Differentiated care refers collectively to flexible service models designed to meet the differing needs of HIV-infected persons in resource-scarce settings. Decentralization is one such service model. Retention is a key indicator for monitoring the success of HIV treatment and care programs. We used multiple measures to compare retention in a cohort of patients receiving HIV care at “hub”  and “spoke”  sites in a large public HIV treatment program in north central Nigeria.This retrospective cohort study utilized longitudinal program data representing central and decentralized levels of care in the Plateau State Decentralization Initiative, north central Nigeria. We examined retention with patient- level  and visit-level  measures. Regression models with generalized estimating equations (GEE) were used to estimate the effect of decentralization on visit-level measures. Patient-level measures were examined using survival methods with Cox regression models, controlling for baseline variables.Of 15,650 patients, 43% were enrolled at the hub. Median time in care was 3.1 years. Hub patients were less likely to be LTFU (adjusted hazard ratio (AHR)=0.91, 95% CI: 0.85-0.97), compared to spoke patients. Visit constancy was lower at the hub , where gaps in care were also more likely to occur .Decentralized sites demonstrated better retention outcomes using visit-level measures, while the hub achieved better retention outcomes using patient-level measures. Retention estimates produced by incorporating multiple measures showed substantial variation, confirming the influence of measurement strategies on the results of retention research. Future studies of retention in HIV care in sub-Saharan Africa will be well-served by including multiple measures. Service models designed to address these differing needs in a client-centered framework have come to be known collectively as “differentiated care” ,2. IncreOne such shift has been known as “decentralization.” Decentralization of HIV care means relocating services from centralized sites to peripheral centres, which are geographically closer to patients. Decentralization was introduced in 2002 by the World Health Organization as a strategy for expanding access to antiretroviral therapy (ART) . BesidesRetention is a crucial indicator for monitoring and evaluating HIV care and treatment programs, particularly in the test-and-treat era, when patients may begin ART before experiencing symptoms. High retention levels have been associated with improved ART adherence, slower disease progression, improved survival and reduced infectiousness -7. DespiOur study addresses this gap, employing multiple measures to explore patterns of retention in a decentralized HIV care program in Nigeria. Our objective was to compare patient retention in central versus decentralized treatment sites.To improve access to HIV care in Plateau State, north central Nigeria, the HIV treatment program at Jos University Teaching hospital (JUTH), in the state capital of Jos, launched the Plateau State Decentralization Initiative in 2007. A “hub-and-spoke” model of decentralization was used in which the JUTH-HIV specialty clinic (“hub”) was linked to 13 community hospitals in surrounding semi-urban and rural areas (“spokes”). Community hospitals were empowered to initiate and maintain patients on ART close to their residences . CommuniART eligibility in the program was determined by clinical staging and CD4+ cell count level, following national guidelines at the time ,23. EligPersons included in this study were HIV-1 infected, treatment-naïve patients (aged ≥ 15 years) at the time of enrolment in care. We followed patients between January 2008 (study baseline) and December 2012 (study follow-up end point). Those included were enrolled in the JUTH HIV specialty clinic (the “hub”) or at one of the 13 community hospital “spoke” treatment sites. Only patients enrolled in care for at least six months at the study end point were included, in order to fully examine longitudinal retention. Data were extracted from electronic medical records  and inclUpon entry into the care and treatment program and following informed consent, all patients were assessed for ART eligibility according to current Nigerian national treatment guidelines ,23. All For the analyses reported here, we included patients who were newly enrolled in HIV care between January 2008 and June 2012, to ensure at least 6 months of follow-up time for the evaluation before data censure (December 2012). All patients were at least 15 years of age at enrolment. Patients who had ART experience prior to enrolling in the program were excluded.retention at fixed-time points - reports the proportion of the study population with a clinic visit within 180 days of three fixed time points: 12, 24 and 36 months after enrolment. The second summary measure - loss to follow-up (LTFU) - cumulatively estimates the proportion of the population: (a) without a clinic visit for more than 180 days at the end of the follow-up period; or (b) with only one visit during the follow-up period.We employed a multiple outcome measurement strategy to assess retention in the hub-and-spoke network. Patient-level and visit-level measures were included to ensure comparability with other studies. Patient-level assessment consisted of two summary measures. The first summary measure - gap in care is a period of >180 days between clinic visits, with the patient having subsequent visits following the gap. Visit constancy is defined as six-month intervals (person-periods) for each patient from the time of enrolment through the study endpoint date. The