Breast cancer incidence rates have been continuously increasing in majority nations with significant higher portion of cancer-related mortality in low- and middle-income countries. Developing new biomarker is an emerging field in the breast cancer research. Application of a promising minimally invasive biomarker, circulating microRNA, for additional improvement of diagnosis, prognosis, and therapeutic monitoring in breast cancer is not well corroborated. miR-155 expression as a clinical biomarker in breast cancer, we analyzed 102 breast cancer patients at diagnosis and after treatment as well as 15 healthy women. Total RNA was isolated from patient’s plasma and expression of circulating miR-155 was measured with quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression levels of circulating miR-155 were compared according to the effect of treatment, clinicopathological variables, and progression-free survival. To uncover the potential use of circulating miR-155 were significantly diminished after patients completed surgery and chemotherapy . Patients older than 40 years old expressed higher circulating miR-155 than those younger than 40 years-old . Circulating miR-155 was significantly higher in patients with tumors larger than 5 cm . MiR-155 expression levels were not significantly different according to various tumor grades, subtypes, and clinical stages. Although longer follow-up is required, progression-free survivals of patients with upregulation of circulating miR-155 were significantly longer  test p=0.038). In comparison to the healthy women, expression of circulating miR-155 levels were significantly higher . The expression levels of miR-155 expression was significantly elevated in breast cancer patients and was decreased after treatment. Therefore, circulating miR-155 is potentially applicable as diagnostic therapeutic monitoring marker in breast cancer.Expression of circulating  The incidence rates of breast cancer have been increasing almost in all countries to reach more than 2 million cases in 2018 . In addition, breast cancer has emerged as the most frequent cause of cancer-associated mortality in developing countries and the second after lung cancer in more developed countries . The implementation of national screening program in developed countries has shifted the diagnosis into more early stages . However, breast cancer in developing countries including in Indonesia is mostly still diagnosed in the late stages . Low cancer awareness and lack of national early detection and screening program  have been associated with late presentation and delayed diagnosis in low- and middle-income counties . In addition, the high proportion of mortality rates is associated with late stages at diagnosis, relapse, and distant metastasis. Given the substantial health burdens of breast cancer, there is an increasing need to develop reliable biomarkers to improve the chance of early detection, to determine patient prognosis, and to predict drug resistance and relapse . The new biomarker is expected to detect breast cancer before the disease spreads beyond the breast or the axillary lymph nodes  and to accurately detect any relapse or metastasis before the emerging of clinical manifestation. Currently, only Cancer Antigen 15-3 (CA 15-3) has been used as circulating marker to predict relapse in the clinical practice although lacking of sensitivity and specificity . To overcome this limitation, new reliable non-invasive biomarker for breast cancer patients is needed.A new class of small noncoding RNAs, known as microRNAs, is a master regulator of gene expression that can target multiple protein encoding genes . MicroRNAs inhibit gene expression post transcriptionally through direct interaction causing mRNA degradation or translational inhibition depending on the cross-matching of the seed sequence and the 5’ or 3’ mRNA targets . Unique patterns of microRNA expression in primary tumor tissue samples have been revealed as a potential biomarker in various cancers including breast cancer . The deregulation of micoRNA in primary cancer tissues results from defect of microRNA biogenesis and transcription factors , epigenetic regulation , genetic amplification/deletion , and interaction of SNPs at the flanking region . Interestingly, microRNAs can be exported from malignant cells into the extracellular fluid and circulating through several modes including micro-vesicles, lipid vesicles, exosomes, and apoptotic bodies . Because RNAs are easily degraded by endogenous RNase, extracellular microRNAs are viably observed forming a protein complex with ribonucleoprotein or high-density lipoproteins . As circulating microRNA reflects biological characteristics of the underlying tumor, there is substantial interest to use it as a non-invasive marker in breast cancer. MiR-155 is an important oncogenic microRNA in human cancers including in breast cancer . Although the upregulation in primary tumor tissues of breast cancer patients has been frequently reported, the deregulation of circulating miR-155 and the application as clinical biomarker have not been completely revealed. In this study, we measured circulating miR-155 in breast cancer patients at diagnosis and after surgery and chemotherapy and correlated with the clinicopathological variables and progression-free survival.Patient cohortPatients diagnosed with breast cancer in 2014-2015 were recruited to participate in this study according to a protocol accepted by the Ethics Commission of the Faculty of Medicine, Universitas Gadjah Mada, Indonesia (Number: 601/EC/2014). In total, periphery blood samples were collected from breast cancer patients at diagnosis (N=102) and at follow-up after completion of surgery and chemotherapy (N=40) as well as healthy controls from females without a history of tumor (N=15). Diagnosis of invasive breast cancer was confirmed with the histopathology and only patients that were older than 18 years-old and were able to provide inform consent were than recruited. Vulnerable patients and participants including those with dementia, terminally ill, and in clinical emergency were excluded. Estimation of sample size was calculated using effect size 0.5 of Cohen’s approach with α=0.05 and statistical power of 0.9 reveling sample size needed of 49 patients. Clinicopathological variables including, tumor size, lymph node infiltration, status of local and distant metastasis, immunohistochemistry, and demographic data were collected at the time of diagnosis. Patients were followed-up according to the hospital recommendation. Progression-free survival (PFS) was calculated as a period between time at diagnosis to the time of any relapse or local/regional/distant tumor spreading was observed (until December 2017). Baseline clinicopathology characteristics of breast patients participated in this study was summarized in the RNA isolation from plasmaoC. The plasma was collected from the supernatant and then stored at -80oC until analysis. Total RNA was extracted from 200 µL plasma of each study participant using miRCURYTM RNA Isolation Kit-Biofluid  following recommendation from the manufacturer. Briefly, plasma was mixed with Lysis and Protein Precipitation Solution and then centrifugated at 12,000 rpm. The RNA was precipitated by mixing the supernatant with 270 µL isopropanol and was then separated using microRNA Mini Spin Column (Exiqon). After 2 washing steps, the RNA was eluted using 25 µL RNA-se free HPLC water. Internal control of RNA isolation was controlled using spike-in of Sp6. After peripheral venous blood was drawn from the study participants and collected at the EDTA-contained tubes, fractionation was performed by centrifugation of 1,500 rpm for 10 minutes at 4Quantification of miR-155 expressionReverse transcription using Universal cDNA Synthesis kit II, 8-64 rxns  was performed to produce cDNA from the RNA derived from plasma. Expression levels of circulating miR-155 were quantified using ExiLent SYBR Green master mix  and specific primers for miR-155 , Sp6 , and MIR-16 . Statistical analysis miR-155 expression levels between breast cancer patients and healthy women as wells baseline and after treatment were compared using the Mann-Whitney-U test. Upregulation was categorized as mean of circulating miR-155 expression + 2xSD. Comparison and analysis of survival was performed using Kaplan-Meier plot and Log-rank (Mantel-Cox) test. All statistical tests were performed with 2-sided and a p-value < 0.05 was considered as statistically significant. Statistical analysis was performed using GraphPad Prism  and SPSS 17.0 software .The expression levels of miR-155 were calculated from Cq values and were normalized with Cq values of spike-in Sp6 and miR-16. Relative expression was then compared with the ΔΔCq in healthy individuals. Fold expression values were calculated using the 2 ΔΔCq method. Comparison of circulatingUpregulation of plasma miR-155 in breast cancer patientsmiR-155 were also not statistically different between early stages (Stadium I-II) and late stages (Stadium III-IV) , Quantification of circulating miR-155 in breast cancer patients (n=102) revealed a significant upregulation in comparison to the healthy women (n=15) , miR-155 in patients older than 40. After treatment, expression levels of circulating miR-155 were not significantly different between patients with tumor size larger and smaller than 5 cm .We quantified circulating Upregulation of circulating miR-155 and the correlation with the progression-free survivalWe determined upregulation of circulating miR-155 expression at diagnosis as expression above the average expression in healthy women plus 2xSD as explained at the Material and Methods. More than 75% of patients (n=76) showed circulating miR-155 upregulation at diagnosis. Patients with circulating miR-155 upregulation have significant longer progression-free survivals . MicroRNAs are shown to be relatively stable against degradation both in primary tissue samples and body fluids, relatively easy to be detected, and the levels in the tissues and body fluid often reflect the tumor burden and the biological as well as clinical characteristics . Circulating microRNA appears in the plasma through several extracellular transfer modes as a result of high biogenesis and degradation in the malignant cells . MiR-155 is frequently upregulated in breast cancer and the higher expression is related with several clinicopathological variables, tumor subtypes, higher tumor grades, and poor survival rates . Iorio et al. have shown that among 29 