The role of personality traits in modulating the incidence and progression of medical disease conditions are well documented, however, there is a paucity of information for its effects on dental health conditions and specifically on the prognosis of restorative dental materials. This study aims to evaluate the clinical performance of Micro-hybrid and Nano-ceramic composite restorations among patients with different personality traits. A total of 323 patients, indicated to receive operative treatment at a University Dental College Hospital, were invited to participate in this study. Consenting patients were requested to complete the Big Five Inventory (BFI-44 Item) personality questionnaire and were evaluated by a psychiatrist for categorizing the participants based on their personality traits. Out of the recruited patients, 124 patients falling in to the dominant trait of Agreeableness (n = 62) and Neuroticism (n = 62) were included in the study for further investigation. Next, patients from the Agreeableness (Group A) and the Neuroticism personality trait group (Group N) were randomly divided into two subgroups each—sub group Am (n = 44) and Nm (n = 48) for Micro-hybrid composite restorations and Sub group An (n = 42) and Nn (n = 47) for Nano-ceramic composite restorations. Two trained and calibrated dentists prepared the cavities according to previously published methodology. The restorations were evaluated at baseline (immediately after restoration), 6-months, 12-months and 24-months intervals by two blinded independent dental professionals for anatomical form, secondary caries, color match, retention, marginal adaptation, surface texture, marginal discoloration and post-operative sensitivity. There is no statistically significant difference noted in various parameters of restoration performance between Micro-hybrid composite and Nano-ceramic composite compared among ‘agreeableness’ personality group and among ‘neuroticism’ personality group after controlling the personality trait factor. Higher ‘Neuroticism’ individuals had higher restoration deterioration in color matching and surface texture when compared to higher ‘Agreeableness’ trait individuals. Regression analysis showed no effect of gender or cavity size on the outcome of results. Assessment of personality traits may serve as a useful tool during treatment planning which would aid clinicians in choosing suitable restorative dental material and prosthesis design according to individual patient’s physiological and functional needs, thereby overall improving the quality of treatment provided. Among various models of personality assessment, Costa and McCrae’s five-factor model is one of the widely used model in personality evaluation, it divides personality traits in to five dimensions which include neuroticism, extraversion, openness, agreeableness, and conscientiousness3.Personality can be defined as the "dynamic organization of the psychobiological systems that modulate adaptation to changing environments through several personality traits, which are long-lasting patterns of how we perceive, relate to, and think about oneself, other people and the world as a whole"4. Whereas, higher agreeableness individuals are warm, cooperative, kind, considerate, trustworthy and selfless natured thus maintaining positive interpersonal relationship with others5.Individuals with higher levels of neurotic traits experience higher amount of stress, anxiety, irritability, low self-esteem, poor emotional control, minor depressions, excessive smoking and alcohol addiction. They are unable to control cravings and urges6. Malouff et al.7 in their meta-analysis examined the relationship between the personality traits and clinical disorders and observed that higher neuroticism had a strong correlation with the incidence of mental and physical health problems than other personality traits. However people with higher agreeableness are better in self-regulating their behavior and experience better subjective mental and physical health8.Higher levels of neuroticism trait has numerous health implications9. Bruxism is proved to be a risk factor in the survival of prosthetic and implant restorations10. Thus, the current study examines the significance of higher neuroticism trait’s impact on restorative treatment procedures.From a dental viewpoint, there is strong association between the characteristics of bruxist and higher neuroticism trait individuals12. However still there is confusion regarding choice of suitable restorative materials. Recently, nanotechnology has been introduced in the dentistry that contains small particles of nanometers ranges. These small particles are favorable to obtain good wear resistance, polishability and better esthetics13. Many researchers observed that Nano-hybrid composites performed better in all mechanical properties tested than Micro-hybrid composites15. However, few other systematic reviews found no difference between Micro-hybrid and Nano-hybrid composite17, and no clear consensus has still been achieved.Physical characteristics of composite materials are supremely important in restorative dentistryThere are many studies done in investigating the role of personality traits in modulating the incidence and progression of medical disease conditions; however, there are hardly few studies for the same in dental health conditions and a literature search revealed that there are no studies for the same in restorative dentistry.Hence, the main objective of the study is to clinically evaluate the performance of restorations using United States Public Health Service (USPHS) clinical evaluation criteria in patients with different personality trait. Also, the current study aims to observe the influence of newer materials like Nano-ceramic on the outcome of the restoration over a period of 24-months after controlling and standardization of various factors, which can affect the restoration’s prognosis.All subjects gave their informed consent for participation in this study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the institute where the study was carried out. The inclusion criteria were age between 20–35 years and presence of full set of permanent dentition (at least 28 excluding third molars) with a bilateral angle class I molar and canine relationship. Patients having large occlusal restorations, presence of endodontically treated teeth, fixed prostheses or active periodontal disease was excluded. Also, presence of local or systemic osseous or neuromuscular disease, presence of spontaneous orofacial pain, temporomandibular joint disorders, large facial asymmetry and pregnancy were not considered for the study.19 to fill and were evaluated accordingly with the help of a psychiatrist. The patients were subsequently allotted to the various personality trait group as per their dominant trait score on Table . As ther124 patients falling into the Agreeableness (n = 62) and Neuroticism (n = 62) traits were included in the study for cavity preparation, restoration and USPHS evaluations.The selected 62 patients of each dominant personality group were numbered. They were further divided in to odd and even numbered sub groups. The choice of micro-hybrid and nano-ceramic restoration between the odd and even sub groups was decided by tossing a coin. Hence, patients from the Agreeableness (Group A) and the Neuroticism (Group N) were divided (Irrespective of gender difference) into two subgroups each—Sub Group Am  and Sub Group Nm  for micro-hybrid composite restorations and Sub Group An  and Sub Group Nn  for Nano-ceramic composite restorations.*Group Nomenclature: Agreeableness(A), Neuroticism(N), Micro-hybrid(m), Nano-ceramic(n).25. The outlines of the preparations were limited to the removal of caries/defective restoration. The teeth were etched , self-priming adhesive resin bonding agent  was applied and cured as per manufacturer instructions. Sixty seven patients received one restoration and fifty seven patients received two restorations but of the same generic category either the micro-hybrid  or the Nano-ceramic composite  [Table 2 and 800 mW/cm2) for 40 s by uniform continuous curing technique followed by finishing and polishing procedures .Two clinical dentists performed the operative procedure. Class I cavities of Small size  and moderate size  were prepared in molar teeth according to previous methodology28 and any variation over evaluations were solved through discussion to reach consensus by both examiners (Appendix II). The study's Principal Investigator conducted calibration of the evaluators prior to the study evaluation. Excellent inter-examiner reliability (kappa > 0.80) for USPHS scorings were observed, as assessed in a subsample of 20 patients not included in the study.At the baseline (immediately after restoration), 6-months, 12-months and 24-months; the restorations were evaluated by two double blinded  independent dental professionals for anatomical form, secondary caries, color match, retention, marginal adaptation, surface texture, marginal discoloration, post-operative sensitivity using the Modified USPHS evaluation criteriaAs the evaluation of restorations provided only ordinal structural data, non-parametric statistical analysis (Mann–Whitney U-test) was performed using an SPSS software program and a probability value of (P < 0.05) was considered to be statistically significant. Mean Rank Scores were compared among groups at different time points and lower mean rank score denotes better restoration performance and Vice-Versa. Regression analysis was performed to observe the effect of gender and cavity size on study results.Guidelines documented in the Helsinki-2013 Declaration of experiments on humans were adopted for this study. The ethics committee of the Ragas Dental College Hospital, Tamil Nadu Dr. M.G.R Medical University, approved the study protocol . Participating individuals were mandated to read and signed a consent form. Prior to signing the consent form, all participating patients were informed that they could withdraw from this study at any stage without any penalty; and were invited to ask questions.Total of nine patients were lost to follow up Table . At 24 m29. Studies indicate high prevalence of dental caries and poor oral health are common among neurotic people31. Among the five personality traits, we selected only two groups (higher neuroticism and higher agreeableness) since they are largely contrasting in nature and experience opposite patterns in disease incidence and progression. Additionally, this study compares the new generation ormocer based Nano-ceramic and universal Micro-hybrid composite in occlusal Class I cavity preparation among patients with different psychological traits.Numerous researchers have established that personality traits have an impact on various health-related outcomes, out of which neuroticism is the most studied personality trait from the health point of view32. However, all previous