number of visits within each interval was counted. Visit constancy estimates continuity in care by determining the proportion of six-month person-periods with at least one clinical visit.We also used two visit-level longitudinal measures to assess patterns of retention over time. A This study was approved by the Institutional Review Board (IRB) at the Harvard T.H. Chan School of Public Health and the Committee on Human Studies at Harvard Medical School, Boston, MA. All consent forms were approved by the Harvard School of Public Health IRB, the National Health Research Ethics Committee (NHREC) of Nigeria, and the institutional review boards at the decentralized treatment sites. Only patients in the treatment program who provided written consent for care and for use of their stored data in future studies were included in this analysis.Descriptive statistics were used to assess baseline demographic and clinical characteristics of the study sample overall, and stratified by type of site (hub vs. spokes). Chi-square and Wilcoxon rank sum tests were used to examine differences in patient characteristics and retention outcomes by site type, including fixed time-point retention at 12, 24 and 36 months, LTFU, gaps in care and visit constancy. We examined the proportion of contiguous visits associated with a gap (>180 days since last visit), the median number of days since last visit among those with more than one clinical visit, and the gap duration for individuals with gaps in care. Finally, we examined the proportion of person-periods associated with at least one clinical visit.Regression analysis was performed separately for each retention outcome. We first examined bivariate associations between the main exposure of interest  and each outcome. Adjusted models included the main exposure of interest (site type) and all available demographic and clinical characteristics. A time-varying indicator was included in survival and longitudinal models to indicate pre or post-ART initiation time for each patient. A time-fixed indicator of any ART use was used in person-level models.Logistic regression models with robust variance for clustering by site were used to examine retention at fixed time points . Baseline time-fixed covariates were included in the model in addition to the main exposure (hub vs. spoke).LTFU was examined using survival analysis methods. The Kaplan-Meier estimator was used to estimate time from enrolment in care to LTFU, comparing patients who enrolled at hub vs. spoke sites. Censoring was defined as having had at least one clinical visit within 180 days from the study end date. Cox regression analysis was used to examine the differences in LTFU for hub vs. spoke sites, adjusting for covariates. Survival analysis was restricted to those with >1 visit.Visit constancy was examined using linear regression with generalizing estimating equations (GEE) for repeated measures among patients with exchangeable correlation structure. Visit constancy was time-updated at each six-month person-period and expressed as a percentage.Gaps in care were examined using logistic regression with GEE with exchangeable correlation structure for repeated measures among patients. Gaps in care were examined only among patients with >1 visit.Statistical significance was set at p<0.05. Stata statistical package version 13  was used for analyses.A total of 16,816 patients were enrolled at hub and spoke sites during the study period. We excluded 1,166 patients because they had fewer than 180 days in care at the time of data censoring on December 31, 2012. The final sample included 15,650 patients with 128,017 total visits, of which 6,761 patients (43%) were enrolled at the hub.3 (interquartile range (IQR): 100- 337). Sixty-six percent of the cohort was female. Median age at baseline was 33 years (IQR: 28-40) and median time since enrolment in care was 3.1 years (IQR: 1.9-4.1).Comparisons across site type showed that a higher proportion of spoke versus hub patients were female (p<0.0001). They were also slightly younger (p<0.0001), more likely to be married (p<0.0001), more likely to have no formal education (p<0.0001) and less likely to have WHO stage III/IV illness at baseline (p<0.0001) than those from the hub site. There was no difference in median baseline CD4+ cell count between hub and spoke sites (p=0.45).Results of analyses examining differences in retention by site type using the four retention measures included in this study appear in Using the summary measure of retention, 65.5%, 53.6% and 46.4% of the sample respectively were alive and in care at 12, 24 and 36 months from the time of enrolment. Hub patients were more likely than spoke patients to be alive and retained in care at 12, 24 and 36 months following enrolment . The proportion of patients LTFU at the end of the observation period was 56.4% vs. 58.0% (p=0.05) for hub and spoke sites respectively, with hub patients having longer median time since loss at the study end date , than for patients at spoke sites (35 days) , compared with spoke patients .Retention at 12, 24 or 36 months did not differ between hubs and spoke sites after adjusting for patient-level factors (data not shown). Females  and those who had ever initiated ART (compared to those who had not) were more likely to be retained at 12, 24 and 36 months.This large longitudinal cohort study evaluated patient retention in the Plateau State Decentralization Initiative, a hub-and-spoke network of HIV treatment and care in Plateau State, north central Nigeria. We used multiple patient- and visit-level measures to assess retention in the central “hub” and the decentralized “spoke” sites. Our measure of retention at fixed time points showed retention overall ranged from 65.5% at 12 months to 46.4% at 36 months of ART, with the hub performing better than the spokes at each time point. The hub also performed better on the loss to follow-up measure. Decentralized sites had better retention rates using the visit-level measures . Visit-level results suggest a more continuous pattern of visits and greater engagement in care among retained patients at the spokes, compared with the hub site. Additional research is warranted to understand the clinical significance of differences in visit constancy measures in this setting.To our knowledge, this study is the first to use multiple outcome measures to evaluate patient retention in routine HIV care in sub- Saharan Africa. Visit-level measures, in particular, have not been used previously to evaluate retention in African HIV treatment programs. Results have shown that patient retention in this setting is comparable (using the gaps in care and visit constancy measures) to that obtained in public health care settings in North America, with site of care being a strong predictor of retention. For example, our results show fewer gaps in care at both central (43%) and decentralized (28%) sites, over a longer follow-up period, than reported in a study of retention among HIV-infected veterans receiving HIV care at US Veterans Health Administration (VHA) facilities (56%) . Rates oPrevious studies comparing rates of retention in decentralized vs. more central facilities have consistently revealed better retention at decentralized care sites ,26-29. TOne reason retention “as continuity of visits” was better at decentralized sites may be because these treatment facilities were closer to patients’ residences. Clinic visits are easier to keep when transport distances are shorter, reducing the time and expense required to keep appointments ,32. In cA useful framework for considering results from multiple measures lies in the medication adherence literature. Vrijens et al.  describeLoss to follow-up rates were higher in this study than reported for either other HIV treatment settings in sub-Saharan Africa or a larger evaluation of HIV treatment programs in Nigeria ,34-36. HWe did not include patient medication refills as part of our definition of a visit in this analysis. Future research that includes medication refills, as well as an examination of the relationship between measures (e.g. the association between visit constancy and fixed-time retention) may be important to fully characterize retention in this setting. Finally, there may be some unmeasured confounding driving the differences in retention by hub and spoke sites reported here.The retention estimates produced by incorporating multiple patient- and-visit level measures into our analysis showed substantial variation, clearly confirming the impact of measurement strategies on the results of retention research and yielding insights that would have remained hidden had we relied upon summary patient-level measures alone. The higher retention levels observed in spoke sites using visit- level measures suggest decentralization results in a more continuous pattern of clinic visits – an observation wholly consistent with the experience, for patients, of being able to access care closer to home.Our study does not include any biological measures reflecting the impact of retention on patient clinical outcomes. While viral suppression is the ultimate goal for patients and programs, viral load data were not sufficiently complete to warrant their inclusion in this analysis. Viral suppression can be thought of as a proxy measure of both patient retention and medication adherence, since both are necessary conditions to achieve suppression. However viral load data are sparse in resource-constrained environments. Future research aimed at understanding patient retention over time should consider how to incorporate frequently missing data on viral suppression as a measure of patient retention in resource-limited settings.A large sample size, use of routine program data, and a study setting reflecting an innovative decentralization model are among the strengths of this study. Several limitations must also be considered. First, there may have been misclassification due to under-ascertainment of outcomes for those who dropped out of care and were considered lost to follow-up, as described above. We did not incorporate mortality estimates or consider death as a competing event for our retention analyses. Insufficient patient-tracing systems at all sites and incomplete or unreliable mortality data likely resulted in overestimates of patients considered not retained using the retention at fixed time points and loss to follow-up measures. Undocumented self-transfers across treatment sites within the hub-and-spoke system may also have contributed to overestimation of true patient losses . FinallyThe impact of decentralization upon retention in HIV treatment and care is complex and varies depending on how retention is operationally defined and measured. The operational definitions and measures chosen can have far reaching effects on results, with each having its own value and utility. Future studies aimed at understanding retention in HIV care will be well served by including multiple measures as a means of strengthening validity and precision. In view of the UNAIDS global 90-90-90 targets for HIV prevention, multiple measurement strategies that include viral suppression will enable programs to more accurately evaluate this important outcome.


