This study was a meta-analysis of the literature on the efficacy and safety of tenofovir disoproxil fumarate (TDF) in preventing vertical transmission of hepatitis B in pregnancies with high viral load. Four observational studies and one randomized controlled trial involving 585 pregnant women and 595 newborns were included in the meta-analysis. TDF was more effective than the placebo in reducing vertical transmission in HBeAg-positive chronic hepatitis B (CHB) pregnancies with high serum HBV-DNA levels  at 4–12 months, infant HBV DNA seropositivity at delivery , and a severe flair in maternal alanine aminotransferase (ALT) levels  during pregnancy. In addition, TDF showed more improvement in HBV DNA suppression at delivery . No significant differences were found in HBeAg seroconversion or ALT normalization; or in rates of cesarean section, emergent cesarean section, postpartum hemorrhage, prematurity, congenital malformations, or infant death. However, TDF induced more drug-related adverse events  and elevated creatine kinase (CK)  than in controls. The available evidence suggests that TDF is effective and safe in preventing vertical transmission of hepatitis B in pregnancies exhibiting a high viral load. Vertical transmission of HBV from hepatitis B surface antigen (HBsAg)-positive and hepatitis B e antigen (HBeAg)-positive pregnancies to infants remains the predominant mode of CHB infection, with up to 90% of infants from these pregnant women becoming chronically infected with HBV without immunoprophylaxis4, which may be partially due to the active replication and HBeAg positivity of women during their reproductive years6. Programs combining postnatal active and passive immunoprophylaxis and universal vaccination have successfully reduced the transmission rates of HBV from > 90% to about 10%8; however, there are still about 8%-30% of newborns that fail active HBV immunoprophylaxis (particularly those born to pregnant women with high serum HBV-DNA levels of > 2 × 105–107 IU/mL13), mainly due to a further increase in the global prevalence of CHB. Thus, preventing vertical transmission of HBV is an effective way to reduce the global burden of CHB.Infection with chronic hepatitis B (CHB) virus remains a major challenge to global health, with approximately 240 million carriers and an estimated > 600,000 deaths annually due to CHB-related disease15. In contrast to lamivudine (LAM), telbivudine (LdT) and adefovir (ADV) manifest potency limitations and low threshold to resistance, and TDF is the only approved NA showing greatest potency but without any associated clinical resistance17. The favorable efficacy and safety profile of TDF have been well demonstrated for the treatment of HIV mono-infected and HIV/HBV co-infected mothers20, but the data on the use of TDF in HBV mono-infected pregnancy are limited. Since Pan et al. published their first case series in 201210 where they evaluated the efficacy and safety profile of TDF in HBV mono-infected pregnancy, there have been several observational studies published in this area24. Recently, an excellent systematic review involving 26 studies was conducted by Brown et al.25, in which these authors comprehensively evaluated the efficacy and safety of LAM, LdT and TDF used in pregnancy; but only 3 observational studies on TDF  were included in the study, and the results were limited23. Since then, a multi-center, randomized controlled trial was published to powerfully demonstrate the efficacy and safety profile of TDF in highly viremic CHB mothers8; however, according to the FDA drug category for pregnancy, TDF is still classified as category B for use in pregnancy, and therefore, guidelines form the WHO14, AASLD17 and APASL (2015 update)16 made this issue a priority for the development of clinical practice guidelines and the synthesis of evidence. To maximize sample size and add the latest evidence, we performed a systematic review and meta-analysis to comprehensively evaluate the efficacy of oral TDF therapy in preventing vertical transmission of hepatitis B in pregnancies carrying a high viral load.An increasing number of studies have suggested that antiviral therapies for CHB with perinatal oral nucleotide analogues (NAs) significantly reduce the risk of vertical transmission of HBV in pregnancies with high serum HBV-DNA levels; however, these studies have also shown some conflicting resultsA flow diagram summarizing the process of study selection is illustrated in Fig. 23 and 1 RCT8, involving 585 pregnant women and 595 newborns, were included in the present meta-analysis. These very recent studies were published from 2013 to 2016; 2 studies were conducted in China23, and another 3 studies were conducted in North American22, Australia2 and Turkey3, respectively. The participants in all studies in the TDF group received 300 mg of TDF once daily, with a baseline HBV DNA level of > 7.7  log10 IU/mL, and an average baseline ALT level of 30.5 U/L. In 4 studies TDF treatment was initiated from gestational weeks 30–32 until 4–12 weeks following delivery23, while in the remaining studies TDF treatment was initiated from gestational weeks 18–27 until 4 weeks following delivery3. The pregnant women in the control group in all studies did not receive antiviral therapy. In 4 studies the investigators compared TDF versus control23, while in 1 study researchers compared TDF versus LAM or control2. All infants in the included studies received a 3-dose HBV vaccination and hepatitis B immune globulin (HBIG) administration after delivery, and all mothers in these studies were instructed not to breast-feed their infants during the period in which they were receiving TDF treatment. More characteristics of the studies and baseline information on HBIG + HBV vaccine administered to infants are summarized in Tables Five studies, including 4 observational studies23. However, in the study conducted by Celen et al.3, 61.9% (13/21) of mothers still showed HBV DNA < 50 IU/mL at 28 weeks postpartum. Regarding the influence of maternal serum ALT levels, synthesis results from 3 studies suggested that TDF generated a less-severe flare in ALT levels  during pregnancy   Fig. , or post2, there was no difference found in any of several maternal outcomes , infant death , or congenital malformations . The congenital malformations observed in these studies were congenital chromosomal disorder syndromeThe quality of the evidence regarding infant outcomes was low to very low due to the observational nature of the studies, imprecision, and indirectness  has not yet been proven; and there have been no identifiable antiviral drugs recommended to reduce vertical transmission of HBV. Thus, characterizing the efficacy and safety of these medications for mother and infant during pregnancy would help inform optimization and potential treatment choices for women of childbearing age.CHB infection remains a major challenge to global health, and by blocking the routes of HBV transmission, global health rates should improve. Vertical transmission is currently the predominant mode of CHB infection, and a linear association between vertical transmission and maternal viral load levels has been demonstrated in several studies25 recently carried out an excellent systematic review involving 26 studies that comprehensively evaluated the efficacy and safety of LAM, LdT and TDF used in pregnancy; and their results revealed that use of LAM and LdT significantly reduced vertical transmission and they were safe in pregnant women, with no increase in adverse maternal or fetal outcomes. These same authors identified no safety issues with TDF use in maternal or fetal outcomes, possibly because their meta-analysis contained an insufficient amount of controlled outcome data. In the present systematic review, we added the latest evidence to clearly delineate the benefits and risks associated with TDF use in pregnant women infected with CHB at a high viral load: synthesis results revealed that TDF was effective and safe in improving maternal HBV DNA suppression at delivery and in reducing fetal HBsAg seropositivity at 4–12 months or HBV DNA seropositivity at delivery. Most importantly, TDF significantly reduced vertical transmission at 4–12 months postpartum, without increasing rates of cesarean section, emergent cesarean section, postpartum hemorrhage of the mother; and prematurity, infant death, and congenital malformations in infants. In the current study, we also found that 1.36%-18% of newborns failed HBV immunoprophylaxis when their mothers were untreated, which was consistent with previously existing evidence13. In addition, maternal virus rebounded and became comparable to levels of the control group at 1 to 6 months after the discontinuation of TDF. The favorable safety profile of TDF has been well demonstrated in HIV mono-infected and HIV/HBV co-infected mothers20; and all of these results, then, support the use of antiviral therapy to reduce maternal viral load and rescue infants from failed immunoprophylaxis.Brown and colleagues27. In fact, in recent studies, TDF has been demonstrated to be effective and safe in pregnant women with CHB who have LAM or LdT resistance28. According to the FDA drug category for pregnancy, TDF and LdT are classified as category B for use in pregnancy; while LAM, ADV and ETV are classified as category C drugs based primarily on animal data16. Although breast-feeding by women who are on antiviral therapy is still controversial and not recommended, data from small studies in HIV-infected women have demonstrated that the median amount of TDF ingested from breast milk was only 0.03% of the recommended pediatric dose30, and thus oral TDF appears safe and is recommended before using other NAs.There were insufficient controlled outcome data to compare TDF with other NAs in CHB-infected pregnant women with high viral load, and results from one included study suggested that there was no statistically significant difference between TDF and LAM on maternal and infant outcomes; however, due to the limitations of potency and low threshold to resistance of LAM and LdT, TDF remains a promising NA used in preventing vertical transmission. TDF is also approved as possessing the highest potency and is the only approved NA without any associated clinical resistance after up to 6 years of monotherapy for CHBThe conclusions of this systematic review should be treated with caution due to several limitations. First, studies warranting high confidence are absent from the present meta-analysis, with only one RCT; and most of the data are derived from observational studies, which are subject to significant biases–especially selection bias. Second, the quality of the evidence for all outcomes was low to very low due to the observational nature of the studies, imprecision, and indirectness. Third, several questions addressed in the previous AASLD HBV Guidelines were not resolved in this systematic review: a) What is the exact viral load threshold and the exact week within the third trimester at which therapy should be initiated?and b) Is it safe for infants to be exposed to antivirals late in pregnancy from longitudinal follow-up and breast-feeding while the mothers are on antiviral therapy? It has been speculated that the risk of HBV vertical transmission (despite active and passive immunoprophylaxis at birth), could be partially attributable to an unsatisfactory approach in terms of the timing of prophylaxis and quality of the vaccine adopted, and thus, c) Is there a relationship between the timing or quality of immunoprophylaxis and the risk of vertical transmission? None of these subjects has been well documented presently because data from controlled studies for these patient populations were sparse and require further corroboration in the future, especially