studies so far were done in populations without the control of personality trait, patient’s age, cavity type, temporomandibular joint (TMJ) and occlusion statuses unlike the present study, hence the results of this study are more reliable.The results demonstrate that the clinical performance of both Micro-hybrid (Spectrum TPH) and Nano-ceramic (Ceram X mono) composites among same personality groups during the 24-month follow-up period was found to be excellent with no statistically significant difference Tables , which i34. The primary objective of this study is to find the patient related factor of personality’s influence on the prognosis of restoration while controlling other above-mentioned factors. In a systematic review conducted by Shisei Kubo35, no effect of gender, age or cavity size  was observed from many articles that studied the prognosis of composite restoration that is similar to the multi-variate analysis results of the current study  and also generate increased speed and frequency of chewing strokes45. Although many studies found Nano-hybrid performed better than Micro-hybrid in wear15, it is also inferred that wear resistance and surface roughness of dental composite resin available in market is more of material dependent rather based on their generic categories usually classified on the basis of filler loading and resin matrix46.Micro-hybrid composites performed better than Nano-ceramic among neuroticism patients. The current study results are contrary to research that demonstrate that larger particle sizes have an unfavorable effect on the wear resistance of dental composites47 which are highly notable characteristics of higher neuroticism individuals. Ceram X composites contain Nano-fillers, but their wider-diameter glass fillers (mean size 1.2–1.6 µm) can easily rip out leading to surface imperfections48. However it should be noted, Nano-ceramic (Ceram X) performance in spite of its material limitations were similar in performance to Micro-hybrid spectrum TPH when compared among agreeableness personality groups (Table Ormocer based Nano-ceramic composite (Ceram X mono), because of its smaller particle size could have easily succumbed to the effects of altered chewing dynamics, increased masticatory forces and consumption of dense abrasive foods49, however these interpretations cannot be generalized but can serve as a predictor on the outcome of results. Acid and alcohol molecules penetrate in to the resin matrix causing softening of the composite surface. This could potentially affect the surface integrity of composite resins leading to its increased discoloration and staining50.Neuroticism patient’s Nano-ceramic restoration had more discoloration than Agreeableness patient’s Nano-ceramic restoration. Increased surface roughness of Ceram X mono restorations among higher neuroticism patients led to their accelerated surface discoloration as well. Also individuals with higher neuroticism trait are vulnerable to lead an unhealthy lifestyle and are more prone to adverse habits such as substance abuse 51. Although best efforts were taken to minimize the effect of these confounding factors by stringent sample selection, nevertheless these confounder’s influence cannot be completely eliminated which is one of the limitations of this study. Clinical performance of composite materials is also influenced by individual patient related unique factors such as dietary habits and oral hygiene which is difficult to be standardized among the participants of this study which is another limitation of this study.Overall higher deterioration and total failure rate of restorations were observed among neuroticism group when compared to agreeableness group. Longer follow up is needed to evaluate the performance of restoration among these group. The complex action of chewing and bite force is influenced by many factors such as age, gender, craniofacial morphology, periodontal support, temporomandibular joint health and dental statusAssessing patient’s personality before the dental treatment may provide valuable information. As high neurotic trait individuals are often associated with significant dental risk factors, counselling can be given explaining the importance of lifestyle habits and oral hygiene procedures on the treatment prognosis. Clinical Psychologist can be included in the dental team for treating patients to offer psycho-counselling and to intervene on stress management for patients with high neurotic traits to control dental related parafunctional habits. Hence, from a clinical point of view, assessment of personality traits will be useful in recommending a suitable restorative dental material or appropriate prosthesis design, thereby overall improving the quality of treatment provided. However, this is a hypothesis generating study and further studies should be done to test its reliability among different populations.Supplementary Information.Supplementary Tables.


Subclonal heterogeneity and evolution are characteristics of breast cancer that play a fundamental role in tumour development, progression and resistance to current therapies. In this review, we focus on the recent advances in understanding the epigenetic and transcriptomic changes that occur within breast cancer and their importance in terms of cancer development, progression and therapy resistance with a particular focus on alterations at the single-cell level. Furthermore, we highlight the utility of using single-cell tracing and molecular barcoding methodologies in preclinical models to assess disease evolution and response to therapy. We discuss how the integration of single-cell profiling from patient samples can be used in conjunction with results from preclinical models to untangle the complexities of this disease and identify biomarkers of disease progression, including measures of intra-tumour heterogeneity themselves, and how enhancing this understanding has the potential to uncover new targetable vulnerabilities in breast cancer. Tumour heterogeneity (TH) can exist at different levels  and refers to the observation that cancer cells can have distinct molecular profiles from one another. This diversity of tumour cells’ profiles, can be either observed between tumours, known as inter-tumour heterogeneity (inter-TH), or within the same tumours, known as intra-tumour heterogeneity (intra-TH)2.Breast cancer is a heterogeneous disease, driven by a myriad of genetic and non-genetic alterations that govern its clinical behaviour4. The seminal studies using gene expression profiling, have further subdivided breast cancers into molecular and transcriptomic subtypes, which are of prognostic and predictive importance9. Together, these studies highlight the innate inter-TH amongst breast cancer patients.Inter-TH in breast cancer has led to the classification based on histology and expression profiles of the molecular markers; oestrogen receptor (ER), progesterone receptor (PR) and the overexpression or gene amplification of human epidermal growth factor receptor 2 (HER2). The expression profiles of these markers have led to three main clinical breast cancer subgroups for which treatment regimes are based; ER + /HER2-, HER2 + or triple-negative breast cancer (TNBC) which lack ER, PR and HER2 expression10, which are extensively reviewed elsewhere13. Despite the development of next-generation sequencing having enabled in depth analysis of intra-TH and tumour evolution, these two factors contribute to systemic treatment failure by initiating phenotypic diversity enabling drug resistance to emerge.Intra-TH escalates the complexity of the disease in many aspects including our understanding of cancer development, progression, metastasis, therapy response and acquired drug resistance. Two non-mutually exclusive models of intra-TH have been proposed, the cancer stem cell model and the clonal evolution modelIn this review, we summarise the importance of next-generation sequencing in identifying intra-TH in breast cancer at a genetic level, identify the clinical impacts and challenges of intra-TH to patients’ treatment outcomes and how genetic intra-TH can be assessed by single-cell approaches. Furthermore, we explore the non-genetic forms of intra-TH during breast cancer evolution, drug resistance and more importantly how single-cell approaches in conjunction with appropriate preclinical models can be used to untangle the complexity of the disease.3. For instance, through large-scale genome analysis of primary human breast cancers, the complex mutational landscape of breast cancer has been mapped, highlighting the existence of clonal tumour subpopulations in breast cancer22. These studies in general have highlighted that a large majority of mutations detected in primary breast cancers are subclonal17 and not necessarily evenly distributed spatially among individual tumours highlighting the need for multiple sampling21. In primary untreated breast cancers, multi-region sequencing has highlighted that the extent of subclonal diversification varies considerably among individual tumours with no strict temporal order being evident, and genomic alterations such as point mutations and rearrangements affect the most common breast cancer genes, including PIK3CA, TP53, PTEN, BRCA2 and MYC, occurring early in some tumours and late in others21. This is in contrast with other tumour types that show a specific temporal order of mutations26. This may be due to the fact breast cancers inherently are thought primarily to be driven by an array of copy number driver alterations, with most somatic mutations occurring later in tumour development19 or may reflect a situation wherein some tumours that grow to a certain size, clonal sweeps occur in which an especially fit subclone replaces the majority of others in the tumour21. As the different subtypes of breast cancer are thought to arise from different cells-of-origin, this may additionally explain the differences in heterogeneity observed in the different subtypes of breast cancer5.In the past decade, through the development and implementation of next-generation sequencing, numerous studies have reported the existence of intra-TH in breast cancer at the genetic level. This has been attributed to chromosomal or genomic alterations affecting biological processes and functions30. Drug resistance can be pre-existing (where therapy does not impact subclone frequency), selected (arising to the selection of rare pre-existing subclones that are able to expand), or acquired (where new genomic/transcriptomic/epigenetic aberrations are introduced contributing to the drug-resistant phenotype). Relapsed or metastatic breast cancers have been found to largely share the vast majority of their genomic alterations with the corresponding primary disease indicating pre-existing resistant clones, however many metastatic tumours also exhibit additional mutations that were not previously detected or are subclonal in the primary disease37. As