Current approaches to stratify psychiatric patients into groups based on violence risk are limited by inconsistency, variable accuracy, and unscalability.Based on a national cohort of 75 158 Swedish individuals aged 15–65 with a diagnosis of severe mental illness (schizophrenic-spectrum and bipolar disorders) with 574 018 patient episodes, we developed predictive models for violent offending through linkage of population-based registers. First, a derivation model was developed to determine strength of pre-specified criminal history, socio-demographic, and clinical risk factors, and tested it in external validation. We measured discrimination and calibration for prediction of violent offending at 1 year using specified risk cut-offs.A 16 item model was developed from criminal history, socio-demographic and clinical risk factors, which are mostly routinely collected. In external validation, the model showed good measures of discrimination (c-index 0.89) and calibration. For risk of violent offending at 1 year, using a 5% cut off, sensitivity was 64% and specificity was 94%. Positive and negative predictive values were 11% and 99%, respectively. The model was used to generate a simple web-based risk calculator (OxMIV).We have developed a prediction score in a national cohort of patients with psychosis that can be used as an adjunct to decision making in clinical practice by identifying those who are at low risk of violent offending


The American Cancer Society  estimateBiomarkers are tools that can be used clinically to help guide treatment decisions. A biomarker is "a biological molecule found in blood, other body fluids or tissues that is a sign of a normal or abnormal process, or of a condition or disease" . Medicalwww.asco.org/guidelines. This article will review five of the major biomarkers found within the ASCO guideline in an attempt to acquaint APs with these available biomarkers.The American Society of Clinical Oncology (ASCO) has recently published an evidence-based clinical practice guideline that provides recommendations on the appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer and known ER/PR/HER2 status . The guiOncotype DX is a 21-gene polymerase chain reaction assay that has been validated to predict recurrence risk and chemotherapy benefit in hormone receptor–positive invasive breast cancer . The recA prospective trial involving 10,253 women with hormone receptor–positive, HER2- negative, axillary node–negative breast cancer with tumors of 1.1 to 5.0 cm in greatest dimension used the Oncotype DX to calculate a risk score. Those with a low risk of recurrence  were assigned to endocrine therapy alone without chemotherapy. Approximately 99.3% of node-negative, hormone receptor–positive, HER2-negative patients had no distance recurrence at 5 years after endocrine therapy alone .Genomic Health and the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute joined in an effort to electronically supplement the SEER registries with Oncotype DX results. The first report characterized breast cancer survival in node-negative hormone receptor–positive-invasive breast cancer. The 5-year breast cancer–specific survival outcomes were 99.6% in over 21,000 patients with low recurrence scores .Based on high-quality evidence, ASCO strongly recommends the use of Oncotype DX to guide decisions on adjuvant therapy for patients with ER/PR-positive, HER2-negative, node-negative breast cancer. However, in patients with node-positive or HER2-positive breast cancer, ASCO does not recommend the use of Oncotype DX in clinical decision-making . OncotypEndoPredict is an RNA-based assay of eight disease-relevant genes and three reference genes as expressed within tumor tissue. It is used to predict the risk of metastases for patients with ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. It incorporates genomics, tumor size, and nodal status . TogethePatients with an EPclin score of < 3.3 were classified as low risk for distant recurrence, whereas patients with an EPclin score ≥ 3.3 were stratified as high risk for distant recurrence . Scores The formal ASCO recommendation for use of EndoPredict to help guide decisions on adjuvant systemic chemotherapy is moderate and intended for use in patients with ER/PR-positive, HER2-negative, node-negative breast cancer. The ASCO panel does not recommend the use of EndoPredict in patients with ER/PR-positive, HER2-negative, node-positive