deregulated microRNAs in breast cancer primary tissues, only miR-155 and miR-21 are upregulated . There are 147 validated miR-155 target genes that have been identified forming interconnecting biological pathways of apoptosis , cell proliferation (SOCS1), cell differentiation , angiogenesis (HIF), and epithelial-mesenchymal transition (TGFβ) . Although deregulation of miR-155 and the relevant target genes in primary breast cancer tissues have been compiled , only few studies have reported expression of circulating miR-155 and the correlation with clinicopathological variables, effects of treatment, and clinical outcomes in breast cancer. We reported upregulation of circulating miR-155 expression in breast cancer patients compared to healthy women. A meta-analysis involving 25 studies with total number of 1,896 breast cancer patients and 1,226 healthy controls showed the diagnostic accuracy of circulating miR-155 . Higher level of plasma miR-155 in early stage breast cancer has also been reported rendering its potential as early detection marker . We confirmed recent report showing upregulation of plasma miR-155 in breast cancer with a larger number of patient cohort . In accordance with our results, Lu et al., (2012) reported elevated plasma levels of miR-155 in breast cancer up to 5-fold times than healthy individuals but were higher in patients with small tumor size (2-5 cm) and lymph node infiltration (N1). Liu et al., (2013) also showed upregulation of serum miR-155 in 20 breast cancer patients compared to 10 healthy controls. Using different setting, Gezer et al., (2014) demonstrated significant changes of circulating miR-155 expression upon neoadjuvant treatment in breast cancer, although their findings were contradictory with ours. In accordance with these previous studies, we showed higher circulating In response to surgery and chemotherapy, we showed significant decreased circulating miR-155 in breast cancer. Circulating miR-155 levels were also significantly decrease after surgery, chemotherapy, and radiotherapy as reported by a study involving 30 Luminal A breast cancer patients and 10 healthy controls . Different from this study , we included larger number of breast cancer patients with different subtypes, although we did not show different expression of circulating miR-155 between Luminal and triple-negative breast cancer subtypes. Further study is required to confirm the potential role of circulating miR-155 as therapeutic monitoring marker in breast cancer including analysis of response into specific chemotherapeutic agent, hormonal therapy, radiotherapy, as well as targeted therapy. With the frequent implementation of neoadjuvant treatment in breast cancer particularly in locally advance stages, the use of circulating miR-155 expression to monitor complete and partial responses to neoadjuvant treatment is also an interesting question in the future. miR-155 in primary breast cancer tissue has been associated with the prognosis, limited study has reported the correlation of circulating miR-155 with clinical outcomes . In contrast to our previous study using circulating miR-21 , our current study showed that upregulation of circulating miR-155 was associated with better PFS. Although miR-155 is commonly associated as oncomir, our study showed the upregulation with better PFS. In addition, we showed that significant reduction of circulating miR-155 in patients older than 40 years old after surgery and chemotherapy. Potential future research question is whether breast cancer patients with upregulation of circulating miR-155 might response better to the standard treatment. Larger study might be required to explore the potential use of circulating miR-155 as a prognostic marker in breast cancer with particular analysis depending on the subtypes, specific molecular characteristics, and tumor heterogeneity. Although breast cancer in younger women has been associated with more biologically aggressive behavior , we showed higher expression of circulating miR-155 in patients older than 40 years old. Expression of miR-155 interacts with hormonal receptor status and metabolic reprogramming in breast cancer . However, the direct correlation with age at diagnosis was not previously explained. Mutations at BRCA1 genes have been associated with younger breast cancer cases and Chang et al., (2011) have shown that particular variations of BRCA1 is able to repress miR-155 expression through modulation of histone acetylation. Although expression of  miR-155 expression after treatment, direct comparison with current biomarker such as CA-15-3, and the correlation with progression-free and overall survival is required. The major constraint of this study is high degree variability of circulating miR-155 expression that might reflect the diversity of clinical stages, tumor subtypes and biological characteristics of breast cancer patients at diagnosis.The strengths of this study are relatively larger cohort than previous report , evaluation of the potential marker after treatment, and thorough analysis in correlation with tumor characteristics and clinical outcome . Upcoming study evaluating the magnitude decreased circulating


A recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine has been reported as safe, immunogenic, and highly protective in a ring vaccination trial. We aimed to identify transcriptomic immune response biomarker signatures induced by vaccination and associated signatures with its immunogenicity and reactogenicity to better understand the potential mechanisms of action of the vaccine.5 to 1 × 108 plaque-forming units [pfu]). Longitudinal transcriptomic responses  were measured in whole blood using a targeted gene expression profiling platform  focusing on 144 immune-related genes. The effect of time and dose on transcriptomic response was also assessed. Logistic regression with lasso regularisation was applied to identify host signatures with optimal discriminatory capability of vaccination at day 1 or day 7 versus baseline, whereas random-effects models and recursive feature elimination combined with regularised logistic regression were used to associate signatures with immunogenicity and reactogenicity.354 healthy adult volunteers were vaccinated in randomised, double-blind, placebo-controlled trials in Europe  and North America , and dose-escalation trials in Africa  using different doses of the recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein  of rVSVΔG-ZEBOV-GP exhibiting the largest amplitude. The most differentially expressed genes that were significantly upregulated following vaccination consisted of type I and II interferon-related genes and myeloid cell-associated markers, whereas T cell, natural killer cell, and cytotoxicity-associated genes were downregulated. A gene signature associated with immunogenicity  was identified correlating gene expression profiles with ZEBOV-GP antibody titres and a gene signatures associated with reactogenicity (Geneva cohort) was identified correlating gene expression profiles with an adverse event .Our results indicated that perturbation of gene expression peaked on day 1 and returned to baseline levels between day 7 and day 28. The magnitude of the response was dose-dependent, with vaccinees receiving a high dose (≥9 × 10Collectively, our results identify and cross-validate immune-related transcriptomic signatures induced by rVSVΔG-ZEBOV-GP vaccination in four cohorts of adult participants from different genetic and geographical backgrounds. These signatures will aid in the rational development, testing, and evaluation of novel vaccines and will allow evaluation of the effect of host factors such as age, co-infection, and comorbidity on responses to vaccines.Innovative Medicines Initiative 2 Joint Undertaking. Ebola virus disease is characterised by a high fatality rate  and can result in uncontrolled epidemics. The severe 2014–16 Ebola virus disease outbreak in west Africa registered more than 11 300 deaths among 30 000 suspected, probable, and confirmed cases. Moreover, the recent epidemic in the Democratic Republic of Congo, with an approximate 65% fatality rate among more than 3000 confirmed Ebola virus disease cases, confirms the urgency for protective vaccines to prevent disease spread.2 Challenge studies in animal models showed high efficacy and specificity of the rVSVΔG-ZEBOV-GP vaccine against Ebola virus,7 and numerous clinical trials (phase 1–3) in North America, Europe, and Africa,8 provided further evidence that the vaccine is safe, efficacious, and fast-acting after a single injection. Moreover, a phase 3 ring vaccination trial of rVSVΔG-ZEBOV-GP was successfully completed in Guinea during an outbreak in 2015, confirming vaccine efficacy after a single injection.8 More recently, in 2018–19 in the Democratic Republic of the Congo, over 250 000 people were vaccinated in a ring vaccination approach using rVSVΔG-ZEBOV-GP in an expanded access  protocol. Preliminary data corroborated earlier findings from the ring vaccination trial in Guinea that rVSVΔG-ZEBOV-GP confers rapid and robust protection, reinforcing its suitability in outbreak situations.9 As a result, the procedure for licensing the vaccine was markedly accelerated leading to its licensure by the European Medicines Agency  and the US Food and Drug Administration  as well as prequalification by WHO .12The recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔGZEBOV-GP) is a live-attenuated vaccine in which the vesicular stomatitis virus glycoprotein encoding gene has been replaced with the Zaire Ebola virus glycoprotein (ZEBOV-GP).13 An innate (non-specific) response-associated signature was identified in a European cohort  and validated in an African cohort .14 This signature was vaccine dose-dependent and correlated with viraemia and adverse events, including arthritis. Additionally, the analysis of rVSVΔG-ZEBOV-GP-specific adaptive cellular and humoral responses revealed strong B-cell responses together with potent Ebola virus-neutralising antibodies15 whereas the overall T-cell responses were weak to moderate.16 To further unravel the mode of action of the rVSVΔG-ZEBOV-GP vaccine, we investigated at the transcriptional level the longitudinal and dose-dependent kinetics of the innate and adaptive immune responses following vaccination of healthy volunteers in Europe, North America, and Africa.Although the rVSVΔG-ZEBOV-GP vaccine is highly effective against Ebola virus, only a few studies have explored its underlying immune mechanisms and its ability to induce long-term