in the form of high-quality, larger-scale RCTs.5 IU/mL). The limited safety data suggest that there is no increased risk for adverse maternal or fetal outcomes; and therefore, in order to prevent vertical transmission, we conservatively recommend the use of TDF in pregnant women with CHB and who manifest high HBV DNA levels of > 2 × 105 IU/mL in the third trimester.In conclusion, oral TDF lowers HBV DNA levels in CHB-infected pregnant women as it does in non-pregnant women, and it reduces the rates of vertical transmission. These effects were demonstrated in CHB-infected pregnant women with high viral loads  Daily and Ovid MEDLINE(R) 1946 to Present, Scopus, Chinese National Knowledge Infrastructure (CNKI), Chinese Biomedical literature database (CBM), and WANFANG databases, with no language limitations. The search strategy was designed and modified according to existing reviews25. In the case of recent studies, we included those entailing the largest number of patients, especially when studies had overlapping (or the same) sample sizes. Incomplete data or missing data and additional studies were obtained by contacting authors by email, telephone, or other mode of communicationWe included randomized controlled trials (RCTs) and controlled observational studies in this meta-analysis if they (a) enrolled pregnant women monoinfected with chronic HBV (characterized by the presence of HBsAg for more than 6 months), with high serum HBV-DNA levels > 6 log copies/ml before antiviral therapy; (b) compared the efficacy and safety profile of TDF with placebo or other NAs; (c) reported the outcomes of interest, including prevention of vertical transmission of HBV, clinical efficacy, and adverse outcomes from antiviral therapy to both mothers and newborns; and (d) provided sufficient information for estimating odds ratios (OR) and 95% confidence intervals (CI). However, studies that enrolled infants who did not receive immunization during the first week postpartum; or that enrolled patients who received steroids, chemotherapy/immunotherapy, liver transplantation, or hemodialysis; or unpublished reports and abstracts or published as abstracts only; or uncontrolled studies, were excludedTwo reviewers (JZC and ZWL) independently and in duplicate screened the titles and abstracts from the literature and identified eligible papers based upon the inclusion criteria. Next, the full texts of potentially eligible papers were further reviewed following the same procedure. Three reviewers  independently extracted the following data using a standardized, pretested Excel form: study characteristics, participant baseline characteristics, intervention details, and outcomes of interest. Discrepancies were resolved through discussion with a third author (ZH).8; rates of cesarean section, emergent cesarean section, postpartum hemorrhage, and adverse events; and elevated levels of creatine kinase (CK). Infant outcomes included vertical transmission (defined as HBsAg seropositivity at 6–12 months or HBV DNA positivity at 6–12 months), prematurity, congenital malformations, and infant death.Outcomes of interest were as follows: maternal outcomes including HBV DNA suppression, HBeAg seroconversion, alanine aminotransferase (ALT) normalization, severe flares of ALT levels 32.Two reviewers (JZC and ZWL) independently assessed the bias risk of RCTs with the Cochrane Risk of Bias assessment tool, and the Newcastle-Ottawa Scale for observational studies. The quality of evidence was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation approach (GRADE); and criteria used to evaluate the quality of evidence were risk of bias, including indirectness (surrogate outcomes), imprecision , inconsistency (heterogeneity), and publication bias2: a value < 25% was considered low; 25–50%, moderate; and > 50%, high. We planned to calculate the pooled effect size using the (I-V heterogeneity) random-effects model when I2 > 50%, and a Mantel-Haenszel (M-H) fixed-effects model otherwise34. The significance of pooled effect size was determined using the Z-test, with a 2-tailed P < 0.05 defined as the significance threshold.We calculated the odds ratios (ORs) and 95% confidence intervals (CIs) using the binomial distribution for dichotomized outcomes, and we planned to use the mean difference (MD) or standardized mean difference (SMD) between the baseline and the longest duration of follow-up for each study of continuous outcomes. Heterogeneity across studies was assessed by calculating I25. All statistical tests for this meta-analysis were performed using STATA, version 12 .If appropriate, we planned to repeat the meta-analysis after excluding individual studies to assess the stability of the results. If the included studies were unsuitable or insufficient, studies were identified for meta-analysis in terms of certain outcomes of interest, and we summarized the results qualitatively; if the included RCTs were insufficient, we planned to synthesize the results of RCTs with controlled observational studies. Publication bias was assessed using the Egger regression asymmetry test and Begg’s funnel plots when a sufficient number of studies (>20) per outcome were available and if heterogeneity was lowAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


In recent years, progress has been made in the characterization of natural killer (NK) cells in lung malignancies, and we have now gained a better understanding of the frequency, localization, phenotype, and functional status of NK cells infiltrating these tumors. NK cell subset recruited in lung cancer is mainly capable of producing relevant cytokines rather than exerting direct cancer cell killing. Thus, the relevance of NK cells in tumor microenvironment might also go beyond the killing of tumor cells, being NK cells endowed with regulatory functions toward an ample array of immune effectors. Nevertheless, boosting their cytotoxic functions and redirecting the migration of cytotoxic NK cell subset to the tumor site might open new therapeutic avenues for lung cancer. Also, we believe that a deeper investigation into the impact of both conventional  or new therapies  on NK cell homeostasis in lung cancer patients is now required. Several lines of evidence support the notion that natural killer (NK) cells play an important role in the control of tumor growth. Notably, along with the large array of results derived from experimental mouse models, a reduced NK cell lytic activity has been associated in human with an increased risk of cancer. Nevertheless, only a very limited number of NK cells can be detected within the majority of human solid tumors.The discovery that NK cell can easily recognize and lyse tumor cells, immediately translated into hope that NK cells might find a place in the clinic as therapeutic tools. Extensive efforts have been made to exploit NK cells into the clinic and many of these attempts have mainly relayed on knowledge obtained by studying peripheral blood (PB) NK cells. However, the environmental context and cytokine milieu in which NK cells are present, and perceive potential activating signals, may strongly impact on their ultimate effector activity, thus offering one of the possible explanations of the inconsistent outcomes of the majority of NK cell-based therapies. As a matter of fact, several studies have aimed to investigate the biology of NK cells present within the neoplastic tissue and the clinical relevance of this infiltrate: in this context, lung cancer, the main cause of cancer-related mortality, represents a major topic for studying the anti-tumoral role of NK cells.+ NK cells at early stages of the disease with the clinical outcome (+CD16+ cells) positively correlated to the survival of lung cancer patients  and inhibitory (ILT-2 and KIRs) receptors, as well as upregulation of NKG2A when compared to the normal counterpart . Along with CD69, CD103 has also been found to be expressed on a subset of NKp46+ cells within the TM  [ [ and persbright NK cells was also observed in pleural effusions (PEs) from different type of primary and metastatic tumors. In contrast with NK cells isolated from solid lung cancer tissues, PE-NK cells express normal levels of both main activating receptors and MHC Class I-specific inhibitory receptors and they rapidly release cytokines upon exposure to neoplastic cells  that is in agreement with the accumulation of non-cytotoxic CD56bright NK cells recruited from PB , which are, on the contrary, preferentially expressed by CD56bright NK cells. This might represent a further mechanism of cancer immunoediting with implications for both immunosurveillance and tumor escape from NK cell attack. Remarkably, also breast cancer, another tumor type characterized by enrichment in non-cytotoxic CD56bright NK cells, displayed upregulation of genes coding for chemokines attracting this subset, when compared with gene expression profile of healthy breast tissues. Since NSCLC are often associated with the presence of intratumoral tertiary lymphoid structures  showed upregulation of KIRs on their surface  able to give rise to both CD56dim and CD56bright NK cells in vitro and in vivo and expressing high levels of CD62L on their surface  pathway, it would be interesting to further investigate the effects of these classes of drugs on NK cell compartment. Anti-PD1 (nivolumab and pembrolizumab), anti-KIR (lirilumab), and anti-NKG2A  may act by removing a brake on the effector functions of NK cells. Anti-PD1 agents have been already approved for the treatment of advanced NSCLC and, very recently, have shown promising results when used in the neoadjuvant setting . Anti-PDWhile we are now accomplishing groundbreaking results by anti-PD1 agents, anti-KIR agents are currently in phase II; nonetheless, they may be important in various settings, in neoadjuvant and advanced settings but also as adjuvant therapy in order to reduce risks of recurrence. Along with KIRs, targeting of the inhibitory receptor NKG2A  is currently under investigation as single agent or in combination with other agents in different types of solid tumors . The ratbright NK cell population infiltrating lung cancers, endowed with low ability to kill cancer cells, can spontaneously acquire KIR receptors and thus further differentiate, suggesting that they might convert in cytotoxic killer cells. Remarkably, CD56bright NK cells, the main NK cell subset infiltrating lung cancer, can undergo robust proliferation upon appropriate cytokine stimulation. At the same time, factors able to efficiently activate NK cells, induce their proliferation and to terminally differentiate them into highly cytotoxic NK cells are currently available as clinical-grade agents   Figure . FinallyWhile further basic investigation on the biology of lung cancer NK cells is clearly required, we recently gained a better insight in this field. Novel immunotherapeutic strategies for lung cancers, conceivably in combination with established anticancer therapies, should now aim at increasing the amount of cytotoxic NK cells in lung cancers: that could be achieved not only recruiting them from PB but also driving their proliferation and differentiation within TM.PC and GF equally contributed in writing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human papillomavirus (HPV)-associated cervical carcinoma is preceded by stages of cervical intra-epithelial neoplasia (CIN) that can variably progress to malignancy. Understanding the different molecular processes involved in the progression of pre-malignant CIN is critical to the development of improved predictive and interventional capabilities. We tested the role of regulators of transcription in both the development and the progression of HPV-associated CIN, performing the most comprehensive genomic survey to date of