an example, ESR1 activating mutations are rarely present in primary ER + breast cancer, even in those 15–20% patients that show intrinsic resistance to hormonal therapies39, whereas they are highly enriched in ER + metastatic tumour samples from patients with acquired endocrine resistance (30–40% of ER + patients) and in particular, are a known mechanism of resistance to aromatase inhibitors (AI)42. Recent large studies cataloguing the mutational repertoire of primary and relapsed metastatic breast cancers from patients have highlighted that multiple mechanisms of endocrine therapy resistance exist in ER + BC cancers43 including evidence that pre-existing low-frequency mutations can be responsible for endocrine resistance45.Subclonal changes can additionally occur upon the selective pressure of therapy Fig. 19,21,27–46 highlighting that while a proportion of patient’s tumours undergo clonal selection, others maintain a similar genomic architecture after chemotherapy47. It is worth mentioning that poly(ADP)-ribose polymerase (PARP1/2) inhibitor resistance is also observed in patients with homologous recombination defects including TNBC52. Although PARPi-based therapy is standard of care for patients with germline BRCA1/2 mutations including breast and ovarian cancer, resistance has been observed in these patients, where reversion mutations of BRCA1 or BRCA2 are a well-documented resistance mechanism54 and can be assessed through profiling of circulating free DNA (cfDNA)55. Although a plethora of potential resistance mechanisms to PARPi have been identified through high throughput functional genomic screens, many of these have yet to be validated in patients relapsing on PARPi treatment57. Overall, whether these reversion mutations are spontaneous, pre-existing and selected for under PARP1/2 inhibition, or induced by other therapeutic agents such as platinums previously administered to patients is still to be determined.Genetic mechanisms of resistance to chemotherapy are less well-documented in TNBC, and in general subclonal alterations are not enriched upon chemotherapy treatment58 and in early breast cancer59 to monitor response to therapy and are predictive of relapse61. Importantly, ctDNA analysis can provide important genomic information that cannot be traced by a single biopsy at a specific cancer site and are becoming increasingly used in clinical studies as a non-invasive way of assessing the emergence of progressive disease and to direct therapy64. Although further research is necessary to determine whether ctDNA can fully represent tumour genomic profiles at any given time, recent data from a seminal clinical trial, plasmaMATCH, has assessed the validity and utility of ctDNA for targeted therapies without previous tissue testing in advance breast cancer patients65. Novel methylation profiling methods  can allow tracking of evolutionary changes of single CpG sites to track clonal haplotypes in ctDNA  at single-molecule resolution without a priori knowledge of the molecular make-up of the disease66. Implementation of such methodologies will likely overcome the limitations in terms of cost and prior knowledge of a patients’ tumour mutational repertoire for successful longitudinal patient tracking.As well as bulk profiling of tumour biopsies, assessment of circulating tumour (ct) or cfDNA of cancer patients is a useful evolutionary methodology to track the evolution of intra-TH of cancer, the presence of treatment-resistant clones and to predict response and resistance. These tools have been shown to be useful in both metastatic patients67. However, these patterns were unchanged between pre- and post-treatment biopsies, suggesting perhaps resistance to NAC is not driven by particular genomic alterations. Interestingly, this study did identify intra-TH based on the expression of cancer stem cell markers such as CD44 and CD24, that correlated with NAC response, suggesting phenotypic evolution plays a role in chemotherapy resistance67. Such studies have also highlighted that the spatial pattern of PIK3CA mutations together with HER2 amplified cells play an important role in chemotherapy resistance68.Single-cell approaches to assess intra-TH have traditionally used florescence in situ hybridisation to measure copy number using probes against frequently amplified loci in breast cancer. Using diversity measures one study seeking to address whether copy number alterations drive neoadjuvant chemotherapy (NAC) resistance revealed that tumours that respond to NAC have a low diversity of copy number alterations before therapy, whereas those that had a poor response showed a higher diversity71.Recent advances in single-cell sequencing technologies now allow the assessment of intra-TH of tumours, as well as the non-genetic changes that may occur upon metastatic disease progression and upon selective pressures such as chemotherapy and targeted therapy in breast cancer. Single-cell sequencing overcomes the need for computational deconvolution of the theoretical contribution of individual clones but instead can profile the DNA, RNA, methylation and chromatin accessibility of each individual cell72. This study revealed that polygenomic tumours likely represent sequential clonal expansions and that seeding of metastasis from a monogenomic tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. Interestingly, the authors found that an unexpectedly abundant subpopulation of genetically diverse ‘pseudodiploid’ cells that did not travel to the metastatic site, suggesting that tumours grow by punctuated clonal expansions with few persistent intermediates in contrast to more gradual models of evolution72. A follow-up study looking at mutations at the single-cell level identified that copy number alterations occurred early in tumour evolution and remained highly stable suggesting clonal expansion, whereas, in contrast, point mutations evolved gradually, generating extensive clonal diversity, with many mutations seen in less than 10% of the tumour cells30. Application of this technology to patients with ductal carcinoma in situ and concurrent invasive disease using topographical single-nucleus sequencing has revealed that the majority of genomic alterations developed in the ducts prior to the invasion, supporting a multiclonal invasion model, whereby more than one clone escapes to establish the invasive disease73. A similar study using single-cell profiling of formalin-fixed paraffin-embedded clinical tumour samples to assess copy number of the transition from ductal carcinoma in situ to invasive disease also found extensive intra-TH in pre-invasive lesions with evolution to invasive disease occurring via multiple mechanisms74. Together, these studies suggest genomic diversification of copy number alterations occur early during tumorigenesis due to clonal expansions and subclonal mutations occur later on, generating substantial mutational heterogeneity.Selective pressures such as targeted therapy can also alter the transcriptome of cancer cells, leading to drug resistance and patient relapse. In the first seminal single-cell sequencing study from Navin and colleagues, single-cell profiling was used to assess copy number heterogeneity in two breast cancer patients78. Drug resistance can be through the selection of pre-existing alterations and/or acquired due to transcriptional and epigenetic mechanisms such as histone modifications and DNA methylation affecting gene expression or communication of tumour cells with the microenvironment  to evaluate intra-TH, as discussed above, there is emerging evidence that non-genetic factors also play a major contributing role. Numerous studies have suggested the existence of differential transcriptomic make-up between primary tumour and the corresponding metastatic lesionsent Fig. 15,46,47,86. Single-cell sequencing of circulating tumour cells (CTC’s) of patients with metastatic breast cancer versus patients with primary breast cancer, highlighted increased transcriptomic heterogeneity in those patients76. Of note transcriptional signatures associated with epithelial to mesenchymal transition were enriched in CTC’s derived from patients with metastatic disease76. Importantly, a single-cell study of patient-derived TNBC primary tumours in vivo and their corresponding lung and lymph-node micro-metastases exhibited a distinct transcriptome at a single-cell level. Interestingly, the mitochondrial oxidative phosphorylation (OXPHOS) pathway was upregulated in micro-metastases75. Notably, pharmacological inhibition of OXPHOS attenuated lung metastasis in vivo, highlighting the importance of this pathway in a metastatic spread as well as its potential in metastatic prevention in breast cancer patients75. These findings are in line with bulk RNA sequencing of primary tumours and their matched metastases of TNBC patients, highlighting consistent gene expression differences including enrichment of hypoxia, cellular metabolism, and fatty acid β oxidation pathways in the metastases, and were independent of the metastatic site86. Collectively, the above studies indicate the importance of transcriptomic heterogeneity in metastatic breast cancer as well as its potential role in the aggressiveness of this disease.Transcriptomic analyses from primary and metastatic cancers at both bulk and single-cell level has further highlighted the complexity of tumour evolution. Several reports have shown that metastatic breast cancer lesions show a higher transcriptomic heterogeneity than the corresponding primary disease47. Although this study only looked longitudinally in a small number of tumours, some patients harboured enrichment of genomic alterations in residual disease post-NAC, however, overall, there was no evidence that specific genetic differences contributed to chemotherapy resistance. Single-cell RNA sequencing of a subset of these tumour samples, however, highlighted that the transcriptomic profiles of pre-treatment and post-treatment samples were distinct, suggestive of a model of therapy resistance in which selective and acquired modes of evolution are responsible for the resistant population, in agreement with previous in situ based studies67.In a seminal study from Navin and colleagues, single-cell DNA and RNA sequencing was used to analyse the contribution of genetic and non-genetic heterogeneity in TNBC patients receiving NAC and treatment with the anti-VEGF therapy (bevacizumab)87. This appeared to be driven by copy number alterations within the cells, suggestive that at least in the primary disease setting, the genotype can influence transcriptomic heterogeneity of individual subpopulations. Perhaps most interesting from this study was the identification of distinct subclonal populations of malignant cells that were shared between the different patients characterised by multiple signatures of treatment resistance and metastasis, including glycosphingolipid metabolism and associated innate immunity