protection.We did a pre-planned transcriptomic analysis on blood samples collected during four clinical trials testing the safety and immunogenicity of the rVSVΔG-ZEBOV-GP vaccine. The trials were conducted in Europe , North America , Africa . Details of the study design and participant groups have been summarised in the 5 − 1 × 10⁸ plaque-forming units [pfu]; 17 comprising four housekeeping genes and 144 selected immune-related genes  from all participants .Ingenuity pathway analysis  was used to explore interactive networks between the DEGs of each cohort. To evaluate the effect of time and dose on the gene expression perturbation, molecular degree of perturbation (R package mdp) and principal component analysis (function prcomp from R package stats) were used. Signatures with the best discriminatory capability of vaccination at day 1 or day 7 versus baseline were identified using logistic regression with lasso regularisation. Leave-one-out cross-validation and train-test split were used to assess the performance of the trained regression models. When pooling cohorts, a random down-sampling approach was applied (if required) to obtain a balanced distribution of the number of individuals within the pooled cohort. To identify immunogenicity-associated signatures, gene expression data were correlated with ZEBOV-GP-specific IgG antibody titres using Spearman correlation in each cohort. Significant correlations obtained in single cohorts (p<0·05) were subsequently integrated using the random-effects models for meta-analyses (R package metaphor). Arthritis was selected for correlation with reactogenicity because of its late onset (day 10–14 post-injection) and prolonged duration (>1 week), compared with other adverse events . Moreover, our analysis was limited to the Geneva cohort, because the low frequency of individuals developing arthritis in the other cohorts precluded comparable analyses . SamplesThe funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report.20 We first evaluated host gene expression kinetics in the Geneva (Switzerland) cohort  did not exhibit a complete separation until day 3 compared with their own baseline, suggesting slower kinetics in low-dose vaccinees.We investigated the longitudinal transcriptomic profiles in response to rVSVΔG-ZEBOV-GP vaccination in four cohorts from different genetic and geographical backgrounds  cohort .18 Princt day 28 . Althougt day 28 . Moreovet day 28 , whereasCXCL10), pattern-recognition receptors , and the myeloid subset marker CD163 were also significantly upregulated. By contrast, the downregulated DEGs included primarily T-cell markers  and the cytotoxicity marker GNLY, whereas other T-cells markers , cytotoxicity markers , and natural killer (NK)-cell markers  were downregulated without reaching statistical significance  of which CCL3 remained differentially expressed until day 28 , several ISGs displayed slow kinetics . Interestingly, together with a lower response magnitude, several genes in low-dose vaccinees displayed slower expression kinetics than in the high-dose-2 vaccinees, with ISGs preferably displaying a peak response at day 3 instead of day 1 ; gene expression perturbation was transient and gradually returned to baseline levels at day 28; and the magnitude of the vaccine-response was dose-dependent . Similarificance . The DEGl day 28 . Detaile20 Similarly, in the USA cohort, both molecular degree of perturbation and principal component analysis analyses identified the transcriptomic perturbation peak at day 1 after vaccination and a dose-dependent response magnitude, evidenced by less gene perturbation with intermediate-dose (3 × 106 pfu) than with high dose 1 (9 × 106 pfu), high dose 2 , and high dose 3 ; most DEGs were transiently regulated and gradually return to baseline levels over the next 7 days; and the magnitude of the response was dose-dependent  and fast-kinetics genes . Differently to the Geneva cohort, GBP5 showed a slow kinetics , Th1-associated genes , and Th2-associated genes  were detected in the Lambaréné cohort (CCL2 and IFIT5) for the Geneva cohort and five genes for the USA cohort  and seven genes in the USA cohort . Biomarker signatures at day 7 post-vaccination with the highest discriminatory power encompassed eight genes in the Geneva cohort  exhibited excellent discriminatory values (AUC=94·8%) and excellent classifying capabilities in the balanced pooled cohort and in each single cohort , we were able to identify 18 genes whose expression significantly correlated with ZEBOV-GP-specific antibody responses in all four cohorts . The topoduction .CD4, CCR7, IL12A, FCGR1A, and GATA3 , have been shown to block viruses at the level of translation, replication, or both,24 potentially contributing to the rapidly induced post-vaccination protection in humans and non-human primates, even after exposure. Furthermore, IFN-inducible transmembrane family members  are known to block viral entrance and to inhibit early life-cycle steps of several viruses, including vesicular stomatitis virus, Ebola virus, and Marburg virus.26 Fast-kinetics ISGs  are known to mediate host defence against different pathogens, including viruses,27 whereas CXCL10 was observed to be upregulated not only after rVSVΔG-ZEBOV-GP vaccination ,22 but also after yellow fever28 and influenza29 vaccination. CXCL10 is known to stimulate the activation and migration of immune cells, such as monocytes, NK-cells, and T cells to the site of infection.30 Corroborating these data, NK markers (NCAM1), T-cell subsets markers , and cytotoxicity markers  were all downregulated in blood at the peak of the response, probably reflecting lymphocyte migration or marginalisation out of peripheral blood, as previously reported,31 whereas myeloid-associated genes  were transiently upregulated.Targeted gene expression profiling in adult healthy volunteers from Europe, North America, and Africa vaccinated with different doses of the rVSVΔG-ZEBOV-GP vaccine, identified (1) vaccine-related transcriptomic signatures, (2) a five-gene signature at day 7 after vaccination shared between all cohorts and associated with immunogenicity (ZEBOV-GP antibody titres), and (3) a five-gene signature at day 1 after vaccination in the Geneva cohort associated with development of arthritis. Our findings showed that the transcriptional response to vaccination is dependent on dose and time. Although we observed a prompt peak of gene expression perturbation at day 1 after vaccination, longitudinal transcriptomic profiling in rVSVΔG-ZEBOV-GP-vaccinated non-human primates showed a peak in the transcriptomic response at day 7 after vaccination, indicating that the vaccine response occurs with slower kinetics in non-human primates than in humans. Notwithstanding, DEGs identified at the peak of the response in both humans and non-human primates were dominated by shared ISGs and innate immunity-associated genes, suggesting a similar mode of action.34 In humans, long-term persistence of ZEBOV-GP-specific IgG antibody responses (at least 1–2 years) following a single injection of the rVSVΔG-ZEBOV-GP vaccine has been reported,35 but early transcriptomic signatures correlating with immunogenicity have not yet been defined. We identified a five-gene signature shared between all cohorts that significantly correlated with immunogenicity of the rVSVΔG-ZEBOV-GP vaccine. Interestingly, only the IFN-inducible gene IFI6 correlated positively with ZEBOV-GP-specific antibody titres. Consistent with our results, enrichment of IFN-signalling transcripts correlated with antibody titres after vaccination against influenza,36 malaria,37 dengue,38 rubella,39 and Ebola.22 These results might be explained by the fact that IFNα and IFNβ, normally activated during infections or immunisations, also execute immunoregulatory activities, being able to modify the adaptive immune system by direct T-cell and B-cell activation, thereby enhancing the production of specific antibodies.40 Alternatively, it was recently shown that IFI6 reduced replication and transcription of modified Ebola virus,41 indicating that vaccine-induced expression of IFI6 might have direct protective effects in addition to its correlation with antibody responses. Other innate immune response genes were among the genes that negatively correlated with the antibody response . A significant correlation between transcriptional apoptosis or survival, inflammasome markers, and pattern-recognition receptors with the humoral response was reported in two studies investigating the immune response following vaccination against hepatitis Band influenza.42 Characterising an early gene signature that might predict arthritis after rVSVΔG-ZEBOV-GP vaccine was one of our aims, given also the occurrence of arthritis following other vaccinations such as the measles, mumps, and rubella vaccination43 and Lyme disease vaccination.44 The five-gene signature included T-cell subset genes CD4 and CCR7, IFN-signalling gene FCGR1A, myeloid-associated gene IL12A, and Th2-associated gene GATA3.Studies in animal models, including rodents and non-human primates, have underscored the importance of humoral immunity in protection from Ebola virus disease. ZEBOV-GP-specific total IgG levels have been shown to correlate with protection against a lethal ZEBOV dose.14 identified a plasma signature that correlated with several adverse events, including arthritis, after rVSVΔG-ZEBOV-GP vaccination. Interestingly, several studies focussing on arthritis pathogenesis revealed that CD4+ T cells together with inflammatory and innate immune responses play a central role. In a mouse model for rheumatoid arthritis, CCR7+ CD4+ T cells accumulated and homed to lymphoid organs where they survived and maintained autoreactivity. Also, CD4+ T cell migration to peripheral tissues and their polarisation into Th1-cells was reported to be orchestrated by the proinflammatory cytokine IL-12.46 Moreover, inflammatory pathways are characterised by involvement of immunoregulatory genes, including FCGR1A.47 Although the classical pathways described in rheumatoid arthritis promote the differentiation of CD4+ T cells into Th1-cells, expression of Th2-cell marker GATA3 might protect against severe joint inflammation by inhibiting differentiation of Th17-cells, but not Th1-cells, as reported in mice.48Previously, Huttner and colleaguesOur study has limitations. In the African cohorts, we probably did not capture the peak of the response because the earliest samples for gene expression profiling were collected at day 7 after