DNA methylation in HPV-associated cervical neoplasia, testing ~2 million loci throughout the human genome in biopsies from 78 HPV+ women, identifying changes starting in early CIN and maintained through carcinogenesis. We identified loci at which DNA methylation is consistently altered, beginning early in the course of neoplastic disease and progressing with disease advancement. While the loss of DNA methylation occurs mostly at intergenic regions, acquisition of DNA methylation is at sites involved in transcriptional regulation, with strong enrichment for targets of polycomb repression. Using an independent cohort from The Cancer Genome Atlas, we validated the loci with increased DNA methylation and found that these regulatory changes were associated with locally decreased gene expression. Secondary validation using immunohistochemistry showed that the progression of neoplasia was associated with increasing polycomb protein expression specifically in the cervical epithelium. We find that perturbations of genomic regulatory processes occur early and persist in cervical carcinoma. The results indicate a polycomb-mediated epigenetic field defect in cervical neoplasia that may represent a target for early, topical interventions using polycomb inhibitors. Despite the effectiveness of screening programs for cervical cancer in developed countries, it remains the third most common cancer in women, affecting 500,000 worldwide each year, leading to 275,000 deaths in 2008 . Human pIn vitro studies of cervical carcinoma cell lines with DNA methyltransferase inhibitors have shown reversal of silencing of genes and restoration of sensitivity to chemotherapeutic agents [Abnormal regulation of gene expression has been studied as a possible complementary mechanism of development of cervical carcinoma –13. HPV c agents –21.http://cancergenome.nih.gov) allowed us to validate our findings in an independent cohort, leading us to studies of polycomb expression that provide support for an epigenetic field defect in the cervical epithelium of women with CIN who progress to develop cervical cancer.The current study builds upon these prior studies by performing genome-wide DNA methylation studies on biopsies from 78 samples from women across the progressive stages of cervical carcinoma. Our study design is cross-sectional, but used samples collected from women followed up over time, allowing us to focus on individuals with early stage (CIN1) disease that persisted rather than resolved spontaneously. The availability of data from The Cancer Genome Atlas  or decreasing  DNA methylation during disease progression. A further K-means clustering analysis was performed on 4 groups , leadingfindings .−10) representation of genes known to be targets of polycomb-mediated repression , targeting loci known to be polycomb targets, with associated increases in polycomb protein expression as a concurrently early event. Furthermore, the similar findings in the independent TCGA data indicate the robustness of our observations across independent cohorts. The results suggest the cervical epithelium to undergo a ‘field defect’ involving the acquisition of DNA methylation and polycomb-mediated repression, making the cervical cells susceptible to the subsequent effects of mutations that drive growth of cells within an epigenetically dysregulated field of cervical epithelium. Our findings support and extend those of prior studies based on exfoliated rather than biopsy samples of premalignant lesions, implicating acquisition of DNA methylation at developmental targets of polycomb repressive complex as an early and persistent event in cervical neoplasia [This study is the most comprehensive genomic regulatory profiling to date of the progressive stages of cervical carcinoma. Unusually among cancers, cervical carcinoma has a well-documented natural history that permits testing of recognizable precursor states, allowing us to identify pre-malignant molecular events in the cervical epithelial cells. Our findings show that DNA methylation is acquired at eoplasia , 18.EZH2 expression by releasing E2F from pocket proteins like pRb [HOTAIR has been found to recruit the PRC2 polycomb group complex [The relationship between DNA methylation and polycomb-mediated gene silencing is complex, initially believed to be a simple co-repression based on the association of DNA methyltransferases with polycomb group protein complexes , but latlike pRb . The lon complex , and has complex , suggest complex ) may be As a prognostic biomarker of cervical carcinoma, the expression of EZH2 has been previously found to be informative. Consistent with our results, EZH2 was found to be rarely expressed in normal cervical epithelium but increasingly with disease stage progression . This anWe recognize that there are limitations to our study design. We tested varying degrees of CIN and cancer, including what we would consider clinically relevant disease; persistent CIN1, high-grade CIN2/3, and CxCa. However, given that we treat all high-grade CIN and cancer, the development of biomarkers for these groups is of less value than for the preceding CIN1 stage, which is therefore the practical focus of the study. Moreover, we would need a substantially larger study sample size to adjust for potential confounding effects of immune status, HPV genotype and other putative factors that could potentially influence DNA methylation. Additionally, we employed only one secondary validation test, p16/Ki-67, which, while widely used, could be reinforced by additional validation studies.in vitro was found to reduce cell proliferation, motility and invasiveness [in vivo of the agent is unknown, while the in vivo use of DNA methyltransferase inhibitors is recognizably associated with toxicity [The implication of epigenetic dysregulatory events in an epithelial tumor like cervical neoplasia offers a strategy for intervention. Both EZH2  and DNA siveness . The phasiveness , so the toxicity . It is tWe obtained cervical biopsies from 78 HIV-seropositive and negative patients attending gynecology clinics at the Montefiore Medical and North Bronx Hospital Centers in the Bronx (NY), corresponding to normal cervix (19 subjects), persistent low grade cervical intraepithelial neoplasia , high grade CIN2 or 3 (16 subjects), and invasive cervical carcinoma . CD4+T-cell counts and HIV viral load levels in the HIV-seropositive patients assessed within six-months of their CIN/cervical cancer diagnosis were abstracted from the electronic medical records.Patients with low grade CIN1 were sequentially and serially biopsied for 6 months to define a group of women with disease persistence. All 78 biopsies were taken from women who were at least 18 years old. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  or with the Helsinki Declaration of 1975, as revised in 1983. This study was approved by the institutional review board (IRB) at the Albert Einstein College of Medicine and is in accordance with Health Insurance Portability and Accountability Act (HIPAA) regulations. Written informed consent was obtained from all subjects prior to participation.DNA extracted from liquid-based cytology smears was digested with proteinase-K/Laureth-12, precipitated and purified in ethanol, and amplified by PCR with Gold-Taq using a well-described the MY09/MY11 protocol , 36, folcis-regulatory elements than other commonly-used survey assays of DNA methylation [For all 78 cervical biopsies , we extrhylation . To minihylation , 40 to gComBat package in R [multinom function available through the nnet package. The odds of DNA methylation at each disease grade relative to normal cervix were calculated, adjusting for the sequencing batch and the HIV control. Overall model significance was assessed by adjustment for multiple comparisons using a false discovery rate (FDR) <0.05, and two criteria were used for further determination of model significance: (a) CxCa compared to normal cervix methylation effect significance <0.05 and (b) an average methylation difference between CxCa and normal cervix of 10%.To generate high-confidence associations between DNA methylation and the cervical neoplasia disease grades, we used principal components analysis (PCA) to identify biological and technical covariates that were associated with the variability in DNA methylation . Covariaage in R  to obtain loci of interest with a particular annotation X with 100 samples of n loci from all HpaII sites tested 100 times, comparing the observed frequency of X with the distribution of simulated annotation overlaps.We explored where the significant DNA methylation changes were occurring relative to genomic annotations. We tested RefSeq gene promoters (the 2 kb region flanking the gene transcription start site (TSS)), the remaining gene body and intergenic regions, and CpG islands and their 2 kb flanking region (shores ). We meakmeans function in R. We determined an optimal number of clusters by plotting the total within cluster sums of squares against the number of clusters and selecting a cluster value that occurred at the first inflection point.To study the dynamic changes of DNA methylation with progression of the disease, we used K-means clustering, an unsupervised clustering approach, using the Using the GSEA  tool froMethPrimer tool, we designed primers to amplify bisulphite-converted target DNA sequences. BiSearch was used to avoid primer sets that generate off-target amplicons. Bisulphite treated DNA was pre-amplified with an equimolar primer mix, then unique dual indexed adapters were added and libraries amplified using a Fluidigm Access Array. The resulting amplicon library was sequenced using 150 bp paired end sequencing with the Illumina MiSeq platform. Reads were aligned to the human genome and DNA methylation ratios were calculated using BSMAP 2.7.3.We performed large scale targeted bisulphite sequencing to verify DNA methylation results from 66 individuals  at 70 different loci. These HpaII loci were chosen to test the full range of HELP-tagging values in each of the four disease groups. Using the UCSF Using The Cancer Genome Atlas (TCGA) datasets for cervical squamous cell carcinoma and endocervical adenocarcinoma, we identified 196 samples with Cervical Squamous Cell Carcinoma, for which 183 samples had DNA methylation tested using Infinium HumanMethylation450 Methylation arrays, 192 had RNA-seq gene expression and 155 had somatic mutation data available. We associated DNA methylation at sites located ±2 kb from the RefSeq TSS with that gene, while RNA-seq data were already associated with genes. For DNA methylation and RNA-seq, we plotted the density of signal for all genes, all PRC2 target genes and the subset of PRC2 genes implicated by K-means clustering to have early, progressive promoter DNA methylation. For somatic mutation data, we plotted the density of the number of samples that contained at least one nonsense mutation occurring in all genes, all PRC2 target genes and all PRC2 genes implicated as having early, progressive promoter DNA methylation.We used immunohistochemistry (IHC) on 15 of the primary samples used for our DNA methylation studies to test expression of the candidate EZH2 protein implicated by our DNA methylation studies. We identified regions within each biopsy in which there was an area of low grade CIN and, when possible, an area of normal epithelium, and compared between samples the presence of EZH2, its expression location within the epithelial layers and the level of expression per cell. A relative scoring system was used for EZH2 signal intensity, and the proportion of epithelium with EZH2 signal was also assessed by the pathologist.All sequencing data generated in this study are deposited at the Gene Expression Omnibus and are available in Series GSE76986.