pathways. Moreover, these signatures identified from the subclonal cellular populations were able to predict long-term prognosis in independent cohorts of primary TNBC from bulk RNA-profiling. This suggests that minority populations of cells have already acquired the transcriptomic features to allow them to become metastatic. Moreover, if this signal can be elucidated from bulk tissue once identified, this heralds a paradigm shift for identification and specific targeting of these subpopulations a priori to circumvent metastatic spread.A recent study performing single-nucleus RNA sequencing of six therapy naïve primary TNBC fresh tumours highlighted that in the absence of chemotherapy therapeutic pressure, TNBC’s show extensive intra-TH at the transcriptomic level79. Additionally, single-cell sequencing of ER + cell lines has identified genetically and transcriptionally distinct subpopulations including rare populations of MCF7 cells highly expressing an apoptosis-related signature, positively correlated with a pre-adaptation signature to oestrogen deprivation88. These results suggest a multi-step mechanism of drug resistance to endocrine therapy where both genetic and nongenetic alterations contribute to this phenotype. In a similar study, authors used molecular barcoding coupled with single-cell transcriptomic sequencing to track the ER + cell line, MCF7, upon long-term endocrine therapy in vitro. Their findings suggested that the majority of fulvestrant or tamoxifen resistance clones were pre-existing and highly selected during treatment80.A number of recent studies have attempted to address whether drug resistance including endocrine therapy or chemotherapy in breast cancer is selected for and/or acquired. Upon endocrine therapy in ER + breast cancer cell line, MCF7, a rare subpopulation of pre-adapted cells with high plasticity have been found to have district transcriptomic signatures , hypoxia and cell polarity) with features of dormancy as well as mixed epithelial and mesenchymal characteristics. Interestingly, single-cell sequencing highlighted that these pre-adapted cells that display features of dormancy and mixed epithelial and mesenchymal traits are able to survive upon short-term endocrine therapy, however, these pathways are not seen in fully resistant cells, suggesting that the cells undergo further transcriptomic reprogramming to become fully resistant to endocrine therapy and to generate subsequent metastasis89. Moreover, single-cell profiling of CTC clusters identified that these clusters are oligoclonal, and although rare have up to a 50-fold increase in metastatic potential and are characterised by high expression of the cell junction component plakoglobin, suggesting this could be a useful biomarker to detect cells with high metastatic potential in the primary tumour90. Profiling of CTC’s from metastatic breast cancer patients has also highlighted the intra-TH of ESR1 mutations in patients with endocrine resistance suggesting independent mechanisms of resistance to these agents, and highlighting the clinical applicability of single-cell approaches for detecting genomic alterations mediating targeted therapy resistance91.Single-cell RNA profiling of CTC’s from liquid biopsies have further revealed the extent of intra-TH, with rare CTC’s displaying divergent gene expression signatures of EMT, cancer stemness, DNA repair, kinase signalling and immune-tumour cross-talk82. More recently, enhancer mapping of H3K27 acetylation states in 47 matched primary and metastatic endocrine-resistant tumour biopsies has allowed the inference of tumour evolution and highlighted that endocrine therapies select for phenotypic clones under-represented at diagnosis92. These studies suggest phenotypic heterogeneity governed by inheritable cell-type-specific transcription is maintained through the activation of epigenetically defined regulatory regions including promoters and enhancers92.It is now well established that epigenetic mechanisms play a vital role in driving phenotypic heterogeneity and evolution and contribute to treatment resistance in breast cancer patients. In endocrine resistance in ER + MCF7 breast cancer cells, for example, drug-induced epigenetic states have been shown to involve large topologically associating domains and the activation of super-enhancers. In particular, MCF7 cells resistant to AI have been shown to upregulate cholesterol biosynthesis through stable epigenetic activation, which leads to constitutive ER-activation in AI-resistant cells93. Indeed, alterations in epigenetic mechanisms through the induction of several transcriptional states, contribute to intra-TH and thus to the aggressiveness of the disease and drug resistance94. In a recent pivotal study by Hinohara and colleagues, the epigenetic enzyme, KDM5B, was found to be a regulator of transcriptomic heterogeneity in a panel of ER + breast cancer cell lines, promoting resistance to endocrine therapy. Its high expression in ER + breast cancer cell lines was found to be associated with higher transcriptomic heterogeneity80 and was reversed upon treatment with KDM5 small molecule inhibitors, providing proof of concept that modulating the activity of epigenetic enzymes, can lead to improved responses to the standard of care treatment. Interestingly in cellular models, resistance to KDM5 inhibitors was acquired rather than selected for as in endocrine therapy resistance. Additional studies have also identified epigenetic reprogramming of the transcriptional repressive mark, H3K27me3, in cell populations resistant to chemotherapy in TNBC and to tamoxifen in ER + breast cancer patient-derived xenografts (PDXs)83.Evidence also suggests epigenetic intra-TH governs resistance to novel therapeutic agents such as Bromodomain and Extra-Terminal motif inhibitors96. Gene expression signatures derived from the tumour stroma can be predictive of patients’ clinical outcome97. Thus, there is increasing interest in addressing the contribution of the microenvironment in terms of tumour development, progression, drug resistance as well as its potential as a prognostic factor and a therapeutic target. The use of single-cell RNA sequencing has enabled deeper insights into intra-TH as well as the contribution of tumour microenvironment which are not detectable by other methods100. Recently, the immune atlas in breast cancer has been mapped. Using unbiased single-cell RNA sequencing analysis, the immune cells from eight primary breast cancers patients, as well as their matched normal breast tissue, blood and lymph nodes were profiled in order to investigate the role of immune cells in tumour microenvironment and its contribution to cancer progression and immunotherapy response98. The authors identified significantly increased phenotypic expansion of intra-tumoral immune cells in comparison to normal breast tissue. Similarly, the tumour microenvironment of primary TNBC tumours has been profiled by high throughput single-cell RNA sequencing100. The authors identified two cancer-associated fibroblast and two perivascular-like subpopulations, where each population clustered into different states. These signatures were found to be strongly associated with cytotoxic T-cell dysfunction, leading to immune evasion100.Increasing evidence suggests that epigenetic and subsequent transcriptomic alterations in cells within the tumour microenvironment  contribute to tumour initiation, progression and metastasis mediated through paracrine signalling with the tumour cells102. Tissue micro-dissection paired with gene expression analysis has provided valuable insights into tumour–microenvironment relationships. For example, it was found that in TNBC T-cell infiltration within a tumour mass, but not peripheral localisation of T cells, was associated with good patient outcome103. Digital pathology coupled with computational assessment allows a unique insight into how the tumours are organised and is an exciting emerging field. For example, the spatial heterogeneity of immune cells, stromal cells and epithelial cancer cells assessed by haematoxylin and eosin routine histopathology sections has been shown to be associated with a poor response to standard therapies in both ER + and ER- primary breast cancers, suggesting that having a mixture of these cells within the tumour microenvironment allows adaptation as the cancer evolves102. Moreover, increased immune cell spatial clustering has been shown to predict both early (0–5 years) and late (5–10 years) recurrence in ER + breast cancer patients after endocrine therapy, whereas total immune cell abundance showed no such an association101. A recent study of 20 primary breast cancers subjected to single-cell RNA coupled with epitope sequencing (CITE-seq), has revealed new PD-L1/PD-L2 + macrophages associated with clinical outcome100. Using spatial transcriptomic sequencing on the corresponding tissue sections of these tumours further revealed that stroma-immune niches were spatially organised. Moreover, single-cell signatures were able to stratify large breast cancer cohorts at the bulk RNA level into nine “ecotype” clusters associated with unique clinical outcomes which were distinct from the classical intrinsic molecular subtypes of breast cancer100. Recent technological advances in proteomics have additionally allowed assessment of the epithelial and stromal cell make up of primary breast cancers whilst maintaining the spatial architecture. Using imaging cytometry time of flight (CyTOF) to measure multiple proteins in a single experiment now allows unprecedented assessment of the tumour microenvironment. These studies have revealed the extensive tumour and immune ecosystems in primary breast cancers105 and highlighted associations between genomic alterations such as TP53 mutations and distinct epithelial and stromal phenotypes, that hypoxic environments may induce immune tolerance, and identified that single-cell pathology subgroups have distinct clinical outcomes105. Further assessment of these during tumour evolution will be important going forward to validate these as biomarkers of disease progression.It is becoming clear that the spatial organisation of tumour microenvironments plays an important role in therapy response and patient outcome in breast cancer107, its prognostic association in breast cancer is less clear. Multi-regional sequencing analysis of 75 TNBC primary tumours suggested genomic intra-TH of copy number status in several driver oncogenes is correlated with metastasis108. In the context of evolution from primary to metastatic disease, high depth bulk profiling of primary and matched metastatic tumours from the same patient have revealed that most distant metastases acquire genomic driver mutations not seen in the primary tumour, drawing from a wider repertoire of cancer genes than early drivers, including a number of clinically actionable mutations inactivating SWI-SNF and JAK2-STAT3 pathways36. In the