vaccination, which might have affected the overall comparisons between the African, European, and North American cohorts. Furthermore, differences in the sizes of the cohorts might have penalised the smaller cohorts in identifying robust gene predictors that discriminate among the classes (vaccinees before versus after vaccination). Additionally, we employed a high-throughput targeted gene expression profiling technique (dcRT-MLPA) using preselected immune-associated markers. Although this technique allowed profiling of large numbers of samples and proved successful, signatures might be refined in the future with more global techniques.In summary, we have profiled and cross-validated the immune response-related, immunogenicity-related, and reactogenicity-related transcriptomic biomarker signatures induced by rVSVΔG-ZEBOV-GP vaccination in healthy adult participants. Our findings are novel, as they expand the analysis to four cohorts from different genetic and geographical backgrounds and report early shared transcriptomic signatures of immunogenicity and adverse events in response to Ebola vaccination. This study provides valuable insights into biological mechanisms that probably underlie the extremely high protective efficacy of rVSVΔG-ZEBOV-GP vaccination. Integrating additional high level omics data generated by different disciplines  will be required to further unravel the immune mechanisms responsible for early protection and reactogenicity following a single vaccination, enabling early interventions. Additionally, correlating early gene expression profiles with ZEBOV-GP IgG subclasses, antibody affinity, and neutralising titres in addition to total IgG might further improve the performance of immunogenicity-associated signatures, aiding in the rational design of new vaccines.Supplementary Material


The ubiquitin-proteasome system (UPS) has been recognized for regulating fundamental cellular processes, followed by induction of proteasomal degradation of target proteins, and triggers multiple signaling pathways that are crucial for numerous aspects of cellular physiology. Especially tripartite motif (TRIM) proteins, well-known E3 ubiquitin ligases, emerge as having critical roles in several antiviral signaling pathways against varying viral infections. Here we highlight recent advances in the study of antiviral roles of TRIM proteins toward influenza virus infection in terms of the modulation of pathogen recognition receptor (PRR)-mediated innate immune sensing, direct obstruction of influenza viral propagation, and participation in virus-induced autophagy. Influenza virus is an enveloped RNA virus that harbors a genome comprising eight segments of negative-sense, single-stranded RNA encoding at least ten distinct proteins . It is oThe ubiquitin-proteasome system (UPS) is a network of proteins dedicated to the ubiquitylation of cellular targets and the subsequent control of numerous cellular functions. Therefore, viruses must be subverted or utilized for this cellular machinery to reshape the cellular environment for establishing productive viral infection. In line with this point of view, accumulating evidence has highlighted emerging roles for the UPS during the influenza virus infection by targeting specific tripartite motif (TRIM) proteins ,13. The C-terminal carboxyl group. Subsequently, this activated ubiquitin is transferred to the active cysteine site on the ubiquitin-conjugating E2 enzyme. E3 ubiquitin ligase enzymes finally transfer E2-conjugated ubiquitin to substrate proteins via forming an isopeptide bond between the C-terminal glycine residue of the ubiquitin molecules and ε-amino group of a lysine in the substrates [Ubiquitination is one of the well-characterized post-translational modifications by which ubiquitin, 76 amino acid small protein, is covalently attached to lysine residues on a target protein . Three cbstrates ,21   domain, PRY-SPRY domain, and C-terminal subgroup one signature (COS) domain. Particularly, this C-terminal region affects subcellular localization and interaction with target proteins. For example, the SPRY and PRY–SPRY domains contribute to protein–protein interaction, exhibiting diverse roles in innate immune responses as observed in TRIM25 [TRIM proteins have been identified as a family of RING-domain containing E3 ubiquitin ligases that are involved in various biological processes including growth, apoptosis, morphogenesis, and transcription, as well as oncogenesis. In addition, more interestingly, recent studies have highlighted their emerging roles in antiviral functions via targeting directly viral proteins, innate immune systems, or autophagy pathway ,32,33,34in (CCD) b. The RIn TRIM25 . The COSn TRIM25 ,31.The TRIM proteins play a crucial role in regulating viral infection and the antiviral strategies which are classified by three broad categories: (1) modulation of pathogen recognition receptor (PRR)-mediated innate immune sensing, (2) direct obstruction of viral propagation, and (3) virus-induced autophagy. Herein, we describe these three functions of TRIM proteins in terms of intrinsic antiviral activities.As noted earlier, the TRIM proteins attract attention as a positive and negative regulation of the PRR-mediated innate immune signaling pathway. There are several types of PRRs that recognize different pathogen-associated molecular patterns (PAMPs) and activate specific signaling molecules participating in innate immunity. Among them, the RIG-I-like receptor (RLR) belongs to the DExD/H-box RNA helicases and functions as an essential sensor of viral double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) in the cytoplasm of the infected cells ,36. In pSeveral TRIM proteins operate as positive regulators of PRRs-mediated innate immune signaling via targeting RIG-I, MDA5, and MAVS . Among tIn addition to modulation of cytosolic RNA stimulate response, TRIM family proteins also regulate cyclic GMP-AMP synthase (cGAS)-STING-mediated cytosolic DNA sensing pathway. This cGAS-STING-mediated DNA sensing pathway has been recently identified as a component of the innate immune system of which functions to detect cytosolic viral DNA . Upon seSome TRIM proteins, including TRIM5 and TRIM22, have the ability to effectively restrict the pathogens by direct interaction with viral proteins . InterfeAutophagy (self-eating) is an extremely conserved intracellular catabolic pathway that induces the degradation of cytoplasmic components within the lysosomes . This prIt is worth mentioning that TRIM proteins have been directly linked to the multiple steps of the autophagy pathway from its induction to autophagosome formation ,69; FirsIn contrast to the action of negative regulation regarding most TRIM proteins for the life cycle of the influenza virus, it has been reported that ubiquitination stimulates polymerase activity of the influenza virus via ubiquitination of all the vRNP components without altering the protein levels, which eventually enhance replication of the influenza virus . In addiTRIM family members serve as a commander for antiviral restriction factors and immune responses. As reviewed here, we have highlighted their critical roles in combating the influenza virus replication either by directly targeting various influenza viral proteins or by regulating either PRR-mediated innate immune response or viral infection-mediated autophagy. Despite this viral antagonism of TRIM proteins, many elegant mechanisms to evade TRIM’s action by the influenza virus have yet to be elucidated. In fact, NS1, which is the only viral protein that has been shown to subvert TRIM25-mediated immune signaling, highlighted its importance in efficient establishment of the influenza virus infection. Therefore, going forward in understanding the mechanism of antagonizing TRIMs by the influenza virus, we should address significant gaps in knowledge regarding the context-dependent regulation and specificity of both TRIMs and DUBs. By adding to our growing knowledge of TRIM proteins, we gain important insights into not only the diverse aspects of virus-host interactions, but also the inner workings of ubiquitination. It is clear that TRIM-mediated ubiquitin modification is a key to coordinating physiological and pathological processes in influenza virus-infected cells. Further insights and developments into how influenza virus thwarts TRIM-mediated antiviral mechanisms will likely yield new approaches to the treatment of viral infection.


We present a case study of a 32-year-old female patient with ALK-rearrangement-positive LCNEC, who had multiple distant metastases including mediastinal lymph-node, bilateral breasts, multiple bones, liver and brain. Multiple biopsy samples obtained from primary lung and three metastatic tumors were analyzed by fluorescent multiplex immunohistochemistry. Tissue localizations of tumor-infiltrating lymphocytes in the tumor nest and surrounding stroma were evaluated. T cell and B cell infiltrations were decreased with distance from primary lung lesion. Although each tumor displayed a unique TIME, all tumors exhibited concomitant regression after treatment with an ALK-inhibitor. This study provides the first evidence of the coexistence of distinct TIME within a single individual with ALK-rearrangement-positive LCNEC. The present study contributes to our understanding of heterogeneous TIMEs between primary and metastatic lesions and provides new insights into the complex interplay between host-immunity and cancer cells in primary and metastatic lesions.Evolution of tumor-immune microenviroments (TIMEs) occurs during tumor growth and dissemination. Understanding inter-site tumor-immune heterogeneity is essential to harness the immune system for cancer therapy. While the development of immunotherapy against lung cancer with driver mutations and neuroendocrine tumors is ongoing, little is known about the TIME of large cell neuroendocrine carcinoma (LCNEC) or  Evolution of tumor-immune microenvironments (TIMEs) occurs during tumor growth and dissemination . AccumulALK fusion genes has been detected in approximately 2–5% of non-small cell lung cancer (NSCLC), most of which are adenocarcinoma ), CD3+ Nuclei+ (representing T cells), and CD20+ Nuclei+ (representing B cells) immune cells. Cytokeratin of tumor cells and PD-L1 were simultaneously stained to evaluate the complex relationship among tissue architecture, spatial distribution of immune cells and expression of PD-L1. High-speed scanning of whole slide images was performed on stained tissue sections using 20× objective . For comparison of quantitative marker expression, images were analyzed with Strata Quest . These TIL subsets were separately counted in the tumor nest  and surrounding stroma under high-power fields. For nuclei and tumor detection, size and staining intensity of DAPI and pan-Cytokeratin were adjusted. Then, cutoff thresholds of CD3, CD8, CD20 and FOXP3 were determined by two independent observers. Finally, all images were analyzed and statistics including number of each subset cells was generated automatically. As for Tregs, cells express both CD3 and FOXP3 were recognized as Tregs. Cells were classified as tumor nest or tumor stroma according to the relationship with pan-Cytokeratin-positive tumor cells.Four subsets of tumor-infiltrating lymphocytes (TILs) were identified: CD3