Despite administration of novel therapies, multiple myeloma (MM) remains incurable with resistance to drugs leading to relapse in most patients. Thus, it is critical to understand the detailed mechanisms underlying the drug resistance of MM and develop more effective therapeutic strategies. Genetic abnormalities are well known to play a central role in MM pathogenesis and therapy resistance; however, epigenetic aberrations mainly affecting the patterns of DNA methylation/histone modifications of genes  and miRNAs have also been shown to be involved. Importantly, while epigenetic silencing of miRNAs in MM is well documented, some epigenetic markers are known to be direct targets of miRNAs particularly the recently described “epimiRNAs”. Drugs targeting epigenetic modifiers  can sensitize MM-resistant cells to anti-myeloma drugs and reversibility of epigenetic changes makes these drugs promising therapeutic agents. Therefore, combination of miRNA mimics with inhibitors of epigenetic modifiers would be a more potent therapeutic strategy in MM patients in relapse or refractory to treatments. In this review, we will discuss the findings of recent investigations on epigenetics/miRNA regulatory axis in development of drug resistance in MM and highlight possible approaches for therapeutic applications of such interaction. Multiple myeloma (MM) is a clonal expansion of plasma cells that is characterized by proliferation of malignant clones producing defective monoclonal immunoglobulins in the bone marrow. Current therapies such as proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) have improved the outcome of patients. Nevertheless, not all patients respond well to the drug, and even in responding patients usually relapse occurs. Thus, multidrug resistance is still the major problem for the effective treatment of multiple myeloma with conventional drugs , 2. ReseAlthough the molecular mechanisms of DR in MM are not fully understood, epigenetic abnormalities have been suggested to play an important role . In factIn addition, DNA hypermethylation has been detected in some tumor suppressor, cell signaling, and cell adhesion molecule genes in plasma cell leukemia (PCL) cells . AnalyziFurthermore, alterations in histone methyltransferases can also mediate chemotherapy resistance in MM including cell adhesion-mediated drug resistance (CAM-DR) which is a rather complex and poorly explored form of DR in MM. Kikuchi et al. demonstrated that direct adhesion to bone marrow stromal cells inactivated (phosphorylated) the histone methyltransferase enhancer of zeste homolog 2 (EZH2) which resulted in H3K27 (histone H3-Lysine 27) hypomethylation. This in turn led to sustained expression of anti-apoptotic genes such as IGF1, BCL2, and hypoxia inducible factor 1-α (HIF1A) . The aboOn the other hand, oncogenic functions of EZH2 have also been reported by some studies. For instance, it was shown that silenced polycomb target genes were more frequent in MM and ChIP-seq profiling data revealed increased number of silenced H3K27me3 (Histone H3 lysine 27 trimethylation) target genes in MM patients at advanced stages of the disease, and the expression pattern of H3K27me3-marked genes was correlated with poor patient survival . In addiIt is important to note that most studies concerning epigenetics in MM pathogenesis focused on EZH2-mediated transcription repression of target genes and it is not clear whether somatic mutations causing EZH2 gain or loss of function could also play a role in MMDR. Indeed, both types of mutations have been reported in other hematologic malignancies leading to biologic and clinical outcomes that indicate context-dependent tumor suppressor or oncogenic function of EZH2 . Taken tMany studies have demonstrated that miRNAs could be involved in DR of MM  in many cancer types including MM . MiR-34aStudies from our group , 52 and Moreover, some oncogenes are targeted by hypermethylated miRNAs in MM and hypomethylation of miRNA genes by using DNMT inhibitors can downregulate those oncogenes and inhibit cell growth and induce apoptosis in MM cells. For example, RecQ helicases (DNA unwinding enzymes involved in the maintenance of chromosome stability) are significantly upregulated in MM and protect MM cells from melphalan and bortezomib cytotoxicity. DNMT inhibitor treatment of MM cells results in RECQ1 downregulation through miR-203 demethylation and sensitizes cells to anti-tumor drugs suggesting that epigenetic modifier could be useful for treatment of relapsed cases .Resistance to drugs could also be associated with histone modifications  of miRNA promoters, another epigenetic mechanism regulating expression of miRNAs in cancers , 55, 56,The chromatin remodeling enzyme EZH2 is probably the most attractive epigenetic modifier in cancers , 59, 60 It is interesting to note that a group of miRNAs, termed “epi-miRNAs”, has been reported to reciprocally modulate epigenetic regulators, suggesting the existence of a regulatory circuit between miRNAs and epigenetic modifiers . The besTaken all together, in the setting of MM cells, the mutual interaction between miRNAs and epigenetic markers plays an important role in regulation of drug response of MM cells.Strategies for clinical application of epigenetic inhibitors including DNMT, HDAC, and HAT inhibitors in MM therapy have been reviewed elsewhere , 66. GenCurrent era of MM therapy is witnessing the significant progress of strategies and approaches aiming mostly at overcoming the DR. While most novel treatments including proteasome inhibitors especially in combination modalities have proved to increase the survival of patients, MM still remains to be drug resistant and most patients relapse or become refractory. Studies have demonstrated that miRNA may be applied for the targeted delivery of personalized medicine to improve the outcome of MM patients . FurtherIn conclusion, epigenetics-miRNA axis plays a crucial role in MM pathogenesis and could provide potential therapeutic targets. However; due to limited studies, further in-depth studies in this regard are required to open a novel and exiting venue to understand the underlying mechanism of DR in MM, which tends to be the outstanding obstacle to MM therapy.


Acoustic phonetics methods are useful in examining some symptoms of schizophrenia; we used such methods to understand the underpinnings of aprosody. We hypothesized that compared to controls and patients without clinically rated aprosody, patients with aprosody would exhibit reduced variability in: pitch, jaw/mouth opening and tongue height (formant F1), tongue front/back position and/or lip rounding (F2), and intensity/loudness.Audio-recorded speech was obtained from 98 patients  and 102 unaffected controls using five tasks: one pertaining to describing a drawing (Task 1), two based on spontaneous speech elicited through a question (Tasks 2 and 3), and two based on reading prose (Tasks 4 and 5). We compared the three groups  in terms of variation in pitch, formants F1 and F2, and intensity/loudness.Phonetic values were generally highly correlated across the five speech tasks. Regarding pitch variation, in unadjusted tests, patients with aprosody differed significantly from controls in Tasks 3 and 4; for Task 5, the difference was statistically significant in both unadjusted tests and those adjusted for sociodemographics. For the standard deviation (SD) of F1, the expected pattern was observed in the two reading tasks in adjusted tests . Regarding SD of F2, patients with aprosody had lower values than controls in unadjusted tests across all tasks; in adjusted tests the expected pattern was observed in the two spontaneous speech tasks. Comparisons of variation in intensity/loudness, despite a much smaller sample size of participants with data on this variable, showed the expected pattern in adjusted tests.Although values of each individual parameter across the five tasks tend to be highly correlated, it appears that different types of prompts for obtaining audio-recorded speech may in fact produce some differences across phonetic parameters. For example, whereas loudness appeared to be blunted equally across all of our tasks, variation in both pitch and F1 were blunted most obviously in the reading tasks, and reduced variation in F2 was most apparent in the two spontaneous speech tasks. Small sample size, no measures of negative symptoms in healthy controls and not controlling for patients’ medications are the main limitations of this work. Nonetheless, findings could represent a step toward developing new methods for measuring and tracking the severity of this specific negative symptom using acoustic phonetics parameters. Such work is relevant to other psychiatric and neurological disorders.


Pioneer factors are proteins that can recognize their target sites in barely accessible chromatin and initiate a cascade of events that allows for later transcriptional activation of the respective genes. Pioneer factors are therefore particularly well-suited to initiate cell fate changes. To date, only a small number of pioneer factors have been identified and studied in depth, such as FOXD3/FOXA1, OCT4, or SOX2. Interestingly, several recent studies reported that the PBC transcription factor PBX1 can access transcriptionally inactive genomic loci. Here, we summarize the evidence linking PBX1 with transcriptional pioneer functions, suggest potential mechanisms involved and discuss open questions to be resolved. These processes are carried out by a special type of DNA binding proteins, termed pioneer TFs  gene already in embryonic stem cells (ESCs), although the gene is not transcriptionally activated until hepatocyte differentiation , together with additional co-regulatory proteins. In addition, there is accumulating evidence that efficient s Smale, . Two of s Smale, .Since the 1990s, a number of additional pioneer TFs were discovered. The emerging definition of a pioneer factor comprises the ability to (1) engage its target site(s) in closed chromatin prior to gene activation, (2) increase chromatin accessibility for other proteins at this site, and (3) establish competence for cell fate changes and thus play a key role in cellular (re-) programming , epigenetic modifications that occur on active enhancers  superclass, a separate branch of the homeodomain proteins characterized by the name-giving Three Amino acid Loop Extension (“TALE”) between the first and second alpha-helix of the homeodomain  TFs are evolutionarily conserved, atypical homeodomain proteins. Phylogenetically, they constitute one class of the D. melanogaster, because mutants of the fly PBC protein extradenticle displayed homeotic transformations similar to those seen in Hox-mutant animals, without altering the expression of the respective Hox genes themselves  ] in pre-B cell acute lymphoblastic leukemia (ALL) that resulted in the expression of an oncogenic E2A-PBX1 fusion protein  significantly reduced the production of neurons and increased the generation of oligodendrocytes in vitro and in vivo, establishing Pbx1 as an early lineage regulator of SVZ neurogenesis. Loss of Pbx1 expression in neuronally committed neuroblasts, by contrast, severely compromised cell survival. By chromatin immunoprecipitation from endogenous tissues or isolated cells, we identified the neuron-specific gene doublecortin (Dcx) and the dopaminergic neuron marker gene tyrosine hydroxylase (Th), as direct PBX1 target genes.The subventricular zone (SVZ) in rodents is an adult stem cell niche that provides new neurons and glia to the brain. In brief, adult stem cells residing in the SVZ produce young neurons, termed neuroblasts, via an intermediate population of transient amplifying progenitor cells. Neuroblasts leave the SVZ and migrate into the olfactory bulb where they terminally differentiate to distinct types of interneurons that are continuously replaced in the existing circuitry as part of a life-long remodeling of the olfactory system. A number of TFs, including DLX2 and PAX6, bias progenitor cells toward a general neuronal fate and promote their subsequent maturation to defined types of interneurons programming—have all been demonstrated for PBX1. Yet, each of these features has been investigated in a different physiological setting and with different tools. A unifying model is therefore still missing. In addition, little is known about whether the other three members of the mammalian PBC family, PBX2-4, also possess the ability to recognize their target sites in silent chromatin or even pioneer factor activity. A coherent assessment of PBX pioneering function by standardized approaches is therefore an important next step in research on this protein family.In conclusion, multiple lines of evidence, obtained from DS and BG jointly wrote the manuscript and approved it for publication.Research related to this Review was funded by the Deutsche Forschungsgemeinschaft, grant SCHU1218/3-1 to DS and a Ludwig Edinger Fellowship to BG.