context of treatment response, analyses of relatively small cohorts using multi-regional profiling have revealed contrasting results associated with therapy responses, with one study suggesting correlation67 and another study finding lack thereof21 between the extent of genomic intra-TH with chemotherapy response. In a study assessing the mutational repertoire in primary HER2 amplified breast cancers with heterogeneous HER2 protein expression, the clinical impact of intra-TH is perhaps clearer. Heterogeneous HER2 amplification is seen in around 5% of HER2 amplified breast cancers. Through bulk dissection and genomic profiling of HER2-amplified and non-HER2 amplified components of clinically classified HER2-positive breast cancers, a recent study identified that the HER2-negative component of these tumours harboured additional driver mutations, including one case with an activating HER2 mutation, highlighting convergent evolution in this patient. Overall, however, the HER2-negative component would not be predicted to respond to anti-HER2 therapies, this highlights the need for close monitoring of these patients undergoing HER2 targeted therapeutic interventions109. Further assessment of the role of phenotypic heterogeneity driving the prognosis of breast cancers from single-cell epigenomic and transcriptomic studies is starting to reveal that transcriptomic features are seen in minority subpopulations and novel immune subpopulations in primary disease correlates with patient outcome100. There is a major need in the field to conduct large-scale survival analysis with genome-wide indicators of intra-TH to thoroughly address the question of whether primary tumour intra-TH is truly associated with patient outcomes in breast cancer.Whilst genetic intra-TH has been documented to be associated with poor patient outcomes in tumour types such as leukaemia and lung cancer111. The generation and use of PDXs, PDX derived organoids (PDXOs)114, and patient-derived organoids (PDOs)115 from surgical or biopsy patients’ samples have proven to be a very powerful, preclinical in vitro and in vivo tool to assess drug response and resistance, modelling metastasis, partially elucidate tumour microenvironment and for biomarker development as they more accurately represent the patients’ tumours. Importantly, PDXs118 and PDXOs115 have both been shown to preserve much of the intra-TH found in originating patients’ biopsies, making them attractive models with which to investigate intra-TH dynamics in the laboratory. PDXOs and PDOs are currently attractive models with which to readily probe the activities of hundreds of experimental compounds in patient-derived breast tumour cells119. The primary limitation of PDX models is the lack of a fully intact immune system. Thus, future studies implementing pharmacologic and genetic screening technologies in models with ‘humanised’ immune systems will be of great value to the field.The observed intra-TH over the past years in breast cancer has raised the need for the generation and use of relevant preclinical models mirroring the full spectrum of the disease. Although established cancer cell lines have served as a fundamental in vitro model in breast cancer research over the past decades, they do not fully recapitulate all aspects of breast cancer including the natural structure of tumours, altered cell morphology, microenvironment, molecular and genomic resemblance to breast cancer tumours120 for an extensive review). One methodology in achieving this is lineage tracing by using highly complex barcode libraries, enabling the tracking of individual cells or subclones during both the metastatic cascade and through therapy resistance, using next-generation sequencing as a readout121. These technologies label up to millions of cells with unique molecular barcode identifiers that allow transmission during cell division and hence single-cell tracing over time. This approach allows the investigation of the presence of pre-existing resistant clones in the tumour mass, molecular changes that can occur due to therapy and resistance, or therapeutic approaches to be taken into consideration to successfully target these pre-existing resistant clones through single-cell DNA or RNA sequencing122. This method in particular has been used to study clonal dynamics and therapy response in primary TNBC123. In these elegant studies, patient-derived models of primary TNBC were used and cells were barcoded with a complex lentiviral library. Residual tumours after chemotherapy exposure were found to maintain their barcode complexity rather than show a pattern of enrichment, suggestive of cellular plasticity, rather than pre-existing genomic alterations that emerged. OXPHOS was one of the most significantly upregulated pathways in residual tumours and treatment with an inhibitor of the mitochondrial electron transport chain complex I delayed the regrowth of chemotherapy-treated residual tumours in vivo. Interestingly, residual PDX tumours had differentially activated epigenetic regulators including HDAC7, HDAC10 and SIRT7, suggesting that these epigenetic regulators can act as promising candidates for TNBC tumours with residual disease in combination with metabolic inhibitors46. In vivo barcoding in primary untreated TNBC PDX models has also allowed investigation of the subclonal evolution of metastatic spread. These studies harness the utility of the fact that many TNBC PDX’s spontaneously metastasise to distant organs in the absence of selective pressure such as chemotherapy. Using the same barcoding approach, Echeverria and colleagues showed that metastatic spread occurs through a selective bottleneck revealing an enrichment of shared clonal lineages across multiple metastatic sites, indicative of polyclonal seeding. Interestingly, rather than only a few clones leading to seeding at distant sites, the authors also observed extremely rare populations of cells that were able to seed and were maintained at low levels at these metastatic sites78. Similarly, studies using barcoded syngeneic 4T1 mammary tumour derived cells have demonstrated a bottleneck in metastatic seeding from the primary tumour in CTC’s analysed in the blood and cells at distant sites. Interestingly in this model, these lineages were largely non-overlapping suggestive of independent subclones colonising distant sites and showed tropism for entering the lymphatic or vasculature system. Vascular tropism in particular was correlated with increased expression of Serpine2 and Slpi, which were necessary and sufficient to programme these cells for vascular mimicry124. In addition, molecular barcoding has been used coupled with CRISPR technologies to trace a cell phenotype immediately after the introduction of DNA mutations in cancer126.Many scientists now use PDXs and PDOs as preclinical models to address intra-TH and evolution in cancer  cooperate to promote metastatic progression and generate polyclonal metastases composed of a driver and neutral subclones. This cooperation was indirect and mediated through a specific neutrophil population that was stimulated by the IL11-expressing minor subclone130. Together, these studies demonstrate that non-cell-autonomous cooperative interactions exist between different cellular populations within tumours and highlight potential intervention strategies to circumvent these. In the context of NAC resistance in TNBC in particular, subclonal cooperativity may explain the reason for the lack of recurrent molecular alterations identified in NAC-resistant tumours.Given the polyclonal nature of metastatic spread and the fact CTC clusters have been shown to have a substantial increase in metastatic seedingTaken together, these studies highlight the polyclonal and complex nature of metastatic spread in breast cancer. Further assessment of these in humanised patient-derived models to encompass competent immune systems will be necessary to further elucidate mechanisms of metastatic spread and resistance to particular therapies. One can envision a strategy whereby this could be intersected with an extensive characterisation of clinical tumour sampling at single-cell resolution in matched models to fully assess the intra-TH at a genetic and genomic level in an individual patient. Only then, we will be in a better place to tackle the complexity of intra-TH and evolution in cancer Fig. .Fig. 2MoIntra-TH remains a challenge in terms of appropriate cancer therapeutic strategies. Current biomarker analysis does not fully represent the complexity of TH; the existence of rare subclones or accurately predicting treatment outcomes of cancer patients. Deconvoluting the genomic, epigenomic and functional heterogeneity in breast cancer can improve our understanding of the drivers of the disease, mechanisms of resistance and metastasis, identify more accurate biomarkers and novel therapeutic strategies. The use of advanced methodologies such as molecular barcoding, single-cell RNA profiling together with mathematic modelling have proven to be of high importance in terms of addressing these issues. However, further efforts are necessary in order to comprehensively determine and control intra-TH in breast cancer. Furthermore, the development of novel cfDNA molecular tracking such as the use of ‘molecular epigenetic clocks’, can provide unbiased tools for longitudinal patient monitoring in the clinic.The continuous transcriptional and epigenetic contribution during cancer progression or upon selective pressures have implications on the gene expression of tumour cells, making the tumour more heterogeneous and thus difficult to treat, leading to drug resistance and metastasis. Although the development of advanced methodologies such as single-cell RNA sequencing and single-cell tracing molecular barcoding methodologies in relevant preclinical models have significantly improved our understanding of TH and evolution, further studies in immune-competent models are necessary in order to map the genomic and epigenetic landscape of these rare drug-resistant clones. Moreover, additional studies are needed to fully appreciate the potential inter-clonal cooperativity mediated by both non-tumour cells and between different tumour subclones. This will then allow insights into the identification of novel drugs that can disrupt these interactions and shift these clones from a drug-resistant state to a drug-responsive state. Additionally, the transcriptomic profiles from the studies described above can be applied as tools to predict which patients will respond to specific therapies, provide prognostic signatures for patients’ outcomes besides the histopathological techniques already in use, as well as promising candidates for new therapeutic strategies in breast cancer. These could be used as novel combinatorial approaches together or using intermittent scheduling approaches with existing therapies including immuno-modulatory agents targeting the tumour microenvironment.