Are phosphorylated–focal adhesion kinase (pY397-FAK) expression levels in endothelial or tumor cells and tumor blood vessel density associated with response to chemotherapy and relapse-free survival after neoadjuvant chemotherapy in patients with locally advanced breast cancer?In this prognostic study of 82 women with stage IIA to IIIC locally advanced breast cancer, low endothelial cell pY397-FAK expression levels in prechemotherapy core biopsies were associated with sensitivity to neoadjuvant chemotherapy and improved 5-year relapse-free survival. Combined analysis of high endothelial cell pY397-FAK, high tumor cell pY397-FAK, and high blood vessel density appeared to identify a high-risk population.The findings of this study suggest that endothelial cell pY397-FAK levels could be used as a clinical tool for indicating response to neoadjuvant chemotherapy. Determining the risk of relapse after neoadjuvant chemotherapy in patients with locally advanced breast cancer is required to offer alternative therapeutic strategies.To examine whether endothelial cell phosphorylated–focal adhesion kinase (EC-pY397-FAK) expression in patients with treatment-naive locally advanced breast cancer is a biomarker for chemotherapy sensitivity and is associated with survival after neoadjuvant chemotherapy.In this prognostic study, expression levels of EC-pY397-FAK and tumor cell (TC)-pY397-FAK were determined by immunohistochemistry in prechemotherapy core biopsies from 82 female patients with locally advanced breast cancer treated with anthracycline-based combination neoadjuvant chemotherapy at Nottingham City Hospital in Nottingham, UK. Median follow-up time was 67 months. The study was conducted from December 1, 2010, to September 28, 2019, and data analysis was performed from October 2, 2019, to March 31, 2020.All women underwent surgery followed by adjuvant radiotherapy and, if tumors were estrogen receptor–positive, 5-year tamoxifen treatment.Outcomes were pathologic complete response and 5-year relapse-free survival examined using Kaplan-Meier, univariable logistic, multivariable logistic, and Cox proportional hazards models.P = .04), progesterone receptor positivity , high Ki67 , 4-immunohistochemically stained luminal-B , higher tumor category , high lymph node category , and high tumor node metastasis stage . Of 21 patients with high EC-pY397-FAK expression levels, none showed pathologic complete response, compared with 11 of 61 patients with low EC-pY397-FAK expression levels who showed pathologic complete response . High EC-pY397-FAK expression levels and high blood vessel density (BVD) were associated with shorter 5-year relapse-free survival compared with those with low EC-pY397-FAK expression levels  and low BVD . High TC-pY397-FAK expression levels in 15 of 82 women (18%) were not associated significantly with pathologic complete response or 5-year relapse-free survival. A multivariable Cox regression model for 5-year relapse-free survival indicated that high EC-pY397-FAK expression levels was an independent poor prognostic factor after controlling for other validated prognostic factors . Combined analysis of EC-pY397-FAK expression levels, TC-pY397-FAK expression levels, and BVD improved prognostic significance over individually tested features.A total of 82 women  with locally advanced breast cancer (stage IIA-IIIC) were included. Of these, 21 women (26%) had high EC-pY397-FAK expression that was associated with estrogen receptor positivity (71% vs 46%; The findings of this study suggest that low EC-pY397-FAK expression levels are associated with chemotherapy sensitivity and improved 5-year relapse-free survival after systemic therapy. Combined analysis of high EC-pY397-FAK expression levels, high TC-pY397-FAK expression levels, and high BVD appeared to identify a high-risk population. This prognostic study examines the use of endothelial cell phosphorylated-focal adhesion kinase as a biomarker for chemotherapy sensitivity in women with breast cancer. Preoperative chemotherapy (neoadjuvant chemotherapy) is commonly given to patients with high-risk breast cancer, particularly triple-negative ERBB2 (formerly HER2)-positive, and locally advanced breast cancer. Although chemotherapy reduces the early relapse rate (over the first 2 years) by 50%, the late relapse rate  is approximately 30%, and overall mortality is 20% to 25%.3 Current practice is largely based on assessment of the recurrence risk and overall survival of the patient, using the traditional clinicopathologic prognostic factors  and the multigene tests . Nevertheless, these tests do not determine whether a patient will respond to chemotherapy. Thus, developing biomarkers that will predict responses to chemotherapies is still required, and selecting those for which functional relevance to the biology of chemoresistance exists will likely provide more robust biomarkers.Breast cancer is a heterogeneous disease and more than 2 million new cases of breast cancer are diagnosed worldwide each year in women.8 FAK activation results in FAK-tyrosine (Y)-397-phosphorylation.9 Bulk tumor FAK expression has been correlated with breast cancer progression, but definitive results vary.16 Evidence suggests that endothelial cell (EC) FAK expression levels correlate with the molecular subtype of breast cancer, indicating that EC-FAK expression is potentially more clinically relevant than tumor cell (TC) FAK expression in breast cancer.10Focal adhesion kinase (FAK) is a ubiquitously expressed nonreceptor tyrosine kinase that is upregulated in many carcinomas, including breast cancer.17 FAK-kinase inactivation in the endothelium is associated with reduced vascular leakage,19 whereas the initiation of tumor angiogenesis is reduced by deletion of endothelial FAK in adult mice.20 In established tumors, loss of EC-FAK does not affect blood vessel density (BVD) but alters DNA damage-induced angiocrine signaling 23 and, in particular, reduces EC chemokine production. Reduced production of these angiocrine signals could govern chemosensitization in malignant cells, resulting in improved therapy efficacy in cancer control.24 Given that overexpression of FAK in many cancer types has led to the development of FAK inhibitors for cancer treatment, analysis of the association between endothelial FAK activation and clinical outcome after chemotherapy could be beneficial in selecting patients with breast cancer who may benefit from such novel therapy.Deletion of FAK in K14-positive epidermal cells reduces skin cancer progression.The purpose of this study was to examine whether phosphorylated (p)Y397-FAK expression levels in the endothelium of breast cancer before treatment are associated with response to anthracycline-based combination neoadjuvant chemotherapy and the subsequent risk of relapse.STROBE) reporting guideline for cohort studies.A cohort of 82 women with locally advanced breast cancer  from Nottingham University Hospital, Nottingham, UK, were included in a prognostic study conducted from December 1, 2010, to September 28, 2019. Analysis was begun October 2, 2019, and completed March 31, 2020. The study was approved by the institutional review board or independent ethics committee and the hospital research and innovations department at Nottingham University Hospital. Oral and written consent was obtained from participants before the investigation. Participants did not receive financial compensation. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  stained for the endothelial cell marker, CD31, and for pY397-FAK, using a modified stripping and reprobing multiplex protocol based on previous reportsExpression of pY397-FAK in ECs and TCs of breast cancer sections was quantified . First, CD31-stained images were virtually aligned with FAK-stained images using the tissue align module. Correct alignment was manually checked for all samples. Damaged or nonaligned tissue areas were excluded from the analysis. To assess EC-pY397-FAK expression, CD31 staining was used to identify blood vessels and FAK intensity was measured only in this overlapping aligned area , progesterone receptor (PR), Pathologic complete response rate was defined as the absence of any neoplastic cells in both the primary breast site and lymph node after neoadjuvant chemotherapy. Five-year relapse-free survival was defined as the number of months from diagnosis to recurrence or distal metastasis relapse. Survival was censored if the patient was alive at the end of the study, lost to follow-up, or died from other causes.2 and t tests were used. Cumulative survival probabilities were estimated using the Kaplan-Meier method, and differences between survival rates were tested for significance using the log-rank test. Univariable analysis was used to evaluate baseline risk of 5-year risk of relapse, and both hazard ratios (HRs) and 95% CIs were calculated. Multivariable analyses for survival were performed using the Cox proportional hazards model after adjusting for other validated prognostic factors, such as ER, PR, ERBB2, and Ki67 expression, lymph node stage, T stage, grade, and pathologic complete response. The proportional hazards assumption was tested using standard log-log plots. Hazard ratios and 95% CIs were estimated for each variable.Data analyses were performed using SPSS, version 17, software (SPSS Inc). Where appropriate, Pearson χP < .05 was considered to indicate statistical significance.For pathologic complete response analysis, univariable analysis as well as multivariable logistic regression tests were used and the odds ratio (OR) and 95% CI were calculated. All tests were 2-sided with a 95% CI, and a −2, epirubicin, 75-100 mg m−2, and cyclophosphamide, 500 mg m−2, on day 1 of a 21-day cycle]), and 49% (40/82) of the