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. The reviewer CC and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Epithelial ovarian cancer is a highly heterogeneous disease characterized by multiple histological subtypes. Molecular diversity has been shown to occur within specific histological subtypes of epithelial ovarian cancer, between different tumors of an individual patient, as well as within individual tumors. Recent advances in the molecular characterization of epithelial ovarian cancer tumors have provided the basis for a simplified classification scheme in which these cancers are classified as either type I or type II tumors, and these two categories have implications regarding disease pathogenesis and prognosis. Molecular analyses, primarily based on next-generation sequencing, otherwise known as high-throughput sequencing, are allowing for further refinement of ovarian cancer classification, facilitating the elucidation of the site(s) of precursor lesions of high-grade serous ovarian cancer, and providing insight into the processes of clonal selection and evolution that may be associated with development of chemoresistance. Potential therapeutic targets have been identified from recent molecular profiling studies of these tumors, and the effectiveness and safety of a number of specific targeted therapies have been evaluated or are currently being studied for the treatment of women with this disease. Ovarian cancer is the leading cause of gynecologic cancer death in developed countries and often presents at an advanced stage . The curCurrently, there is no established screening test for ovarian cancer, thereby hindering early stage detection of this disease. An assessment of ovarian cancer screening conducted in 1998 by the National Health Service Health Technology Assessment Programme based on a review of 25 ovarian cancer screening studies was that the routine use of cancer antigen 125 (CA125) serum testing and transvaginal ultrasound was unwarranted . While sOn both histologic and molecular levels, it is becoming increasing clear that ovarian cancer is a clinically diverse and morphologically and molecularly heterogeneous disease . AlthougTraditionally, high-grade serous carcinoma, a common and typically aggressive form of ovarian cancer, has been considered to arise from a well differentiated, low-grade form of serous carcinoma ,15. HoweThese studies have shown that low-grade serous carcinoma is unlikely to be a precursor lesion for high-grade serous carcinoma; rather, the two conditions are generally believed to be separate diseases characterized by differing patterns of genomic variation, with distinctly different prognostic implications ,15,18. UThis new classification scheme not only facilitates more accurate characterization of disease and prediction of patient prognosis, it also provides insight into the mechanisms underlying the development of epithelial ovarian cancers. For example, most type I tumors are believed to arise from endometriosis or borderline serous tumors whereas type II tumors are generally believed to principally originate in the fallopian tube ,14,15,18KRAS or BRAF), as well as a number of other genomic variants , CDKN2A, ,PIK3CA and PTEN have also been found in a number of studies of type I lesions; mutations in TP53 are rarely seen in these tumors, with the exception of mucinous carcinoma in which TP53 mutations occur relatively frequently (ARID1A (a tumor suppressor gene involved in chromatin remodeling) in ovarian clear cell carcinomas [ERBB2 (gene encoding for HER2) in 19% and 14% were reported in mucinous tumors and clear cell carcinomas of the ovary, respectively [BRCA1, BRCA2, CHEK2, ATM, and germline mutations in BRCA1, BRCA2, RAD51D, CHEK2 and BRIP1, although BRCA1/2 alterations were less common compared with serous ovarian cancers [TP53 , occurring within the fallopian tubes, are being closely scrutinized as possible precursor lesions of high-grade serous ovarian cancer ,18,42, aproposal . Neverthproposal ,45,46.As previously detailed, molecular characterization of ovarian cancer specimens has uncovered a wide range of tumor heterogeneity within epithelial ovarian cancer  that extends to tumors of the same histological subtype. In addition, these types of studies have also demonstrated the existence of both intra- and intertumoral heterogeneity at the level of the individual patient, a phenomenon that has also been observed in other tumor types . SynchroNext-generation/high-throughput sequencing has been used to investigate tumor evolution of specimens of high-grade serous ovarian cancer ,51,52,53ETV4, FOXM1, LSR, CD9, RAB11FIP4, and FGFRL1. However, the authors stated that further work is needed before therapeutic applications of these findings are elucidated.Beyond DNA sequencing, the molecular characterization of epithelial ovarian cancers at the level of mRNA expression has the potential to identify and quantify specific transcripts expressed within the tumor specimen at a given point in time, and to provide an enhanced understanding of tumor evolution on a detailed molecular level. Gene expression profiling studies conducted within the last decade have focused on the elucidation of differential gene expression between epithelial ovarian cancer and normal ovarian tissue using RNA microarray analysis. These studies have provided evidence for the existence of several distinct molecular subtypes of high-grade ovarian cancer  that correlate with clinical outcome ,56,57. ARB1, NF1, RAD51B, and PTEN, and that resistant/refractory disease was frequently associated with a relatively high frequency of CCNE1 amplification. Moreover, the low frequency of point mutations observed in earlier next-generation/high throughput sequencing studies of high-grade serous ovarian cancer was also observed in chemoresistant recurrent disease. In this study, which also involved matched sequencing of germline DNA samples, there were cases of reversions in germline BRCA1 or BRCA2 mutations as well as loss of BRCA1 promoter hypermethylation in the setting of acquired resistance. In addition, the ABCB1 gene encoding for the multi-drug-resistant protein 1 (MDR1) was upregulated as a result of promoter translocation and fusion events in approximately 8% of samples of patients with recurrent disease. The results of this study, as well as several other recent studies utilizing next-generation/high throughput sequencing technology to characterize molecular changes in high-grade serous ovarian tumors over time in response to the selective pressure of chemotherapy suggest that both selection of subclones present in the primary tumor as well as acquisition of a limited number of new genomic alterations may be associated with the development of acquired chemoresistance in patients with recurrent high-grade serous ovarian cancer [BRCA1 or BRCA2 in platinum-sensitive, relapsed tumors after a single line of standard-of-care, platinum-based chemotherapy compared with untreated primary tumors [Genomic heterogeneity can occur as the result of selective pressures  that induce changes to the molecular landscape of epithelial ovarian tumors. A study involving whole genome sequencing of specimens from patients with chemoresistant high-grade serous ovarian cancer has recently been reported . An advan cancer ,51,61,62One of the main objectives of developing a molecular classification of epithelial ovarian cancer that reflects the underlying pathogenesis of the disease is to facilitate individualized treatment selection. However, despite the complexity of ovarian cancer, the current standard-of-care for the treatment of this disease is only minimally dependent on histological subtype or other disease classification scheme, and platinum-based chemotherapy is currently recommended as primary systemic therapy for most patients with epithelial ovarian cancer . FurtherAlthough molecularly-targeted therapies are urgently needed for patients with ovarian cancer, these types of treatment approaches necessitate knowledge of one or more driver mutations that alter the function of a limited number of cellular signaling pathways. In addition, selection of targeted therapy is based on the premise that these genomic alterations are “actionable” in the sense that they have significant therapeutic implications in subsets of patients with ovarian cancer and for specific therapies. At the present time it is likely that only a limited portion of the highly complex molecular information available through the detailed molecular characterization of epithelial ovarian cancers by next-generation/high throughput sequencing will be translatable into targeted therapeutic approaches . MoreovePresently, only two targeted agents have received approval for the treatment of ovarian cancer by the U.S. Food and Drug Administration and the European Medicines Agency (EMA). These are the polyadenosine diphosphate (ADP)-ribose polymerase (PARP) inhibitor, olaparib, and the antiangiogenic agent, bevacizumab, both of which are approved for patients with advanced disease that has been pretreated with chemotherapy ,66; firsMultiple potential therapies targeted against specific molecular alterations are currently being investigated for the treatment of ovarian cancer, although it is likely that they will be useful in particular patient subsets only. The molecular targets and associated targeted therapies listed in the section below illustrate the diversity of therapeutic approaches that are being evaluated in advanced ovarian cancer, although this list is not meant to be an all-inclusive. BRCA1 or BRCA2 tumor suppressor genes since those molecular alterations are also associated with impairment of homologous recombination in DNA repair. More specifically, use of PARP inhibitors in patients with tumors characterized by deficient homologous recombination is an example of synthetic lethality, cell death resulting from a combination of mutations in two or more genes. In addition, there is evidence that the combination of a PARP inhibitor with a platinum agent, which also damages DNA and interferes with its repair, may provide another level of synergism [PARP inhibition has been shown to lead to an accumulation of DNA strand breaks, and is believed to be particularly effective in patients with ovarian cancer who have mutations in the ynergism ,71.BRCA1 or BRCA2 mutations, including 193 patients with recurrent ovarian cancer [BRCA1/2 [BRCA1/2 mutations experienced a substantial PFS benefit with niraparib therapy compared with placebo , 0.17 to 0.41; p < 0.001), perhaps even more notable was the finding that in the niraparib arm, the cohort of patients without a germline BRCA1/2 mutation also experienced a marked benefit vs. placebo . In this latter group, niraparib-associated PFS benefit was more pronounced in patients with somatic mutations in BRCA1/2 or other defects in homologous recombination, emphasizing the potential utility of biomarkers other than BRCA1/2 in selecting patients for treatment with PARP inhibitors. However, long-term benefit of niraparib was also observed in approximately 20% of the patients without either of these biomarkers. Overall, niraparib was well tolerated although grade 3/4 hematologic toxicities were observed in approximately 20% to 33% of patients receiving niraparib. Regulatory approval of olaparib for the treatment of advanced ovarian cancer is based on results of a nonrandomized phase II trial of 298 patients with solid tumors and germline n cancer ,73. In tn cancer ,75. More[BRCA1/2 . AlthougBRCA1 mutations in the trabectedin arm were significantly higher compared with this subgroup of patients receiving chemotherapy alone. However, in the subgroup of patients classified as BRCA1 wild-type, the addition of trabectedin to chemotherapy was not associated with a difference in OS compared with chemotherapy alone [In this context, it is also worth noting the data from a recently published exploratory analysis of patients enrolled in the phase III OVA-301 study investigating the use of pegylated liposomal doxorubicin with or without trabectedin in patients with recurrent ovarian cancer. In this analysis, the response rate, PFS and OS for the subgroup of patients with germline