descent. This level of relatedness (roughly 20% of the genome) corresponds approximately to the minimal expected sharing between first cousins in an outbred population. For the purposes of GWAS, if a skin cancer case was found to be related to a skin cancer control, the case was preferentially kept in the sample.A maximal set of unrelated individuals was chosen for each analysis using a segmental identity-by-descent (IBD) estimation algorithm38. Data for each genotyping platform were phased and imputed separately. Variants that were only genotyped on the “V1” platform were flagged due to small sample size, and variants on chrM or chrY, because many of these are not currently called reliably. Using trio data, variants that failed a test for parent–offspring transmission were also flagged; specifically, the child’s allele count was regressed against the mean parental allele count and variants with fitted β < 0.6 and P < 10−20 for a test of β < 1 were flagged. Variants with a Hardy–Weinberg P < 10−20 in Europeans, or a call rate of <90%, were also flagged. Genotyped variants were also tested for batch effects and variants with P < 10−50 by analysis of variance of genotypes against a factor dividing genotyping date into 20 roughly equal-sized buckets were flagged. For imputed GWAS results, variants with average r2 < 0.5 or minimum r2 < 0.3 in any imputation batch were flagged, as well as SNPs that had strong evidence of an imputation batch effect, using an analysis of variance of the imputed dosages against a factor representing imputation batch; results with P < 10−50 were flagged. Each variant flagged by QC on genotyped or imputation data were excluded from the GWAS analysis.Participant genotype data were imputed against the March 2012 release of 1000 Genomes project reference haplotypesHmisc library in R . To ensure robustness of inference, we duplicated our analyses using the non-imputed dataset, and compared the models. Without phenotype imputation, the number of participants dropped from 103,008 to 74,703 in the training set , and the number of skin cancer free participants (cases at baseline and not reporting skin cancer treatment in the follow-up survey).39. This pipeline uses a clumping and thresholding method to select variants . The BCC, SCC, and melanoma PRS included 47, 14, and 18 variants, respectively  was conducted independently for BCC, SCC, and melanoma using data from the training set. Association with the phenotype was performed using logistic regression, including age, sex, the first five principal components, and variables representing the genotyping platform as covariates. Principal components were calculated using ~65,000 high quality genotyped and trans-ethnic variants that are present on all four genotyping platforms. GWAS analyses were run independently for the genotyped and imputed dosages. About 13M variants passed the pre- and post- GWAS QCs. The genomic control inflation factor was estimated as 1.06, 1.02, and 1.01 for BCC, SCC, and melanoma GWAS, respectively. PRS for each skin cancer was computed for all study participants  using RiskPipe Our construction of risk models for skin cancer consisted of three stages.P-value selection (<0.05), the best models contained a total of 20 factors, the three demographic factors (after combining the 5 PCs into a unique ancestry risk factor) and 17 risk factors , and published risk factors, present in the cancer survey  and the squared correlation (for quantitative variables) between the variables and the center of the cluster which is the first principal component of PCAmix. PCAmix is defined for a mixture of qualitative and quantitative variables and includes ordinary principal component analysis (PCA) and multiple correspondence analysis (MCA) as special cases. A total of 67 phenotypes passed the P-value threshold in at least one of the three skin cancers, and were organized into 16 clusters , fitted a unique GLM for each skin cancer, including the 20-factor model and the 14 newly discovered risk factors, and identified the best model with a P-value selection. The new models contained 31 risk factors  were <2.2, at the exception of BMI and weight (6.9 < VIF < 8.1). We, therefore, decided to keep all the 32 factors in the final models because VIF > 10 is often used as a criteriaWe defined six risk scores by grouping risk factors included within the final 32-factor models Figs. , 2. The We finally constructed two main disease risk scores, called DRS and DRSA. DRS includes all the factors from 32-factor model. DRSA excludes the effects of age. Similar to the PRS computation, the risk scores are calculated as the sum of the factor responses weighted by their effect sizes, estimated by the 32-factor model in the training set . The three skin cancers showed different risk score signatures, in particular for the contribution of Exposure, Susceptibility, and Family risk scores. The PRS and Mole risk scores have the largest and generally orthogonal effects on the first two PCs  curve in both the training and validating sets, using ROCR library in R, and we