patients received 3 cycles of the FEC regimen plus 3 cycles of docetaxel, 100 mg m−2. All patients underwent mastectomy or breast-conserving surgery and axillary dissection, followed by adjuvant radiotherapy and, if tumors were ER-positive (43/82 [52%]), 5-year tamoxifen treatment. For ERBB2-positive cancers (16/82 [20%]), adjuvant trastuzumab was prescribed for a year. The median follow-up time was 67 months .The association between EC-pY397-FAK, TC-pY397-FAK protein expression levels, and BVD and clinicopathologic parameters, response to chemotherapy (pathologic complete response), and relapse-free survival were evaluated in the prechemotherapy core biopsies from 82 women  with locally advanced breast cancer (stage IIA-IIIC), who were treated with anthracycline neoadjuvant chemotherapy at the Nottingham City Hospital. Details of the characteristics of the patient cohort are given in eTable 2 in the P = .04), PR-positivity , high Ki67 , 4-IHC luminal-B , higher T category , high lymph node category , and high TNM stage   in the study cohort . High ECP = .03) .P = .04) (P = .01) (P = .05) (P = .05)  . High ECP = .01) A. High EP = .05) B, and thP = .05) C.P = .02), whereas low BVD compared with high BVD was associated with ERBB2 overexpression  (eTable 3 in the P = .02)  and, compared with low tumor BVD, was associated with 4-IHC luminal-B , 4-IHC luminal-B , higher T category , and high TNM category  (P = .09) or TC-pY397-FAK expression levels and 5-year relapse-free survival were observed (High TC-pY397-FAK expression levels (cutoff ≥127.91) were observed in 15 of 82 patients (18%) and, compared with low TC-pY397-FAK levels, were associated with ER-positivity  . AlthougP = .04) , no statobserved . These robserved .Combined analysis of patients with low EC-pY397-FAK and high BVD showed poorer clinical outcome than those with low EC-pY397-FAK and low BVD D. CombinERBB2, and Ki67 expression, lymph node stage, T category, and pathologic complete response . The interaction term between EC-pY397-FAK and TC-pY397-FAK expression was statistically significant. Also, the interaction between EC-pY397-FAK, TC-pY397-FAK, and BVD was statistically significant  may be an independent prognostic biomarker for chemotherapy response in patients with advanced breast cancer who received neoadjuvant chemotherapy followed by adjuvant therapy. Combining biomarkers for EC-pY397-FAK, TC-pY397-FAK, and BVD may provide an improved relapse risk stratification over individual features alone. These data have potential therapeutic implications for high-risk populations that could benefit from additional novel therapy.Biomarkers that inform appropriate treatment of breast cancer are part of the increase in success of breast cancer treatment.33 and others suggest that FAK expression does not correlate with outcome, showing no correlation with prognosis or that low FAK expression levels are associated with poor prognosis.37 For example, a study with 335 node-negative breast cancers indicated improved patient survival with high TC-FAK expression levels.11 The relevance of this finding to our study is limited since 83% of the cohort studied herein was node-positive. This apparent discrepancy highlights the idea that one biomarker may or may not be relevant in different subtypes of breast cancer and that understanding the biological functions of FAK in different breast cancer subtypes or at different stages of progression is of value.Several studies have linked high tumor FAK expression levels with poor prognosis in cancer,40 although most studies have not focused on FAK expression in blood vessels. A previous study described an association between EC-FAK expression levels and the molecular subtype of invasive breast cancer.10 Loss of EC-FAK is associated with improved outcome in patients with lymphoma after anthracycline-containing combination therapy, and loss of EC-FAK is sufficient to sensitize malignant cells to doxorubicin treatment in transgenic models of cancer.24 Further studies have shown that external signals, including growth factors,43 derived from the tumor microenvironment or tumor cells themselves induce activation of EC-pY397-FAK,44 and that FAK transduces ECM signals in a variety of different cell types,45 including ECs.49 Furthermore, an EC-Y397 nonphosphorylation mutation controls inside-out signaling, resulting in increased Tie-2 expression levels, reduced VEGFR-2 expression levels, decreased β1-integrin activation, and disrupted FAK/Src/PI3K(p55)/Akt signaling.44 These pY397-FAK–specific targets may be used as druggable targets for personalized therapies in patients who present with increased EC-pY397-FAK expression levels. For example, VEGFR-2 directed therapy is used in the clinic setting to target angiogenesis51; Src inhibitors,52 PI3K p55 specific inhibitors,53 and Akt inhibitors54 have all shown promising results in clinical trials.The study of the potential role of FAK expression in predicting response to chemotherapy in human cancer is an emerging field,6 we have seen this association only with high EC-pY397-FAK levels and not with TC-pY397-FAK levels. Our observations appear to corroborate preclinical studies in which EC-FAK regulates blood vessel permeability and metastasis.55 Given that high EC-pY397-FAK expression levels were associated not only with lymph node stage but also with relapse and that the main cause of relapse is metastatic spread, it is plausible that EC-pY397-FAK also contributes to breast cancer metastasis.Although previous reports have linked TC-FAK expression with lymphovascular invasion,59 and the results from this cohort support these findings. Several clinical trials using antiangiogenics or vascular-disrupting agents alone or in combination with chemotherapy and endocrine therapy were conducted, but the therapy did not provide clinical benefit,60 possibly owing to failure in selecting subgroups of patients who would benefit from such therapies. Given that our data suggest that low EC-pY397-FAK expression levels with low BVD are associated with a better outcome than low EC-pY397-FAK expression levels with high BVD, future testing of vascular-disrupting agents in low EC-pY397-FAK expression levels with high BVD may provide added benefit and identify a group of patients who would benefit from targeting tumor blood vessels.Previous studies have associated high BVD with poor prognosis,62 and their benefits in breast cancer preclinical models have been demonstrated.65 Increasing evidence suggests a benefit of combining FAK-kinase inhibitors with chemotherapy in other types of cancer.69 Ongoing and future clinical trials will examine the value of FAK-kinase inhibitor treatment with chemotherapy in breast cancer progression and patient survival. Herein, however, our findings focused on the prognostic value of EC-pY397-FAK expression levels and its utility as a biomarker in identifying high- and low-risk patient subgroups to inform alternative treatment strategies.The upregulation of FAK in many carcinomas has led to the development of FAK inhibitors,This work could impact the management of therapy for patients with ER-positive breast cancer and may lead to the development of novel strategies for more effective management and treatment. For instance, high expression levels of EC-pY397-FAK and TC-pY397-FAK were more common in ER-positive luminal-B tumors and were associated with both chemotherapy and endocrine therapy resistance, providing an opportunity to select these patients for alternative novel therapies. Our findings have the potential to introduce an accurate predictive biomarker for identifying patients with ER-positive breast cancer who would or would not benefit from endocrine therapy and chemotherapy, and could also be used to monitor tumor response during treatment, thereby sparing patients with nonresponsive tumors the burden of adverse effects that would affect their quality of life. The next phase of our work is to develop this protocol into a diagnostic assay, which may prove to be cost-effective, and it will be validated in retrospective and prospective clinical studies and trials. This assay will help to deliver precision medicine for patient benefit.Overall, our observations of poor prognosis in patients with breast cancer with high EC-pY397-FAK expression levels alone or in combination with high BVD open new opportunities for enriching recruitment studies of novel therapeutics for this population. Such novel therapeutics could be given instead of, concurrently with, or after standard of care treatment is completed. These high-risk patients could also benefit from more intensive follow-up. Further work will be required to streamline and accelerate the IHC and analysis processes for large numbers of patients. Given that protein expression is a dynamic and highly regulated process, it is possible that FAK has different roles at later stages of the disease. Thus, in future studies, it would also be of interest to determine the clinical relevance of EC-pY397-FAK in tumors taken at surgery.12 The modest number of patients analyzed is also a potential limitation. To fully define the biological significance of EC-pY397-FAK expression levels as a marker of chemoresistance would require analysis in a series of patients with untreated disease, which is not feasible because withholding treatment would be unethical.This study had limitations. We used digital analysis to calculate EC-pY397-FAK expression levels. Although digital analysis avoids subjectivity and variability across pathologists, it only takes into consideration signal intensity without accounting for staining extent. Cutoff values will need to be standardized for future applications; however, the percentages of patients in each group used herein are comparable with those used in previously published work.The results of this study appear to support the significance of EC-pY397-FAK expression levels as an independent biomarker associated with advanced breast cancer therapy above TC-pY397-FAK expression or BVD. In addition, EC-pY397-FAK expression levels in combination with BVD and TC-pY397-FAK expression levels provide multiparametric biomarker associated with pathologic complete response. This study highlights the potential clinical utility of EC-pY397-FAK expression levels in combination with BVD to guide therapeutic decision-making and testing of novel therapeutics in the high-risk group. Our data also appear to support the usefulness of combining molecular and morphologic features in designing prognostic factors and therapy response.