py alone . A phasepy alone .The molecular rationale for the use of antiangiogenic therapy is based on the results of studies showing that increased levels of vascular endothelial growth factor (VEGF) in ovarian cancer were associated with increased resistance to chemotherapy and a poorer prognosis . BevacizA current drawback with the use of antiangiogenic therapy is the lack of a molecular biomarker to identify patients most likely to benefit from such an approach. However, recent studies investigating response to bevacizumab in molecularly-defined subgroups of women with ovarian cancer have provided initial support for the feasibility of such a molecularly-guided approach . For exap < 0.0001) for the comparison of arms C and A, although toxicity was increased for patients receiving cediranib. Interestingly, results from a recent phase II study have demonstrated that PFS in patients with recurrent, platinum sensitive high-grade serous ovarian cancer was significantly longer  in patients receiving the combination of cediranib and olaparib compared with olaparib alone, suggesting that the two agents may act synergistically [Tyrosine kinase inhibitors that specifically target the VEGF receptor or act as multi-targeted receptor tyrosine kinase inhibitors, including cediranib, pazopanib, and sunitinib, are also under investigation in the setting of advanced ovarian cancer . For exastically .PIK3CA and PTEN), a prototypic survival pathway, have been identified in type I and type II tumors  which has been shown to form a stable complex with mutant p53, preventing the degradation of the latter protein, thereby resulting in its accumulation [Another novel method of targeting mulation ,116. A tmulation .KRAS and BRAF mutations found in many of these tumors  are short, non-coding RNAs that are thought to function as posttranscriptional gene regulators that can either suppress or stimulate tumor growth . DiffereThe concept of targeting the immune system to attack cancer cells has recently emerged as a promising new avenue for the treatment of ovarian cancer. Immune cells can recognize and destroy aberrantly proliferating cells, and this activity is modulated by immune checkpoints expressed on T-cells ,136. IncData from a phase II trial examining the safety and efficacy of nivolumab, an antibody against the programmed cell death-1 (PD-1) immune checkpoint, in 20 patients with platinum-resistant ovarian cancer were recently published . TreatmeAlternative immune-based approaches in development for the treatment of ovarian cancer include vaccines, cytokines, and adoptive T-cell therapy, a treatment involving lymphocyte transfusion . As moreThe high degree of heterogeneity associated with epithelial ovarian cancer has long been an impediment to its effective characterization and to optimizing the treatment of patients with this disease. However, molecular characterization of different histological subtypes of epithelial ovarian cancer, including results of more recent studies utilizing next-generation/high throughput sequencing technology, have facilitated the development of a more unified approach to the classification of these tumors. In conjunction with multiple tumor sampling separated through space and time, this technology is likely to be fundamental in uncovering evolutionary patterns involved in the etiology of tumorigenesis in epithelial ovarian cancer, particularly the molecular processes underlying the pathogenesis of high-grade serous ovarian cancer, as well as the site(s) of precursor lesions and the basis for the development of metastatic disease. The limited effectiveness of most single-agent targeted therapies evaluated thus far and the absence of a standard-of-care for patients with advanced epithelial ovarian cancer that is resistant/refractory to platinum-based chemotherapy represent major unmet needs. While the recent strides made in uncovering molecular details of epithelial ovarian cancer have been considerable, the clinical implications of this information are only beginning to be understood. For example, new avenues being opened up through the use of next-generation/high throughput sequencing technology include approaches to characterize the genes responsible for drug resistance in order to facilitate personalized selection and targeted delivery of treatment to suppress the development of such resistance ,141. It


Similarities on the cellular and neurochemical composition of the amygdaloid subnuclei suggests their clustering into subunits that exhibit unique functional organization. The topological principle of community structure has been used to identify functional subnetworks in neuroimaging data that reflect the brain effective organization. Here we used modularity to investigate the organization of the amygdala using resting state functional magnetic resonance imaging (rsfMRI) data. Our goal was to determine whether such topological organization would reliably reflect the known neurobiology of individual amygdaloid nuclei, allowing for human imaging studies to accurately reflect the underlying neurobiology. Modularity analysis identified amygdaloid elements consistent with the main anatomical subdivisions of the amygdala that embody distinct functional and structural properties. Additionally, functional connectivity pathways of these subunits and their correlation with task-induced amygdala activation revealed distinct functional profiles consistent with the neurobiology of the amygdala nuclei. These modularity findings corroborate the structure–function relationship between amygdala anatomical substructures, supporting the use of network analysis techniques to generate biologically meaningful partitions of brain structures. Although small in total size, its complex composition of structurally and functionally heterogeneous subnuclei supports these multiple functions. Histological studies suggest that the amygdala may be composed of at least twenty subnuclei2 that share cytoarchitecture, myeloarchitecture and chemoarchitecture features. Based on these similarities, schemas have been proposed to segregate these nuclei into subunits based on their functional and anatomical characteristic3. Although the boundaries and even the names of these subunits remain unsettled, one of the most widely accepted classification schemas classifies them as the superficial (SF) (corticoid) amygdaloid nucleus, the centromedial (CM) group and the laterobasal (LB) complex6. A stereotaxic probabilistic map of the human amygdala, based on the superposition of cytoarchitectonic mapping of cell-body stained histological sections, has been developed by Amunts and colleagues5 and classifies the SF group to include the anterior amygdaloid area, the amygdalopyriform transition area, the amygdaloid-hippocampal area and cortical nuclei , the CM group combines the central and medial nuclei, while the LB group incorporates the lateral, basolateral, basomedial and paralaminar nuclei. This mapping is quite consistent with the cellular and neurochemical composition of the amygdaloid nuclei, since the CM, which is continuous with the bed nucleus of stria terminals, is comprised of cells that are morphologically similar to those in the striatum, while the LB and SF resemble cortical area10. Additionally, the LB is the main amygdala division that receives efferent projections from basal forebrain cholinergic neurons projecting mostly to the basolateral nucleus11.The amygdala is involved in a series of emotional processes and cognitive functions including learning, memory, attention and perception12/superficial-cluster13 and inferior12/deep-cluster13, representing the LB and smaller nuclei (inferior/deep-cluster) and the CM and cortical nuclei 14, others found the amygdala subdivided into four main subregions, dividing LB into lateral and basal regions and the CM into central and medial, leaving the cortical nuclei undifferentiated15. Therefore, these findings are not fully in agreement with the amygdala subdivisions shown in the stereotaxic probabilistic map5.The neurobiological composition of these main subdivisions indicates a potentially exclusive functional/anatomical neural interaction that may be captured either in synchronized fluctuation of the fMRI signal, as in rsfMRI, or through anatomical connections, such as, diffusion tensor imaging (DTI). For this reason, different functional and structural MRI based approaches have been proposed to parse the amygdala architecture. As a result, while some studies found the amygdala subdivided in two main regions, which were identified as superior17, using imaging approaches, found three subdivisions of the amygdala forecasted by the probabilistic atlas5. The first study proposed a method that started from a hypothetical topographic model, based on the functional-anatomic organization of brain networks subserving social cognition, by defining three social networks: perception, which would be associated with the ventrolateral sector of the amygdala; affiliation, related to the medial section of the amygdala; and aversion, linked with the dorsal sector of the amygdala16. As a result, three amygdala regions were pre-defined in those locations  and using an iterative seed-target-seed methodology, the border of these subdivisions were established. The second study proposed an imaging-parcellation method based on meta-analysis, where three amygdala subdivisions were identified by computing whole-brain co-activation patterns for each amygdala seed voxel and grouping these seeds based on similarities between their co-activation patterns17. This method required a systematic analysis of a broad range of associated experiments  to increase the robustness of parcellation results and to avoid dependence on any particular user-specified parameter. Therefore, while the first approach16 predefine the initial number of subdivisions, the second approach was a meta-analysis requiring a significant amount of data from different imaging sources.Two recent studies18. Furthermore, by using hierarchical modularity analysis to explore the community structure of brain networks20, previous studies have shown that functionally and anatomically related brain regions are more densely interconnected, with relatively few connections between these clusters, indicating that these networks are intimately related and share common topological features, such as modules and hubs21. Therefore, based on these concepts, we employed the principle of modularity to identify subnetworks within the amygdala. We hypothesized that the modular organization of the amygdala will reflect its internal functional segregation and integration consistent with the neurobiological properties of its nuclei. For this purpose, we applied the community structure algorithm to a high spatial and temporal resolution rsfMRI data from the Human Connectome Project (HCP)23. We further assessed the functional consequence of each identified subnetwork by determining their functional connectivity and then by correlating these circuits with amygdala activation during an emotional processing task. Our findings validate the association between the modular structure of the amygdala and the biological characteristics of amygdala subdivisions.In this work, we propose to use resting state BOLD data and the topological principle of community structure to identify the main subdivisions of the amygdala. Our approach is based on global topological characteristics that has been shown to produce stable brain structural and functional networks, suggesting that the observed hierarchical modular organization coincide with the underlying anatomical communities at different scales5, displayed in the Juelich histological atlas , but better represents the average amygdala size in healthy adults24. Despite size discrepancies, the synergy between the relative size of the subdivisions found in the Juelich atlas (LB > SF > CM) and the modules obtained from our modularity approach , together with the closeness between the subdivision location, suggest labeling the dorsal module as CM, the medial as SF and the lateral as LB.Modular analysis of the amygdala revealed three distinct modules Fig.  that arePatterns of significant correlation for each amygdala subdivision reveal unique connectivity circuits. MRI signal at the CM was mainly correlated with the signal in the middle and anterior cingulate cortices, frontal cortex, striatum, insula, cerebellum and precuneus, while a negative correlation was observed at the occipital gyrus Fig. , Table 1The LB nuclei was positively correlated with the superior, medial and inferior