breast cancer. Additionally, for patients with HER2-positive breast cancer, the assay is not recommended for use.The PAM50 is a validated reverse transcription polymerase chain reaction test based on a panel of 50 genes . PerformIncorporating this technology is NanoString’s Prosigna Breast Cancer Prognostic Gene Signature Assay. In addition to identifying the breast cancer subtype, the test generates a risk of recurrence score and a risk category. The score value is between 0 and 100 and correlates with the probability of distant recurrence within 10 years. The test is indicated for use in postmenopausal women with breast cancer with stage I/II, node-negative or stage II, node-positive (one to three nodes), hormone-positive disease .While providing a strong recommendation for use in patients with ER/PR-positive, HER2-negative, node-negative breast cancer, ASCO does not advocate the use of PAM50-based testing as a guide for decisions on adjuvant systemic therapy in women with ER/PR-positive, HER2-negative, node-positive breast cancer. In addition, ASCO does not incorporate this type of testing in patients with HER2-positive breast cancer or those with triple-negative breast cancer.Although the prognosis for women with early-stage, hormone receptor–positive breast cancer treated with 5 years of endocrine therapy remains good, the risk of distant recurrence is still a concern for all. This concern is what led to the exploration of the concept and currently accepted clinical practice of extended endocrine therapy.The Adjuvant Tamoxifen: Longer Against Shorter (ATLAS) trial found that for women with ER-positive disease, continuing tamoxifen to 10 years rather than stopping at 5 years produced a further reduction in recurrence and mortality, particularly after year 10 . The AdjRecently, Goss et al.  publisheAlthough such findings clearly support a role for extended therapy, deciding which patients may benefit most from extended therapy is an important part of advanced practice. As always, the risk of any therapy must be balanced against it benefits. Risks of extended endocrine therapy include an increased risk of endometrial cancer with continued tamoxifen, hot flashes, deep vein thrombosis, ischemic heart disease, loss of bone mass, cognitive dysfunction, and vaginal dryness . The BreHOXB13 and IL17BR expression; and a 5-gene predictor (the molecular grade index), which recapitulates tumor grade and/or proliferation , which may or may not change the future recommendation for MammaPrint use . The stuFollowing the printing of the ASCO guideline, Piccart et al.  presenteThese biomarkers represent some of the most researched and recognized in clinical practice, along with several other biomarkers for use in patients with early-stage breast cancer. Breast cancer treatment continues to evolve and will no doubt be permanently influenced not only by the microscope but the molecular profile as well. As a result, APs need a working knowledge of all available biomarkers to appropriately apply the information they impart to each individual patient’s case.The ASCO guideline serves as an evidence-based, relevant blueprint for the incorporation of biomarkers into clinical practice. The guideline also clarifies which biomarkers the panel considers are not of clinical utility in guiding the choice for adjuvant therapy see . It is s


We aimed to identify the key miRNAs protecting DCIS from malignant evolution, that may constitute markers for non-invasive lesions.A substantial number of ductal carcinoma We studied 100 archived DCIS samples, including pure DCIS, DCIS with adjacent IDC and pure DCIS from patients with subsequent IDC in contralateral breast or no recurrence. A DCIS derived cell line was used for molecular and cellular studies.in vitro and, in patients with pure DCIS, was associated with later onset of IDC. Survival studies of independent cohorts indicated that both miRNAs play a protective role in IDC. The clinical findings are in agreement with the miRNAs’ roles in cell adhesion, differentiation and proliferation.A genome wide study revealed that pure DCIS has higher miR-126 and miR-218 expression than DCIS with adjacent IDC lesions or than IDC. The down-regulation of miR-126 and miR-218 promoted invasiveness We propose that miR-126 and miR-218 have a protective role in DCIS and represent novel biomarkers for the risk assessment in women with early detection of breast cancer. Ductal