Current cancer immunotherapy has limited response rates in a large variety of solid tumors partly due to the low immunogenicity of the tumor cells and the immunosuppressive tumor microenvironment (ITM). A number of clinical cancer treatment modalities, including radiotherapy, chemotherapy, photothermal and photodynamic therapy, have been shown to elicit immunogenicity by inducing immunogenic cell death (ICD). However, ICD-based immunotherapy is restricted by the ITM limiting its efficacy in eliciting a long-term antitumor immune response, and by severe systemic toxicity. To address these challenges, nanomedicine-based drug delivery strategies have been exploited for improving cancer immunotherapy by boosting ICD of the tumor cells. Nanosized drug delivery systems are promising for increasing drug accumulation at the tumor site and codelivering ICD inducers and immune inhibitors to simultaneously elicit the immune response and relieve the ITM. This review highlights the recent advances in nanomedicine-based immunotherapy utilizing ICD-based approaches. A perspective on the clinical translation of nanomedicine-based cancer immunotherapy is also provided. Cancer immunotherapy has changed the paradigm of clinical cancer therapy by demonstrating that boosting the systemic immune response can lead to tumor regression , 2. DespImmunogenic cell death (ICD) is an alternative approach that activates antitumor immunity to eradicate tumor cells . Tumor cRecently, it was demonstrated that chemotherapeutics , doxorubicin (DOX), and mitoxantrone) and tyrosine kinase inhibitors, such as crizotinib elicit antitumor immunity by inducing the immunogenic death of tumor cells –26. FurtDespite the promising potential of ICD-based approaches for cancer therapy, inadequate induction of ICD by conventional approaches has hampered clinical translation. Shortcomings of conventional approaches include insufficient circulation time, limited intratumoral accumulation and retention, restricted tumor cellular uptake and slow release in tumor cells , 32. MorTo overcome these challenges with conventional ICD approaches, nanoparticle-based drug delivery systems with tunable size and surface properties offer unique advantages for ICD-based cancer therapy Fig. . NanoparThis review discusses recent advances in nanoparticle-mediated ICD and focuses on the effects induced by certain drugs and physical stimuli. Novel insights into engineering nanoparticles to enhance their antitumor efficacy utilizing a combination of ICD and ITM reprogramming are also provided.D, L-lactide-co-glycolide) (mPEG-b-PLGA) nanocarrier enhanced the induction of ICD to a greater extent than did OXA delivered alone via the promotion of DAMPs, enhanced maturation of DCs and elevated levels of intratumor infiltrating CTLs . Th. Th86]. To precisely control drug release at the tumor site to promote antitumor immunogenicity and minimize systemic adverse effects, a light-inducible nanocargo (LINC) was designed for use in combination cancer immunotherapy . A LINC L) led to higher ROS generation efficacy than did LINC alone. In the 4T1 xenograft tumor model, LINCL significantly increased the cell surface expression of CRT, promoted HMGB1 release , anti-programmed death ligand 1 (αPD-L1) or anti-cytotoxic T lymphocyte-associated antigen 4 antibodies has shown potential in the clinic , 90. ThuIn addition to the small molecule inhibitors and monoclonal antibodies for combating ITM, peptide-based immune checkpoint inhibitors have also been exploited for enhancing ICD by ICB. For example, a biomimetic nanoparticle has been designed to integrate PPa, a ROS-activated PTX prodrug, and αPD-L1 peptide (dPPA) into a single nanoplatform . Combina+ T cells compared with that of the control group. HFSIRP-α-DOX efficiently suppressed tumor growth by activating the adaptive immune response and combating the ITM.CD47 is another crucial immune checkpoint protein that is abundantly expressed on the surface of various tumor cells. To prevent phagocytosis of tumor cells by APCs, CD47 acts as a “don’t eat me” signal by binding with signal regulatory protein-α (SIRP-α) on the surface of macrophages or DCs . TherefoTo further demonstrate the potential of ICD induction and CD47 blockade for combination immunotherapy, a tumor microenvironment-activatable nanoparticle was designed to destroy tumor cells and induce ICD. The nanoparticles were constructed from an MMP-2-activatable vesicle (MPV) and 2,3-dimethylmaleic anhydride-modified hexadecyl-oxaliplatindiethylene amine (HOAD), which was termed MPV-HOAD . The MPVIn summary, several emerging therapeutic methods, including chemotherapy, phototherapy, and radiotherapy, have been shown to be effective for eliciting ICD. However, ICD-activated immune responses only moderately suppressed tumor recurrence and metastasis due to insufficient tumor distribution of these therapies. To improve the therapeutic performance of ICD-based cancer immunotherapy, nanoparticle-based drug delivery systems enable codelivery and multiple irradiation regimens to enhance the performance of ICD-based therapies. The combination of nanotechnology and ICD-based cancer immunotherapy shows potential for improved cancer immunotherapy by simultaneously eliciting antitumor immunity and suppressing the ITM.Despite great success using these nanoparticle-based ICD therapies in preclinical studies, their clinical application is still hindered by several challenges. First, the nanoparticle-based ICD strategy is in its infancy, with many novel tactics remaining to be explored. Tumor-specific drug distribution and activation are crucial for eliciting antitumor immunity. Second, simple nanoparticle designs and quality control mechanisms are essential for the clinical translation of nanomedicine-based cancer immunotherapy. Third, further efforts to elucidate the mechanisms underlying ICD-based cancer immunotherapy are required to facilitate its clinical translation.


Possible selves are defined as “personalized representations of one’s self in future states” . Research has also found that thinking frequently about possible selves predicts lower well-being, whereas thinking clearly about possible selves is associated with higher well-being . However, possible selves differ across the lifespan  and life stages can impact exploration of possible identities . Thus we hypothesize that the relationship between thought about possible selves and well-being differs across the lifespan. In a cross-sectional survey, 240 participants (age range: 18-92) reported on frequency and clarity of possible selves, as well as general self-clarity . Results indicate curvilinear moderation by age of the link between possible self-clarity and well-being, with the association being stronger among midlife adults than among younger adults and older adults. Also, as clarity of feared possible selves increases, self-concept clarity decreases. Similarly, frequency of thinking about feared possible selves was negatively correlated with self-concept clarity. However, clarity and frequency of thought about hoped-for positive possible selves had no correlation with self-concept clarity.


We report two cases of coronavirus disease 2019 (COVID-19) in patients who developed pulmonary embolism and transient anti-phospholipid antibodies. At the time of presentation with acute pulmonary embolism, both patients had leukocytosis and increased levels of anti-cardiolipin antibodies, which resolved at testing 12 weeks after initial presentation. Studying cases of pulmonary embolism and increased anti-phospholipid antibodies in the context of COVID-19 could be one of the factors for elucidating the possible connection between severe acute respiratory syndrome coronavirus 2 infection, anti-phospholipid antibodies, and thrombosis. Coronavirus disease 2019 (COVID-19), caused by the novel RNA beta coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can have many clinical and laboratory manifestations. Because SARS-CoV-2 is new to the human population, a range of immunological responses have been noted [A 59-year-old African American man with a history of double heterozygous state with sickle cell trait/beta thalassemia+ presented to the emergency department with shortness of breath and pleuritic chest pain. He was febrile, tachycardic, and hypoxemic on arrival and was hospitalized for further care. Diagnostic investigations showed leukocytosis 27.8 × 103/μL) with normal absolute lymphocyte count, microcytic anemia (hemoglobin 6.8 g/dL) with abnormal hemolysis markers, and a normal platelet count. Reverse-transcription polymerase chain (PCR) test from nasopharyngeal swab was positive for SARS-CoV-2. Computed tomography (CT) imaging of the chest revealed multi-lobar infiltrates and bilateral upper lobe pulmonary embolism. Anti-phospholipid antibody testing showed the presence of cardiolipin Ab IgG 33.5 GPL, cardiolipin Ab IgM 41.7 MPL, and B2 glycoprotein Ab IgG 103 SGU . Diagnostic investigations revealed leukocytosis with monocytic predominance , hemoglobin and platelet counts within reference range, and he had elevated ferritin (2277 ng/mL) and lactate dehydrogenase (365 IU/L). CT of the chest revealed multi-focal infiltrates and bilateral pulmonary embolism, including a saddle embolus with evidence of right heart strain. SARS-CoV-2 PCR test via nasopharyngeal swab was negative. Anti-cardiolipin antibodies were outside of reference range, with cardiolipin Ab IgA <9.0 APL, cardiolipin Ab IgG 83.4 GPL, and cardiolipin Ab IgM 136 MPL (Table The patient was seen in the hematology clinic three months later where he was recovering well without oxygen requirement. Repeat testing showed normalization of anti-phospholipid antibodies and resolution of leukocytosis. A test for IgG SARS-CoV-2 antibodies in peripheral blood was reactive, strongly suggesting that his initial nasopharyngeal swab had been a false negative. He completed three months of anticoagulation therapy with apixaban without any complications.In the cases presented here, both patients had pulmonary embolism and showed clear presence of anti-cardiolipin and anti-beta-2 glycoprotein antibodies during acute illness that resolved in the weeks following recovery.Anti-phospholipid antibodies have been implicated in the pathogenesis of thrombosis and pregnancy morbidity in patients with anti-phospholipid syndrome . Also, cSevere COVID-19 infection has been shown to be associated with increased risk of thromboembolism . Both veWe suspect that the transient anti-phospholipid antibodies seen in our patients were reactive in nature and perhaps had developed from antigenic stimulation caused by SARS-CoV-2 virus and a massive inflammatory response. We wonder whether transient anti-phospholipid antibody production contributed to the development of pulmonary emboli. The patient described in the first case had abnormal hemoglobin electrophoresis that revealed a double heterozygous state with sickle cell trait/beta thalassemia+, possibly placing him at higher risk of venous thromboembolism and explaining his severe anemia at presentation. For the patient described in case two, we could not identify any other triggering events for his hemodynamically significant pulmonary embolism.We hypothesize that COVID-19 had contributed to development of venous thromboembolism in both patients, with transient anti-phospholipid antibodies possibly playing a role in pathogenesis. Studying the relationship between anti-phospholipid antibodies and thromboembolic complications in patients with COVID-19 will be important for understanding the multiple pathogenic mechanisms of SARS-CoV-2 infection and to inform treating physicians about the many clinical complications that this problematic virus may cause.Anti-phospholipid antibody testing might be beneficial in individuals with COVID-19 and venous thromboembolism for better understanding of thrombotic complications. The cause and effect relationship of transient immunological responses with the development of pulmonary embolism in patients with COVID-19 and the long-term implications of these transient findings are uncertain and require investigation.