frontal gyri, precentral gyrus, paracentral lobule, middle temporal gyrus and cerebellum, and negatively correlated with the parietal lobule, precuneus and cingulate gyrus Fig. , Table 1Activity in the SF correlated with signal fluctuations in the orbitofrontal cortex, posterior insula, olfactory cortex, precentral gyrus, paracentral lobule and posterior cingulate, and was anti-correlated in the anterior cingulate cortex, superior and middle frontal gyrus, cerebellum, parietal lobule, inferior temporal gyrus and precuneus Fig. , Table 1Pairwise comparisons show that the connectivity strength of the SF subdivision is higher when compared with CM based circuits in the occipital lobe, PCC, precentral and postcentral gyrus, paracentral lobule, superior frontal gyrus and OFC and higher than the connectivity path of the LB at medial and inferior frontal gyrus, precuneus, PCC, precentral and postcentral gyrus, middle temporal gyrus, precentral gyrus and OFC Fig. .A two-way ANOVA (session x seed location), comparing the connectivity pattern obtained for each amygdala subunit and for each session (subseries), revealed a main effect of the seed location in the striatum, insula, OFC, ACC, superior medial gyrus, occipital gyrus, medial, middle and inferior frontal gyrus, cingulate gyrus, inferior parietal lobule, cerebellum, superior and middle temporal gyrus Fig. , Table 3Altogether, these results indicate a stable pattern of brain connectivity for each modularity based amygdala substructure across sessions Fig.  that gen, while no significant correlation between BOLD and corresponding connectivity maps was observed for the others two amygdala subdivisions.As expected, the entire amygdala was significantly activated when subjects performed the face/shape matching task Fig. 1. HoweveFigs  and 5S,17, but with a method that did not require an extended meta-analysis. Moreover, modularity has shown to be very reliable, even with small datasets; the results from the HCP dataset (98 subjects) reproduced our previous pilot findings from a group of just 18 subjects, acquired in our center with different EPI sequences and different set of parameters , or bottom-up, as in Louvain’s method27, which is a hierarchical clustering approach, where nodes are progressively merged to others nodes becoming larger and larger sets of nodes, a process that is repeated iteratively until no increase of modularity is possible28. By using the Louvain’s method, we were able to identify amygdala modules that resemble the most accepted classification schemes for its anatomical subdivision5, suggesting a degree of functional segregation of these subunits that is consistent with the biological characteristics of the amygdala substructures.Our work further supports that topological properties may have an impact on the study of structural-function relations in brain networks. Whilst it helps to understand the fundamental architecture of connections within and between brain regions, it also provides a way to elucidate how this architecture supports neurophysiological dynamics. Modularity approaches can be either top-down, such as the Newman’s method29 and Optimal Compression method30, each having particular strengths and weaknesses. For example the Surprise method29 is best suited for the study of binarized networks. However, it is vulnerable to noise errors that affect small modules, and in the case of the amygdala, located in a brain area with low SNR, it may not necessarily improve the quality of the partitions. The Optimal Compression method30, on the other hand, is not recommended when “community structure are considered as statistical deviation from the null model in which the degree sequence is held constant but links are otherwise equiprobable among all nodes, wherein the modularity optimization methods by definition provides the optimal partitioning”30. In contrast, by construction, the Louvain method26 is able to unfold a complete hierarchical community structure for the network, with each level of the hierarchy being given by the intermediate partitions found at each pass. In our work, we considered only the top level of this hierarchy, namely the final partition found by the algorithm, since no further meaningful sub-module was found in the amygdala  are identified in the connectivity pathway of the amygdala subdivisions.Temporally correlated patterns of low-frequency fluctuations during rest revealed regions of distinct functional networks associated with each of the three amygdala subdivisions, which were maintained across sessions, demonstrating the robustness and stability of our findings. Notably, their combination reproduces the previously reported33. In particular, the connection between the LB and the temporal lobes was previously observed using rsFC16 and probabilistic DTI34, and is supported by axonal tracing studies in non-human primates, where a connection between the superior temporal gyrus and the ventrolateral nucleus of the amygdala was identified35.Signal variation at the LB subdivision was positively correlated with activity in the superior temporal gyrus, middle frontal and precentral gyri, supporting the involvement of similar circuits in associative learning and emotion regulation17 and rat anatomy37. Similarities of the connectivity pattern of the LB and SF mainly with the temporal lobe38, suggests that these two amygdaloid nuclei may share a common functional organization. Comparative architectonic studies have also suggested a separation of the SF from the CM group, assigning it to the LB group39, which is justified by the fact that these two nuclei also contain similar cortical –like neuronal composition9.Significant correlation was found between the SF region and posterior insula, OFC, subgenual ACC, inferior parietal lobule, olfactory cortex, middle temporal gyrus and PCC. This correlation pattern suggests an involvement of SF in olfaction-related affective processes, consistent with a meta-analysis of human imaging data40, suggesting a possible indirect connection between these regions through the sACC, as previously reported in monkeys41. Nevertheless, a functional connection between the amygdala and the PCC has also been reported42, suggesting an involvement of SF in self-awareness related emotional processes, autobiographical memory, past self-relevant stimuli and future prospection43. The SF was also anti-correlated with the more dorsal part of the precuneus, which is consistent with the pattern of positive correlations, previously observed, between the DMN and the ventral, but not the dorsal precuneus44. In addition, a decrease in regional cerebral blood flow in the PCC, medial frontal cortex and ventral but not dorsal precuneus during a working memory task45, suggests that dorsal precuneus (BA 7) may not be part of the DMN46. Furthermore, the connection between the SF and the precuneus may corroborate an amygdala-precuneus pathway that has been anatomically observed in tract tracing studies in monkeys48. Lastly, our findings showing the SF amygdala as the most activated in response to an emotional task that elicited either fear or anger, corroborates its involvement in directing attention towards affective stimuli49.Despite the biological resemblance between the SF and LB, we also observed unique connectivity patterns between SF and areas of the default mode network (DMN), although anatomical projections from the amygdala to the PCC has not been verified50, is consistent with the central nucleus involvement in facilitating attention to salient stimuli51. In addition, the CM was the only amygdala subdivision in which the rsFC pattern predicted activation in response to fear and angry faces, thus indicating its association with adverse feelings. Even though the SF had the highest average activation for this task, its BOLD signal did not correlate with the mean functional connectivity pattern of this subdivision. Moreover, the most activated voxels occurred not only in the SF, but also in the CM subdivision. Therefore, our findings suggest an involvement of CM and its connected areas (SN and striatum) with negative emotion and is consistent with previous findings. For example, the strongest amygdala functional connectivity within the SN is seen in those with the greatest amygdala activation to and aroused by negative pictures52, which is sustained by the role of the amygdala, SN and ventral striatum on the identification of the emotional significance of environmental stimuli and the production of affective states53. Multiple fMRI studies have also reported activation in the ACC/MCC in anger and fear emotional tasks54, while monkey studies show that this part of the cingulate cortex receives input from the amygdala55 and has been implicated in fear56. The anterior insula has also been linked with such emotional experiences as fear and anger57, supported by a projection from the central nucleus of the amygdala35. The striatum has been associated with anger58, such that functional connections between the CM and the striatum are enhanced during stress59. The CM participates not only in the expression of conditioned fear10 but also in the learning and consolidation of fear conditioning60. Experiments in rats show that lesions of the central, medial and cortical amygdala nuclei markedly increase the number of contacts a rat will make with a sedated cat, demonstrating a decrease of fear in these lesioned animals61. The involvement of CM in anger has also been demonstrated in cats, with stimulation of the central nucleus suppressing defensive rage, while stimulation of the medial nucleus enhances aggressive behavior62. Overall, our findings reinforce the previously observed participation of the CM and connected areas (SN and striatum) in fear and anger.The significant correlation between resting activity of the CM nuclei and signal fluctuation from areas attributed to the salience network (SN), e.g., anterior insula, ACC and middle cingulate cortex (MCC)et al.16 and by Roy et al.6, which used the Juelich atlas as a template. Both found that the CM nucleus correlated with the striatum and insula, that the LB complex correlated with the temporal lobe, and activity of the SF mainly correlated with activity of the OFC. Additionally, Roy and colleagues6 also reported laterality differences of the CM connections, as observed here, that is consistent with those previously reported63. For example, right amygdala lesions in rats have been shown to generate greater deficits in contextual fear than left sided lesions64. Previous electrophysiological studies also showed hemispheric lateralization of pain processing by CM neurons. Specifically, neurons in the left latero-capsular division of the central nucleus of the amygdala (CeLC) did not develop increased responsiveness in a rodent model of arthritis pain, while the right CeLC played a major role in the processing of prolonged nociceptive inputs and develops sensitization65. This suggests a tonic inhibitory mechanism from the prefrontal cortical areas over the left CeLC, which exert a top-down inhibitory influence on the amygdala66. In our work, the lower connectivity strength observed for the CM, besides being consistent with previous work, may also indicate a diminished inhibitory effect from the prefrontal cortical areas over the right CM, since this was the amygdala subregion most connected to these cortical areas. This may explain the unique correlation of its connectivity pattern and the activity on this area during the faces task, once more being consistent with a predominant role of the right amygdala in negative emotions. Finally, while the anatomical and functional basis for this lateralization remains unclear, taken together, these findings highlight the role of the CM on amygdala lateralization.The functional connectivity circuits for the modularity defined amygdala subdivisions reproduce the major findings obtained by Bickart 6, although they are consistent with another resting state study that identified connectivity between the LB and SF with motor areas38. Structural connections between the amygdala and motor areas has also been previously observed in a DTI study34. Studies in cats67 and monkeys68 have shown that the projections to the motor system arise from the magnocellular division of the basal nucleus of the amygdaloid complex. In addition, disruption of the amygdala-motor pathway has been suggested to be responsible for the inability of those with Autism to react to social stimuli69, which is consistent with data from both monkeys70 and humans71 reporting that the LB and SF subregions are especially