carcinoma in situ (DCIS) represents 20–25% of newly diagnosed BC in industrialized countries and up to 40% of DCIS lesions progress to invasive carcinoma (IDC) if untreated , P = 0.03; RFS UK , P = 0.02), while miR-126 only in one  (Table P = 0.03) and UK (P = 0.02) cohorts and longer overall survival in METABRIC (P < 0.001)  , and UK < 0.001) . As a poThe protein coding genes correlated with miR-126 and miR-218 in BC were identified using Spearman correlation, and cross-validation in METABRIC, TCGA, and UK breast cancer cohorts. The use of independent cohorts, each assayed using different technical platforms, ranging from microarrays to RNAseq, ensured a very robust assay by removing major technical and platform related biases. Successively, we performed a Gene Ontology analysis on the correlated coding genes . High miOnce DCIS is detected, the lesion is excised and therapy administered according to the tumor phenotype , 5. Althin vitro for breast and other solid cancers [The presence of miR-126 and miR-218 was also associated with better prognosis in three large cohorts of patients with invasive breast cancer, reiterating that these miRNAs down-modulate malignant properties, as shown  cancers , 21. In  cancers . At variThus, we propose that the increase of miR-126 and miR-218 in DCIS is part of the mechanism activated by non-invasive tumor cells to counteract tumorigenesis and that the failure of this process, resulting in the down-modulation of these two miRNAs, may have a role in facilitating the progression of DCIS to invasive lesions. Modification of methylation levels, related to expression of miR-126 and miR-218 in mesothelioma  and gastin vitro, using MCF10DCIS cells, the only established DCIS model for the study of the malignant evolution of non-invasive breast cancer [We tested and validated this hypothesis t cancer , 26. In t cancer  and the t cancer , 28. Usiin situ to invasive breast carcinoma [The EMT program broadly regulates invasion and metastasis and it is assumed to play an important role in the progression of arcinoma , 29. Thearcinoma . Thus wein vitro assays and Gene Ontology analysis of patient samples highlighted the role of miR-126 and miR-218 in invasion. Our results are in agreement with those by Lesurf et al. [Both Our approach was designed in two stages to identify miRNAs related to cancer progression. First, we identified and validated differentially expressed miRNAs in DCIS compared to IDC, using a patient number larger than previously reported in literature, and then we assessed the expression of those miRNAs in two special cohorts of DCIS patients, one with adjacent IDC and the other with subsequent contralateral IDC. Both cohorts included progression events, either local or delayed and distant, thus encompassing divergent but high-risk types of DCIS patients.In our present work, we aimed to overcome a common limitation in the miRNA studies on DCIS: the very limited size of the cohorts. First, we started with an initial RNAseq cohort of 30 DCIS samples, which is up to now the largest published study on miRNA. Second, in order to further strengthen our findings, we used additional DCIS samples with invasive potential to validate our initial miRNA signature. Thus, instead of looking at miRNAs specific for BC subtypes, our redundant approach allowed us to pinpoint those miRNAs that had a robust pattern of expression across multiple DCIS cohorts with high invasive features. Although we took special care to address the study of small cohorts as outlined above, our conclusions could still be affected by cohorts’ size. Additionally, while we looked for common mechanisms in DCIS, the genotype background and pattern of somatic mutations in DCIS were not investigated. Finally, although we extended the cellular studies to invasive cells, yielding significant results on miRNA roles, repeating the assays on more non-invasive cell lines could lead to further significant results.Over-diagnosis is a recognized major problem in breast cancer and the lack of prognosticators for early lesions hampers the management of patients. miR-126 and miR-218 are highly expressed in DCIS with low-invasive potential and thus represent important novel biomarkers for the risk assessment in women with early detection of breast cancer. In addition, up-modulation of these two miRNAs could constitute the basis of clinical trials aimed to help to make decisions on therapeutic