Dear Editor,We read with great interests the speculation put forward by Mustafa MZ et al.  that stiAlthough much about pathogenesis of SARS-CoV-2 remains unknown, clinically staged progression of COVID-19 is being recognised and molecular mechanisms at each stage are being unveiled and inferred from other respiratory viral infections . In brieIn established pulmonary parenchyma stage II disease, infected epithelial cells undergo pyroptosis, a highly inflammatory form of programmed cell death, and release damage-associated and pathogen-associated molecular patterns, triggering a T helper 1 cell-polarised pro-inflammatory feedback loop involving neighbouring epithelial cells, endothelial cells, alveolar macrophages and T cells via production of pro-inflammatory cytokines and chemokines. Pulmonary damage and recruitment of immune cells manifests as viral pneumonia clinically with bilateral infiltrates or ground glass opacities on chest imaging. Hypoxia may occur in more advanced stage (stage IIb). At this stage, management primarily consists of hospitalisation with close observation, supportive care and antiviral therapy, if available. Only with hypoxia likely to require mechanical ventilation supports that anti-inflammatory therapy can be judiciously administered. About 14% of patients progressed to this stage .Unfortunately, about 5% of patients developed stage III with systemic hyperinflammation syndrome . In thisIn line with the staged progression of COVID-19, anti-inflammatory therapy might play roles in the more advanced stages of COVID-19 rather than in the early stages of the disease. As such, the ideas put forward by Mustafa MZ et al. to treat early infected patient with sting bee honey via its anti-inflammatory properties do not resonate well with the current understanding of COVID-19. Even in the later stages of COVID-19, it is doubtful sting bee honey has similar efficacy as compared to other anti-inflammatory therapies such as glucocorticoid administration.Considering most of COVID-19 patients would manifest stage I disease and recover from this stage, it does not negate the preference of some patients for self-care treatments that may offer some benefits for symptomatic relief. These self-care treatments should be construed within the realms of symptomatic relief rather than specific treatments for COVID-19. In relation to this, guideline for acute cough associated with an upper respiratory tract infection or acute bronchitis, which usually has a viral etiology, states that honey as a self-care treatment may be used to alleviate cough symptoms . Apart f


We developed Pre-Loss Group Therapy (PLGT) for dementia caregivers at risk for Complicated Grief (CG). PLGT is a manualized ten-session multi-modal group therapy that includes elements of cognitive behavior therapy, motivational interviewing, exposure therapy, memory revisiting, meaning-making, and self-care. We implemented and evaluated three PLGT cohorts in three long-term care facilities with family caregivers at-risk for CG whose care recipient had a life expectancy of 6 months or less and resided in a long-term care facility (NT = 24). Evaluation of participant preparedness for the death of the persons with dementia (PWD), self-care and grief outcomes showed significant improvement across multiple domains between pre and post-group, notably a statistically significant decrease in grief as measured by the Inventory of Complicated Grief score from baseline  to post-group  t(21)= 6.280, p<0.001. Clinician-rated grief severity declined  per week and grief improvement increased  per week, as assessed on the Clinician Global Impressions Scale. We subsequently trained two LCSWs to conduct PLGT, and both clinical outcomes and treatment fidelity and skills measures achieved performance levels of master clinician-trainers. Family caregivers at risk for CG may benefit from group therapy targeting preparedness and pre-loss grief experience, as we provide with PLGT. Manualized PLGT is suitable for implementation by LCSWs in the settings of hospice and long-term care.


Identification and clinical translation of routinely tested biomarkers require a complex and multistep workflow. Here, we described a confirmatory process estimating the utility of previously identified candidate tissue miRNAs for diagnosis of prostate cancer (PCa). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) prostate tissue surgically resected from 44 patients with PCa and 24 patients with benign prostate hyperplasia (BPH). Of the 92 RNA samples obtained, 68 represented 42 malignant (PCa) areas and 26 represented nonmalignant (PCa 0%) areas of the prostate tissue sections. The levels of miR-32-5p, miR-183-5p, miR-141-5p, miR-187-3p, miR-375, miR-663b, miR-615-3p, miR-205-5p, miR-221-3p, and miR-222-3p were evaluated using Exiqon chemistry. Five , one (miR-32-5p), and two (miR-32-5p and miR-141-5p) miRNAs discriminated between BPH and areas of cancer-bearing prostate tissue harboring different numbers of cancer cells , with an area under the receiver operating characteristics curve (AUC-ROC) > 0.9. Only miRNA 32-5p discriminated BPH specimens from sections of cancer-bearing prostate tissue with a low percentage, a high percentage, or no dysplastic cells. miR-32-5p could be considered as potential diagnostic biomarker discriminating BPH from noncancerous areas within cancer-bearing prostate tissue. However, further clinical studies are warranted to confirm its diagnostic utility. Identification and clinical translation of routinely tested biomarkers remain the challenge of modern medicine. Due to ease and reproducibility of extraction from biological samples, their stability and measurement accuracy by standard techniques, miRNA have been considered as valuable biomarker candidates. However, more than 50 miRNAs have been described to be involved in the development of prostate cancer (PCa) but their aberrant expression may fluctuate due to the molecular heterogeneity of prostate cancer .Histological evaluation still remains the gold standard for cancer diagnosis. However, the ability to detect cancer, even when neoplastic cells are missed on needle biopsies, is a highly desirable attribute for a PCa biomarker. In cases of suspected PCa in which the initial biopsy is negative, such biomarkers may facilitate the clinical choice between watchful waiting and collection of additional biopsy samples.A clinically and economically feasible biomarker requires an appropriate type of specimen, such as a formalin-fixed, paraffin-embedded (FFPE) tissue sample. Using frozen tissues, we described recently a diagnostic workflow for selecting miRNA biomarker diagnostic for PCa . Deep seHere, we used a set of independent FFPE tissues from Polish patients to assess the diagnostic value of the candidate biomarker miRNAs previously identified by us  and by KThe study examined 68 FFPE prostate tissues surgically resected at the Department of Urology at the Maria Sklodowska-Curie Institute-Oncology Centre, Medical Center for Postgraduate Education and Multidisciplinary Hospital Warsaw-Miedzylesie, Warsaw, Poland. None of the patients had received previous hormonal therapy or radiotherapy to the prostate. The study was performed in accordance with the ethical standards of the local bioethical committee and in accordance with the principles of the 1964 Declaration of Helsinki.Prior to RNA extraction, several sections were prepared from different areas of all malignant and nonmalignant prostate tissues; the upper and lower sections from each set of sections were evaluated by referral pathologists to control for the relative cell type content. Of the 92 RNA samples obtained, 68 represented 42 malignant (PCa) and 26 represented nonmalignant (PCa 0%) prostate areas in tissue samples from 44 PCa patients  and 24 BPH patients .RNA was isolated using a QIAamp RNA FFPE Tissue Kit (Qiagen), according to the manufacturer's protocol. The samples were submitted to QIAGEN Genomic Services, where each RNA sample was reverse transcribed (in triplicate) into cDNA and run on the miRCURY LNA miRNA PCR Custom panel. The levels of the following miRNAs were determined: (1) four miRNAs  selected from our previous study  was  was 3] wMeasurable qRT-PCR signals were obtained in all samples for ten candidate miRNAs and four miRNAs selected for data normalization. Since three miRNAs  selected for the normalization were differentially expressed among samples, the data were normalized only in relation to the level of miR-151a-5p.P value < 0.05) between BPH and PCa > 10% (2–10%), PCA < 10% (15–70%), and PCA 0% samples, respectively  tissue sample. Here, we reevaluated the diagnostic potential of four miRNAs selected by us with the use of massive sequencing of small RNAs isolated from frozen prostate tissues  and six Accurate detection and quantification of miRNA depend on the technology platform used for testing ; therefoThe present study highlights a discrepancy between the results from the initial miRNA biomarker discovery studies , 3 and tAs reported previously, we found that increased expression of miR-32-5p is associated with tumorigenesis; indeed, miR-32 promotes proliferation and migration of both breast and PCa cells , 9. TreaA suspicion of PCa results from elevated levels of serum prostate-specific antigen (PSA) and/or palpable alterations within the prostate upon digital rectal examination. Transrectal ultrasound- (TRUS-) guided prostate core biopsy is a standard procedure for PCa diagnosis. However, PCa is a multifocal disease, and even a 20-core biopsy approach can miss up to 10% of cancers . Thus, mWhile several miRNAs might distinguish cancerous from benign prostate tissue, miR-32-5p seems to be the best PCa biomarker (at least in Polish patients) as it can discriminate benign prostate tissue from noncancerous areas within cancer-bearing prostates. However, before a miR-32-5p-based biomarker will be ready for clinical use, further studies on its clinical benefits (including comparative- and cost-effectiveness) are required. In conclusion, this study confirms that miRNA is a stable biomarker for PCa, even in FFPE tissue samples, which are a suitable source of material for miRNA diagnostic assays. However, the dependency of miR-32-5p on tissue fixation methods and sample age (both of which affect miRNA integrity) should be evaluated.