Liver regeneration is a key compensatory process in response to liver injury serving to contain damages and to rescue liver functions. Hepatocytes, having temporarily exited the cell cycle after embryogenesis, resume proliferation to regenerate the injured liver parenchyma. In the present study we investigated the transcriptional regulation of choline kinase alpha (Chka) in hepatocytes in the context of liver regeneration. We report that Chka expression was significantly up-regulated in the regenerating livers in the partial hepatectomy (PHx) model and the acetaminophen (APAP) injection model. In addition, treatment with hepatocyte growth factor (HGF), a strong pro-proliferative cue, stimulated Chka expression in primary hepatocytes. Chka depletion attenuated HGF-induced proliferation of hepatocytes as evidenced by quantitative PCR and Western blotting measurements of pro-proliferative genes as well as EdU incorporation into replicating DNA. Of interest, deletion of Brahma-related gene 1 (Brg1), a chromatin remodeling protein, attenuated Chka induction in the regenerating livers in mice and in cultured hepatocytes. Further analysis revealed that Brg1 interacted with hypoxia-inducible factor 1 alpha (HIF-1α) to directly bind to the Chka promoter and activate Chka transcription. Finally, examination of human acute liver failure (ALF) specimens identified a positive correlation between Chka expression and Brg1 expression. In conclusion, our data suggest that Brg1-dependent trans-activation of Chka expression may contribute to liver regeneration. Acute or chronic loss of liver function following a wide range of injuries disrupts internal homeostasis and leads to liver failure with life-threatening consequences . Liver rCholine kinases catalyze the biosynthesis of phosphatidylcholine, a key component of cellular membranes . Two isoBrahma-related gene 1 (Brg1) is a chromatin remodeling protein playing diverse roles in orchestrating both physiological and pathophysiological events in the liver. Previously we have reported that Brg1 deficiency attenuates diet-induced non-alcoholic steatohepatitis, concanavalin A-induced fulminant hepatitis, and lipopolysaccharide-induced acute liver injury in mice by regulating the transcription of pro-lipogenic genes, chemokine nephronectin, and pattern recognition receptor TLR4, respectively . On the LKO) mice have been described previously  with freshly added protease inhibitor (Roche) as previously described . WesternChka, GGGTGGTCTCAGTAACATGCT and GAACCCTGGACTCACCATCTT; Ccna2, TGGATGGC AGTTTTGAATCACC and CCCTAAGGTACGTGTGAATGTC; Ccnd1, GCGTACCCTGACACCAATCTC and ACTTGA AGTAAGATACGGAGGGC; Cdk1, CTTTGTCCGAGAGTTT CAGCC and AGTTCCGGGTAGACATCTTTGT; and Ccnb1, CAATTATCGGAAGTGTCGGATCA and CTGGTGAACGACT GAACTCCC. Ct values of target genes were normalized to the Ct values of housekeeping control gene  using the ΔΔCt method and expressed as relative mRNA expression levels compared to the control group which is arbitrarily set as 1.RNA was extracted with the RNeasy RNA isolation kit (Qiagen). Reverse transcriptase reactions were performed using a SuperScript First-strand Synthesis System (Invitrogen) as previously described . Real-tiChromatin immunoprecipitation (ChIP) assays were performed essentially as described before . In brieHistological analyses were performed essentially as described before . ParaffiLiver biopsies were collected from patients with acute liver failure (ALF) referring to Nanjing Drum Tower Hospital . Written informed consent was obtained from subjects or families of liver donors. All procedures that involved human samples were approved by the Ethics Committee of the Nanjing Drum Tower Hospital and adhered to the principles outlined in the Declaration of Helsinki. Paraffin sections were stained with indicated antibodies.post hoc Scheffé analyses were performed by SPSS software . Unless otherwise specified, values of p < 0.05 were considered statistically significant.One-way ANOVA with Ccnd1 encoding cyclin D1 and Ccna2 encoding cyclin A2). Western blotting , started to go up as early as 12 h after the surgery; Chka expression peaked at 24 h and gradually declined until it reached basal levels at 72 h . These cblotting  and immublotting  confirmenjection ; Chka exnjection . In a cenjection . Taken tTo provide support for a role of Chka in hepatocyte proliferation, endogenous Chka was depleted using two separate pairs of siRNAs, which potently and comparably down-regulated Chka expression in primary murine hepatocytes . Chka knWe also measured the direct effect of Chka knockdown on hepatocyte proliferation by EdU incorporation. Immunofluorescence staining showed that Chka depletion markedly decreased HGF-induced EdU incorporation in primary hepatocytes indicative of suppression of proliferation .LKO), there was a significant decrease in Chka expression in the Brg1LKO livers following PHx compared to the WT livers  positive correlation between Chka expression and Brg1 expression in the human livers . Because liver regeneration typically is initiated no earlier than 12 h after CCl4 injection in mice . In the present study, we focused on the transcriptional regulation of Chka in hepatocytes and its implication in the context of liver regeneration. It remains to be determined, however, whether the ability of Chka to contribute to hepatocyte proliferation  is stricin vivo evidence showing the causal relationship between Chka and liver regeneration, (2) the underlying mechanism whereby Chka contributes to hepatocyte proliferation, and (3) the relatively small sample size of human ALF specimens. Additional investigation, harnessing new transgenic mouse models and transcriptomic analyses, is needed to solidify the role of the Brg1-Chka axis in liver regeneration so that novel therapeutics based on this finding, through screening for small-molecule compound boosting Chka activity/expression, could be devised to treat liver failure.In summary, our data unveil Chka as a novel transcription target for Brg1 and implicate Chka as a potential regulator of hepatocyte proliferation . Major lSupplementary Material, further inquiries can be directed to the corresponding author/s.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Nanjing Medical University Ethics Committee on Humane Treatment of Lab Animals.QY, YW, and YX conceived the project. MK, WD, HX, ZF, XM, YG, and CL designed the experiments, performed the experiments, collected data, and analyzed data. YX wrote the manuscript. QY and YW provided funding and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Electrical source imaging (ESI) and especially hdEEG represent a noninvasive, low cost and accurate method of localizing epileptic zone (EZ). Such capability can greatly increase seizure freedom rate in surgically treated drug resistant epilepsy cases. Furthermore, ESI might be important in intracranial record planning.We report the case of a 15 years old boy suffering from drug resistant epilepsy with a previous history of DNET removal. The patient suffered from heterogeneous seizure semiology characterized by anesthesia and loss of tone in the left arm, twisting of the jaw to the left and dysarthria accompanied by daze; lightheadedness sometimes associated with headache and dizziness and at a relatively short time distance negative myoclonus involving the left hand. Clinical evidence poorly match scalp and video EEG monitoring thus requiring hdEEG recording followed by SEEG to define surgical target. Surgery was also guided by ECoG and obtained seizure freedom.ESI offers an excellent estimate of EZ, being hdEEG and intracranial recordings especially important in defining it. We analyzed our results together with the data from the literature showing how in children hdEEG might be even more crucial than in adults due to the heterogeneity in seizures phenomenology. The complexity of each case and the technical difficulties in dealing with children, stress even more the importance of a noninvasive tool for diagnosis. In fact, hdEEG not only guided in the presented case SEEG planning but may also in the future offer the possibility to replace it. Epilepsy surgery represents a valid treatment in drug-resistant epilepsy  showing Analyzing the pertinent literature, the authors found how the increasing interest in hdEEG as ESI enhancer seems to be limited to adult population. Interestingly, few papers address specifically pediatric population even though it is there where it could have the highest value. In fact, children may suffer of more complex forms of epilepsy and in them its prompt resolution can greatly modify short and long-term prognosis.The patient is a 15-year-old boy whose clinical history started at 5 years old. Seizures at onset were (1) imbalance and cold sensation and (2) limbs and face paresthesia followed by loss of consciousness and limbs stiffening. MRI scan showed a right parietal