sensitive to social stimuli. Therefore, together with previous findings, our work indicate that a direct amygdala-motor pathway might provide a mechanism by which the amygdala can influence more complex motor behaviors34.The functional connectivity between the LB and SF with the SMA differ from the results obtained by Roy and colleagues22. The high spatial resolution improves the assessment of small brain regions, such as the amygdala, by limiting partial volume effects, reducing dephasing artifacts and improving in-plane MRI signal uniformity72, while the high scan rate has the further advantage of minimizing the aliasing of physiological artifacts over the low frequencies of interest73. Even though smaller voxels always carry the disadvantage of lower SNR, it was compensated by the large sample size and multiple long rsfMRI runs.Finally our results benefited from using high spatial and temporal resolution data from the HCPIn conclusion, we have applied a community structure approach to characterize the modular organization of the amygdala using resting state fMRI data from the HCP. The anatomical and functional specificity of the results provide compelling evidence of the structure–function association between the subnuclei identified herein and histological anatomical substructures, demonstrating that graph theory based network analysis techniques can generate biologically meaningful partitions of brain structures. Finally, our findings suggest that characterization of regional modular organization may be useful to evaluate disease- or age-induced abnormalities.http://www.humanconnectome.org/documentation/S500/). All experiments were performed in accordance with the relevant guidelines and regulations of the Human Connectome Project74. Detailed description on the standard operating procedures of the protocol is available at the WU-Minn HCP 500 Subjects + MEG2 Data Release: Reference Manual Appendix IV. Subject recruitment procedures and informed consent forms, including consent to share de-identified data, were approved by the Washington University (WU) institutional review board75. The use of the HCP data was approved by the Office of Human Subjects Research Protections at the NIH. All data presented in this paper is not identifiable, only group results are presented overlaid on a MNI template.Analyses were performed on 100 adult healthy volunteer , randomly selected through the 100-unrelated option from the WU-Minn Consortium HCP (WU-Minn HCP 500 Subjects + MEG2 Data Release), obtained through the Open Access agreement. Data is publicly available at the HCP online database . Within each day, acquisitions alternated between phase encoding in the right-to-left (RL) direction in one run and the left-to-right (LR) direction in the other run. Anatomical images were acquired with a high-resolution (0.7 mm isotropic) T1-weighted magnetization-prepared rapid gradient echo (3D-MPRAGE) sequence. Anatomical and functional imaging sequences covered the whole brain. Subjects were given a simple instruction to rest and keep their eyes open with relaxed fixation on a projected bright cross-hair on a dark background (presented in a darkened room)77.Images were acquired on a 3 Tesla Skyra Siemens system using a 32-channel head coil, a customized SC72 gradient insert (100 mT/m) and a customized body transmit coil. Resting state fMRI data were acquired in four runs on two different days (two runs per day); task-evoked fMRI was acquired in two runs on the same day after completion of the rsfMRI. Both used a multi-band gradient-echo EPI sequence33 wherein they were asked to match which of two faces/shapes presented on the bottom of the screen with the face/shape at the top of the screen; faces exhibited either an angry or fearful expression. Each of the two runs includes 6 blocks (3 face blocks and 3 shape blocks), each block was composed of 6 trials of the same task type (face or shape), with trials presented for 2000 ms and an inter-trial interval of 1000 ms. Shape blocks were presented interleaved with face blocks and each block was preceded by a 3000 ms task cue (“shape” or “face”); 8 seconds of fixation was presented at the end of each run77.Participants performed a block design modified face-shape matching task1 bias field corrected and normalized to MNI152 space. FMRI runs (rsfMRI and task-evoked) underwent gradient distortion correction, motion correction, registration to the T1w image, spatial normalization to the MNI standard space, 4D global mean-based intensity normalization, B1 bias field correction; rsfMRI were also subject to independent component analysis (ICA)-based artifact removal. Finally, both task and resting data were whole brain masked77. Local data processing was performed in AFNI78 and MATLAB . The pre-processed task-evoked fMRI time series were smoothed (FWHM = 4 mm) to improve signal-to-noise ratio (SNR)79, censored for time points that exceed a Euclidean distance of 0.3 mm and then modeled in a fixed effect analysis, two runs per subject, with canonical hemodynamic responses time-locked to the shape-face epochs, contrasting face with shape condition; head motion parameters (3 translations and 3 rotations) were also entered into the model as regressors of no interest. Time-series datasets were scaled so that beta weights could be interpreted as percent signal-change. Each pre-processed rsfMRI run had the first four EPI volumes removed to ensure signal equilibrium, following by band-pass filtering (0.01–0.10 Hz) to minimize instrument induced drifts80 and physiological noise81. Multi-linear regression with the 6 time-varying realignment parameters was performed to minimize motion related fluctuations in the MRI signals. The first three principal components (using PCA) of white matter (WM) and cerebrospinal fluid (CSF) signals were also regress out to preclude non-neuronal induced signal fluctuations83. The WM and CSF masks were generated by segmenting the preprocessed high-resolution anatomical images using SPM884 and down-gridding the obtained masks to the same resolution as the pre-processed functional data. Individual subject rsfMRI time series were smoothed (FWHM = 4 mm) to improve SNR79, and censored for time points that exceed a framewise displacement (FD) threshold >0.5 mm and the root mean square variance across voxels (DVARS) > 0.5%85. Time series with excessive number of time points censored  were discarded; two subjects were excluded from resting BOLD and another two from the task BOLD data.The datasets underwent an initial preprocessing by the HCP consortium. Anatomical images were distortion corrected, co-registered and averaged across runs, AC-PC registered, brain extracted, B27 from the Brain Connectivity Toolbox (https://sites.google.com/site/bctnet/) on the remaining 98 subjects. More specifically, Pearson correlation coefficient was computed for each voxel (node) inside of the anatomically pre-defined left and right amygdala templates in MNI space (from Jerne Volumes of Interest database87), which was resampled to 2 mm isotropic to reproduce the image resolution of the processed functional data. Individuals’ correlation matrices were then averaged across subjects and runs to obtain the final connectivity matrix, M. Following, after removing the auto-correlation values from M, a Louvain algorithm26, was used to find the community affiliation vector (C) corresponding to the undirected and weighted correlation matrix (M)27, accounting for the contribution of positive and negative edges. The algorithm finds the optimal number of clusters by aggregating the nodes in the network into groups of modules. More specifically, Louvain method starts from a set of nodes that, through subsequent passes of the algorithm, are clustered to others nodes becoming larger and larger sets of nodes ; next the found communities are aggregated in order to build a new network of communities, finally it is repeated iteratively until it reaches a number of modules that have a maximal possible number of within group connections, and a minimal possible number of between-group connections88, i.e., until the modularity factor, Q, is maximized27. Therefore, no initial information regarding the number of modules is needed. However, the algorithm is non-deterministic, and may produce different solutions each run, so to address this limitation, the calculation was repeated 100 times  and the maximum modularity factor, QM, was registered , then it was repeated another 100 times and another modularity factor was selected, Qm; these two values were compared and if they are different the latest is kept to compare with the results from the next iteration. This process was repeated until no change to the maximum Q was observed (QM = Qm). Note that for every step (every QM and Qm) the number of modules remained constant varying only their size. Next the final community affiliation, vector (C) was entered into the fine-tuning27 modularity algorithm, in order to refine the modules borders. Here, again the process was repeated iteratively, as before, until no change in the maximum modularity factor, Q, was observed; this allowed for the number of optimal partitions (modules) to be determined automatically. The modularity factor, Q, ranges between 0 and 1, and represents the goodness in which a network is optimally partitioned into functional modules27.Modularity analysis of the amygdala was performed using a weighted-connectivity conserving null model in fully connected, undirected networks with positive and negative weightsSeed-voxel partial correlation was used to calculate the FC maps. The final subdivisions of left and right amygdala were used as seeds and correlated with the entire brain for each subject, regressing out the signal contribution from the neighbor seeds. The cross-correlation coefficient maps were then converted to z-score maps using the Fisher’s r-to-z transformation. Final FC maps for each subject were generated by averaging the results across runs. In order to evaluate temporal variations in the connectivity pattern generated for each amygdala seed, the original time-series were divided into four equal subseries (sessions) and FC maps were calculated for each session and averaged across runs for each subject.89. The FC pathways for the different sessions (subseries) and each amygdala subdivision were calculated with t-test, and the difference between sessions across seed location  was evaluated with two-way analysis of variance (ANOVA) using 3dLME89. Results were corrected for multiple comparisons using 3dClustSim, based on Monte Carlo simulations90, including voxels of the entire brain and considering a conservative imaging smoothing (FWHM) of 10 mm; statistical significance was set for pcorrected < 0.05 .One sample t-tests were performed independently on the FC maps generated for each amygdala subset. Differential assessments of the FC maps were conducted to evaluate differences in functional connectivity across amygdala subdivisions by contrasting the connectivity path of one seed against the combination of the others and by pairwise comparison  using a linear mixed-effects modeling approach (3dLME)91; specifically the between subject and residuals mean square values were computed for each voxel using the function 3dICC_REML89 on the FC maps generated for each amygdala subdivision, considering all sessions .The reliability of the connectivity maps obtained for the four sessions (subseries) was also evaluated using a two-way mixed single measures intra-class correlation )One sample t-tests were performed on the beta weights generated for each subject providing a group activation map for the angry/fear faces contrasted against shapes. In addition, region-of-interest (ROI) analyses were carried out on the beta weights using the modularity results as a mask to extract the average BOLD signal in each amygdala subdivision for each subject. Results from the ROI analyses were compared with the average of the positive and significant (p < 0.05) Fisher transformed correlations obtained from the FC maps generated from seed-voxel partial correlations for each amygdala subdivision. Both analyses were carried out for the 96 subjects that survived head motion criteria for both resting and task-evoked datasets.http://www.humanconnectome.org/). Detailed description of the analysis is included in the methods section and in the supplemental material.All data analyzed in this manuscript were obtained from the WU-Minn Consortium HCP (WU-Minn HCP 500 Subjects + MEG2 Data Release), which is publicly available at the HCP online database (Supplemental material