extracted the area under the curve (AUC) values. We also computed the precision-recall (PR) curves , and evaluated different attributes of the three skin cancers, using the baseline survey data. We first calculated the prevalence per bin of participants who reported skin cancer. All risk scores showed an increase of skin cancer with higher risk score values. They also showed long tails, as observed in PRS studies Fig. , with stUsing the prospective data, we computed the number and proportion of incident cases . The number and proportion of cancer free participants  in each percentile bin Table , 12.We explored more complex prediction skin cancer models by including and testing pairwise interaction. We first tested for all pairwise interaction between the 7 risk factors using GLMs (Supplementary Table I) from the prevalence (P) in the validation set. For m-year incidence rate at age a:We computed the incidence rates (I) from the lifetime risks associated to each curve. To model the age distribution in the US population, we extracted the estimated yearly survival rates (S) produced by the CDC for white Americans (sex combined)41. The expected mean and SD of age of diagnosis (m) was obtained with:We compared the observed and expected ages of diagnosis in the validation set. The expected ages of diagnosis in Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Leukemia cutis (LC) is a rare presentation of leukemia. It is characterized by the infiltration of leukemic cells into the different layers of the skin causing varying skin manifestations. It can occur before the hematological presentation of leukemia or during the disease course and carries a poor prognosis. Here, we report a patient with therapy-related myelodysplastic syndrome (t-MDS) whose transformation to acute leukemia was heralded by the development of LC. Worrisome cutaneous lesions should not be overlooked, and a skin biopsy should be pursued to confirm the diagnosis. A high index of suspicion is the key to early recognition of sometimes nonspecific skin findings of widespread systemic disease. Leukemia cutis (LC) is the skin infiltration by malignant hematopoietic cells leading to varying dermal manifestations ranging from a simple rash to nodules and patches. Its incident varies in different leukemias but most commonly reported in acute myeloid leukemia (AML), where the incidence ranges from 5% to 10% . We repoHere, we emphasize the principal role of clinicopathologic correlation through biopsy from the cutaneous lesions with immunohistochemical (IHC) stains to recognize the malignant infiltrates in hematopoietic malignancies, and highlight the chromosomal aberration found upon cytogenetic analysis.3/µL with 39% neutrophils, 23% lymphocytes,0% eosinophils, and 13% monocytes with 6% blasts, but flow cytometry did not show any increased blasts. A subsequent bone marrow aspiration and biopsy reported a hypercellular marrow with some evidence of dysplasia  regimen, and he had been in complete remission since then. The patient was asymptomatic, and the physical examination was unremarkable. His laboratory workup was relevant for hemoglobin of 7.6 g/dL, platelets of 79 K/µL, white blood cells (WBC) of 3.5 x10The cytogenetics showed 46, XY, and (11)p15q23); therefore, he was diagnosed with t-MDS and started on azacytidine. The patient remained relatively asymptomatic for 10 months until he was noted to have lower extremity nodular skin eruptions with purpura and crustation  or mylotarg (gemtuzumab ozogamicin) was offered with the caveat of intensified risk of morbidity and associated mortality with the induction therapy. After detailed consideration, the patient decided to forgo aggressive treatments and was provided with supportive care and blood transfusions. We conducted further discussion regarding the terminal and progressive nature of his condition. The palliative care team was involved in the patient's care, and he was referred to hospice, where he eventually died three months after his LC diagnosis.Leukemias, especially acute leukemias, can present with a spectrum of extramedullary myeloid cell tumors, including skin infiltrates with or without bone marrow disease . LC is aIn our patient, LC was the first manifestation of the transformation of t-MDS to acute leukemia and predated hematological manifestation. The clinicopathologic correlation of cutaneous manifestations in patients with pre-existing hematologic malignancy is crucial in recognizing this condition. Interestingly, this patient had the inv(11)(p15q23) chromosomal aberration via cytogenetic analysis at the time of t-MDS diagnosis. LC can be the first sign of a new leukemia diagnosis or the transformation of an existing hematologic condition (t-MDS in this case) to leukemia. We aim to raise awareness amongst dermatologists and internists as these patients may initially present to these specialists. Maintaining a high index of clinical suspicion, through a complete history and physical examination, is crucial in order to recognize rare cutaneous manifestations of common hematopoietic malignancies.