dysembryoplastic neuroepithelial tumor (DNET). After surgical removal, the boy was seizure free for 2 years. At 7 years old, focal seizures characterized by behavioral arrest, mental confusion, left upper limb paresthesia, and distorted voice sensations, followed by lower limb stiffening and weakness or imbalance appeared. Long-term video EEG monitoring showed background rhythm asymmetry and right centro-parieto-temporal interictal paroxysmal abnormalities. Ictal EEG highlighted large slow right parietal and temporal waves preceded by rapid activity. Seizures control lacked despite poly-antiepileptic drugs (AEDs) with valproate acid, oxcarbazepine, and clobazam. Control MRI excluded tumor regrowth showing only scar tissue on the superficial and deep-middle inferior parietal region extending to the marginal and angular gyrus, to the posterior insula, and to the inferior parietal gyrus behind rolandic cortex. Thus, the child underwent a second surgery, aided by electrocorticography (ECoG) to remove the altered sulci in the postero-medial portion of the previous surgical cavity as well as the right superior temporal gyrus. Seizure freedom was achieved for 3 years.Nonetheless, seizure relapsed after 2 years becoming heterogeneous and multidrug-resistant. The boy experienced anesthesia and loss of tone in the left arm, twisting of the jaw to the left and dysarthria accompanied by daze, lightheadedness sometimes associated with headache and dizziness, and negative myoclonus involving the left hand. Therefore, a new pre-surgical epileptic work up was proposed.The registration was made through 21 copper disc electrodes according to the International System 10-20 . The recording lasted 3 days and included Intermittent Light Stimulation protocol at increasing frequencies (3–50 Hz) and 5 minutes hyperventilation. Data were analyzed using Micromed System View. Interictal EEG was characterized by slow activity and spike and slow wave discharges on right central parietal regions, spreading on the posterior vertex ones. Independent and isolated spikes on right posterior temporal derivations were also observed. Three types of seizures were recorded: focal motor, characterized by discharges of spike-wave on the right parietal regions with subsequent bihemispheric diffusion; focal non-motor, characterized by delta-like slow activity; and spike-wave on the right centro-parietal regions with right hemispheric diffusion and negative myoclonus, characterized by slow wave discharges on the right parietal and frontal regions with diffusion on the contra lateral homologous regions.Cognitive assessment was carried out using the Wechsler Intelligence Scale for Children  which highlighted a normal level (IQ 88), characterized by a disharmonic profile, in the presence of a significant difference (> 12 points) between the indices, to the detriment of the score obtained in working memory (73) and processing speed (82). Visuo-spatial memory was quite impaired . Verbal memory tests showed normal performance in short-term recall and slight difficulty in the long-term one. Furthermore, there was a marked difficulty in lexical retrieval with a phonological facilitator and slight difficulty with a semantic facilitator. Tests performed with Developmental Neuropsychological Assessment  showed a marked deficit in sustained attention and a slight difficulty in visual-motor integration.TM 3D Electrode Digitizer) . The recording lasted about 2 h including wake and sleep. No seizures were recorded. Offline, EEG was analyzed through the Micromed SystemView; band pass filtered between 0.1 and 100 Hz and any paroxysmal anomalies was noted. A head model was built using T1-weighted MRI and scalp electrodes were co-registered with the MRI using a template net of electrodes with standard position that was translated/rotated/dilated with personalized digitalization through scalp navigation system  and 3 platinum electrodes; and 10 contacts with 5 mm interelectrode spacing, with a total recording surface of 47mm (Ad-Tech LTM—Spencer probe depth electrodes). The Medtronic Stealth Station™ S8 was used to carry out the pre-operative planning using MR and CT angiography with sequences for neuronavigation  pars opercularis, S (10) superior parietal lobule, Ps (8) parietal, Pi (10) inferior parietal lobule, V (8) posterior portion of superior parietal gyrus, T (8) median temporal gyrus, and I (10) insula Fig. . From prAfter multidisciplinary discussion a surgical approach was proposed to remove the presumed EZ, including pericavitary area, frontal operculum, superior insular, and mesial parietal regions. In general, anesthesia  reopening of the right temporo-parietal incision and craniotomy was performed. After dural opening, control of the epileptogenic areas identified with SEEG was carried out under magnetic, ultrasound and ECoG navigation. These analyses confirmed the pericavitary area as epileptogenic thus proceeding to its circumferential removal for 1 cm, widening the resection at the level of the posterior parietal area towards the midline. Frontal operculum was identified and removed too under ECoG guidance as well as somatosensory evoked potential and motor evoked potential monitoring. At the end of surgery, a satisfactory ECoG silencing was obtained.Post-operative course was uneventful except for a mild left hemiparesis which gradually recovered during hospitalization. MRI scan showed the extent of resection without complications Fig. .Fig. 5PoSurgical resection or complete disconnection of the EZ  is the objective in patients suffering from drug-resistant focal epilepsy . SeveralWhile MRI scan can easily demonstrate epileptogenic lesions, irritative zone (IZ) and seizure onset zone (SOZ) recognition can be more challenging.Still now, invasive recording remains the gold standard in SOZ definition . In factAlthough these techniques have been increasingly validated, they might fail. Focusing only on the most frequent interictal discharges (IEDs), it is possible to miss SOZ especially whenever it is not the most active IED generator or when IED are multiple. Therefore, recordings and source localization of actual seizures might be very useful especially when interictal ESI is ambiguous or not possible . DirecteFocusing hdEEG on pediatric patients, a possible issue for clinical use regards superficial electrode number. Papers from the literature state how to reach an adequate level of accuracy and sensitivity for hdEEG in adults, at least 128–256 electrode have to be used , 14. In Further problems regarding ESI in children are related to multifocal and diffuse IEDs patterns even in cases of focal epilepsy with a delimited EZ. Analyzing the literature, it seems that in children non-epileptiform abnormalities may be accurate in localizing abnormal cortical function than spiking activity . SimilarIn order to evaluate these problematic, the authors presented the case previously described hoping to underline potential roles for hdEEG in future prospective. In fact, the heterogeneous seizures phenomenology of the child and the lack of video EEG localization failed to give sure targets for surgery. hdEEG and interictal recording analysis, together with clinical data, suggested that the pericavitary area as well as right frontal lobe were involved in seizures onset. These findings also helped to direct the positioning of the SEEG electrodes confirming the presence of epileptic discharges coming from the frontal operculum as well as from the more intuitive parietal EZ. Coming finally to surgical resection, merging hdEEG, SEEG, and ECoG was possible to obtain electrical silence after removal of the defined EZ. The role of hdEEG in our patient’s pre-surgical workup was in agreement with Foged et al. that observed ESI through hdEEG had additional value in one third of the patients analyzed . MégevanOne of the most important debates about ESI is whether hdEEG could identify deep sources. In fact, in a large study that included 152 patients [Finally, analyzing the aforementioned data it was possible to see how hdEEG and ECoG offered a superficial and depth covering of the epileptic discharges overlapping SEEG one. This approach that integrates the information obtained during the pre-surgical workup with the ECoG seems unprecedented as no studies are published in the medical literature.ESI represent a relevant tool in epilepsy surgery workup in order to define EZ together with clinical data, MRI, neuropsychological assessment, and video EEG. ESI via hdEEG increases the localizing power of surface electroencephalographic recordings especially when coupled with intraoperative ECoG, granting a more easily identification of deep sources. This combination might even in the future grant the possibility to further reduce the need for SEEG in selected cases, which is of utmost importance in the pediatric setting and in particular in very young patients.


Cardiac amyloidosis is a rare underdiagnosed condition with increasing morbidity and mortality. Its diagnosis is challenging and requires high clinical suspicion. Several diagnostic tools aid in the diagnosis of cardiac amyloidosis such as electrocardiogram, echocardiography, and, most importantly, cardiac MRI. A wide range of clinical symptoms is associated with cardiac amyloidosis, with shortness of breath and peripheral edema being the most common presenting complaints. Here, we report a case of transthyretin cardiac amyloidosis and discuss the importance of imaging in establishing the diagnosis. Cardiac amyloidosis (CA) is a rare underdiagnosed condition with increasing morbidity and mortality. Transthyretin (ATTR) amyloidosis is currently considered the most frequent form of CA and its incidence is increasing thanks to the advances in diagnostic imaging techniques . ATTR-caA 65-year-old white male with a previous history of hypertension was referred to the Heartland Clinic for evaluation of newly diagnosed pulmonary hypertension. He reported shortness of breath with exertion for one month in addition to leg swelling. His symptoms were so severe that he could no longer work. The patient denied palpitations, dizziness, or syncope. Family history was negative for cardiac disease including cardiomyopathy. A 2D echocardiogram showed markedly increased left ventricular wall thickness without left ventricular outflow tract obstruction, mild mitral regurgitation, mild left atrial dilatation, and mildly reduced left ventricular systolic dysfunction with an estimated ejection fraction of 45-50%.On initial physical examination, blood pressure was 110/70 mmHg with heart rate (HR) of 77 beats per minute (bpm). There was jugular venous distention of 8 cm. Cardiac auscultation showed 2/6 left upper sternal murmur, normal S1S2, and regular rhythm. Lung auscultation was symmetrical, clear bilaterally. There was 2+ bilateral lower extremity edema with intact pulses. Electrocardiogram (ECG) showed normal sinus rhythm with left ventricular hypertrophy.Due to ongoing symptoms and high clinical suspicion of cardiac amyloidosis, contrast-enhanced cardiac MRI Figure  was perfNuclear imaging of 99mTechnetium pyrophosphate (99mTc-PYP) planar scintigraphy was ordered. It showed intense radiotracer uptake by the heart that was greater than normal bone uptake  and a quantitative heart to the contralateral ratio of 1.9 (>1.5 considered positive); both strongly suggestive of ATTR amyloidosis Figure . Serum aA right heart catheterization revealed severe pulmonary hypertension with mean pulmonary artery pressure of 40 mmHg, pulmonary capillary wedge pressure (PCWP) of 26 mmHg, and pulmonary vascular resistance (PVR) of 1.85 suggestive of elevated left-sided filling pressures and cardiac etiology of pulmonary hypertension (post-capillary). Subsequently, a right ventricle biopsy was performed, and six specimens were taken from the endomyocardium. Microscopic examination showed perimyocytic and nodular deposits of eosinophilic amorphous material in several of the fragments. This material stained positive for amyloid on thioflavin S stain examined under fluorescence microscopy. Immunohistochemical staining was performed for amyloid typing. ATTR immunostaining was positive in the amyloid deposits. Lambda and kappa light chains were negative. The histologic and immunostaining patterns were consistent with the ATTR type of amyloidosis.The patient was started on tafamidis 61 mg capsule daily. He has regular clinic follow-ups since diagnosis and has noted improved symptoms of shortness of breath and edema.Amyloidosis is a rare multisystemic disease caused by the deposition of misfolded proteins in various organs such as the kidney, liver, heart, and nervous system. Many types of amyloidosis exist, differentiated by the nature of the deposited proteins, however, the light chain amyloidosis (AL) and ATTR amyloidosis proteins represent the majority of CA .Most commonly, electrocardiography in CA shows left ventricular hypertrophy (LVH). Unexplained left LVH, characterized by low QRS voltage on electrocardiogram despite LVH on echocardiogram, provides valuable clues for the suspicion of CA. However, these electrocardiographic features are actually not sensitive enough to identify CA -8. Less Echocardiography is the modality of the first approach in the diagnosis of CA due to easy accessibility. The amyloid phenotype is typically characterized by biventricular wall thickening with small, non-dilated ventricles and left ventricular (LV) wall thickness, typically greater than 12 mm . HoweverCardiovascular magnetic resonance (CMR) has established a significant role in diagnosing CA when suspected. CMR is especially useful in patients with increased left ventricular wall thickness and/or hypertrophy because it can differentiate different cellular and interstitial pathologies, which is not possible on echocardiography . In CA, Nuclear imaging is a sensitive tool for the identification of the TTR subtype of CA, and it is encouraging in distinguishing between it and the AL subtype which can also involve the heart. There are many radiotracers, and the most common single-photon emission CT tracer is a 99m Technetium-phosphate derivative. The exact mechanism behind the effectiveness of this imaging modality remains somewhat elusive, though there are a few plausible theories. Of these, it suggests that phosphate in the radiotracers binds to high calcium levels in amyloidosis. Another hypothesis is based on the duration of amyloid deposition that occurs in less time frame in patients with AL relative to a more indolent course of patients with ATTR. As 99mTc-PYP preferentially binds to ATTR relative to AL fibrils, this technique can also be used to distinguish the aforementioned amyloidosis subtypes .This case demonstrates the advanced utility of current imaging modalities to better diagnose CA so that treatment options can be introduced at an earlier stage. In the future, these advanced imaging modalities may assist with avoiding invasive procedures such as endomyocardial biopsy which is considered the gold standard. In the end, the early detection of cardiac amyloidosis is critical since treatment options are currently available with the goal of improving the quality of life and survival in affected patients. Current research has shown promise that the aforementioned imaging modalities may also help assess response to therapy.


Plasmodium parasites, restricting disease to merely uncomplicated cases or asymptomatic infections. Does this acquired malaria immunity in endemic areas protect against other diseases, especially outbreak diseases like COVID-19? Does malaria activation of innate immunity resulting in trained or tolerance immunity contribute to protection against COVID-19? In an attempt to answer these questions, this review highlights the components of malaria and viral immunity and explores possible links with immunity against COVID-19. With malaria-endemic areas of the world having a fair share of cases of COVID-19, it is important to direct research in this area to evaluate and harness any benefits of acquired malaria immunity to help mitigate the effects of COVID-19 and any possible future outbreaks.Malaria-endemic areas of the world are noted for high morbidity and mortality from malaria. Also noted in these areas is the majority of persons in the population having acquired malaria immunity. Though this acquired malaria immunity does not prevent infection, it resists the multiplication of None declared Plasmodium parasite, is an endemic disease found in most tropical countries, where environmental and socioeconomic factors foster its perennial persistence.1Malaria, caused by the Plasmodium parasite.Despite the high burden of malaria in these endemic areas, there is some level of malaria immunity among most of the population, acquired through a precarious, repeated lifelong encounter with the COVID-19 is a viral disease caused by a novel type of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).6Plasmodium and viral coinfections, highlighting a possible relationship between acquired malaria immunity and coronavirus immunity.There are several schools of thought for the relatively lower number of cases recorded in Africa, including inadequate testing of suspected cases and the prevailing warmer climate conditions.Plasmodium parasite acquired through the repeated activities involving the clearance and restriction of the multiplication of the parasite.9Malaria immunity is a complex phenomenon, an evolving topic yet to be fully understood.In holoendemic malarious areas, people are repeatedly exposed to malaria infection from childhood and well into adulthood, with children manifesting more disease symptoms than adults.9Asymptomatic infection is the predominant presentation seen in patients with malaria immunity and is believed to confer some form of immunity through premunition.12h1 and Th2, resulting in cell-mediated and antibody-mediated immunities, respectively.h2 CD4 cells activate B cells to produce antibodies, especially immunoglobin G, which play pivotal roles in the immunity against the parasite.h1 promotes cell-mediated immunity by aiding macrophages in the destruction of parasitized RBCs.12The immunity against malaria is quite complex but mainly targets the asexual forms of the parasite employing a timely relay between antibody and cell-mediated immunity.Natural killer cells are activated in malaria infection, a finding seen among infected children in endemic areas.20After the immune cells and their products (cytokines and antibodies) are produced during an immune response to malaria, they can linger for years as memory cells and as circulating cytokines and antibodies if sustained by repeated infections.21Like the immunity against malaria, immunity against viruses begins with innate immunity and chiefly involves dendritic cells, natural killer cells and pro-inflammatory cytokines, especially interferons.32Natural killer cells are the chief immune cells involved in cellular immunity to viruses.37Antibodies are another product of the immune system that is very potent against viruses and efficiently prevent and resolve viral infections.44One of the ways to understand how acquired malaria immunity affects other pathogens like viruses are to understand what happens in coinfections with malaria.47A similar effect was seen in a study on malaria and Epstein-Barr virus coinfection, which established that malaria lowers antiviral immunity in some children resulting in uncontrolled growth of B lymphocytes leading to Burkitt's lymphoma. The Epstein-Barr virus can suppress anti-parasite immunity in some children resulting in noncerebral severe malaria.52Influenza virus, Rhinovirus, human Respiratory Syncytial Viruses and coronaviruses, among others.57A scan of the available literature on malaria and respiratory virus coinfections in malaria endemic areas show that respiratory viruses are common in these areas and include: It is an established fact that malaria is a potent immunomodulator of adaptive immunity62The immune response against malaria involves innate immunity with natural killer cells, monocyte, macrophages, proinflamatory and anti-inflammatory cytokines as the major players.63Mycobacterium tuberculosis.59Cross protection of trained immunity is highly likely because of the non-specific response of innate immunity, evident by BCG vaccination conferring protection against other pathogens aside Malaria activation of innate immunity produces not only trained immunity (hyperresponsiveness) but also tolerance (hyporesponsiveness).66Taken together, while malaria influences adaptive immunity,66Plasmodium as the levels of malaria exposure even in endemic areas is variable.68To answer the question posed by this review, studies need to be conducted among patients with COVID-19 who live in malaria-endemic areas. Acquired malaria immunity must be assessed both in patients with COVID-19 and among healthy individuals. All immune system components characteristically seen in persons with acquired immunity for malaria, like natural killer cells, interferon (interferon gamma and type 1interferon) and immunoglobin G, should be evaluated among symptomatic and asymptomatic patients with COVID-19. The role of malaria-induced trained immunity and immune tolerance in patients with COVID-19 should also be investigated. It is important to note that not all persons living in malaria-endemic areas have acquired malaria immunity.The exposure risk is higher for persons with lower socioeconomic status, especially those living in crowded or riparian communities, compared to those with higher socioeconomic status living in well-organized urban cities.The COVID-19 pandemic presents a great opportunity for scientists, physicians and epidemiologists in malariaendemic areas to investigate the impact of endemic diseases like malaria on COVID -19 and future epidemics.