intervention in low-risk DCIS patients.n = 23). The Cohort 4 included 11 patients who developed a contralateral IDC and 12 control patients who did not recur. The control was selected on the basis of patients’ homogeneity in terms of age , adjuvant therapy and radiotherapy administration. The median follow-up for Cohort 4 was of 94 months (range: 61–120 months). Twenty unrelated IDC samples were used as invasive controls. No samples were excluded from the cohorts.We used four different cohorts of DCIS patients, two with pure DCIS lesions (Cohorts 1 and 2), the third with adjacent DCIS and IDC lesions, and the fourth one with pure DCIS followed or not by the occurrence of IDC in the contralateral breast during follow-up. 30 formalin fixed paraffin-embedded (FFPE) samples of primary pure DCIS (>70% tumor tissue) were selected from a collection of tumors archived at three independent sites: Verona, Padua and Rovigo (Cohort 1) and 17 FFPE breast tissue sections with pure DCIS were from Pisa Archive (Cohort 2). 30 FFPE breast tissue sections with adjacent DCIS and IDC lesions were obtained again from the Pisa archive (Cohort 3). Finally, 20 FFPE samples of primary unilateral pure DCIS from patients with available follow-up data were from the Verona site and 3 from the Rome archive . Successively, we used for validation the raw data for DCIS and IDC in the Norway study by Lesurf et al. [The miRNA expression profiles of DCIS Cohort 1 were generated using next-generation sequencing (NGS) on the SOLiD platform (ABI) as described in the Supplemental Materials and were compared with those published by Farazi et al. , and witf et al. .2 were selected and micro-dissected samples were loaded on Maxwell 16 (Promega) to extract RNA, following the Maxwell 16 LEV RNA FFPE protocol. RNA was reverse-transcribed for miR-126 and miR-218 using the TaqMan MicroRNA RT kit (Applied Biosystems). cDNAs in Cohort 2 and 3 were pre-amplified using TaqMan PreAmp protocol (Applied Biosystems) and subsequently droplet digital PCR performed using the QX100™ System (Bio-Rad) according to the manufacturer's instructions. QuantaSoft (Bio-Rad) was used to convert the data into concentrations using Poisson distribution. The miRNAs in DCIS samples from Cohort 4 were quantified by Real-Time qPCR using TaqMan MicroRNA assays (Life Technologies) and a Bio-Rad CFX96™ system. Statistical analysis was performed using the non-parametric median test or Mann-Whitney U test for independent samples (SPSS version 21). Two-sided tests were always used and P values ≤ .05 were considered statistically significant.Samples with pure DCIS (Cohort 2), with adjacent DCIS and IDC (Cohort 3), and with pure DCIS samples from patients with follow-up (Cohort 4), as well the IDC control samples, were laser micro-dissected (LMD) by using the PALM MicroBeam laser microdissector (Carl Zeiss) on 2 μm thick sections. For each sample, 200.000–600.000 μm2. The BT-474 breast cancer-derived cell line was from ICLC  and was maintained in RPMI 1640 growth medium (Gibco) supplemented with 10% FBS, 1 mM Na pyruvate and 0.01 mg/ml bovine insulin. MDA-MB-231 cells were from the American Type Culture Collection (ATCC) and were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% FBS.The human breast cancer-derived cell line MCF10DCIS, one of the very few established models of DCIS , was kinAll cell lines were monthly tested for mycoplasm and other contaminations and quarterly subjected to cell identification by single-nucleotide polymorphism.For specific modulation of miRNAs, transient transfections were carried out with synthetic inhibitors or mimics for miRNAs  and Lipofectamine 2000 in Opti-MEM I medium (Gibco) without serum. Random sequences were used as negative controls and to check for any contribution from miRNAs in serum. The transfected cells were incubated at 37° C for 48 hours prior to immunochemical and cellular assays.U test for independent samples. Two-sided tests were always used and P values ≤ 0.05 were considered statistically significant.Total cells lysates (25 μg protein) were separated on 7.5% polyacrylamide SDS gels and blotted to nitrocellulose membranes . The membranes were incubated with antibodies directed against Vimentin and β-tubulin (Sigma) and E-cadherin (Santa Cruz Biotechology), and revealed with ECL (PerkinElmer). The chemiluminescent bands were detected on an ImageQuant™ LAS 4000 imager and densitometry was performed with Image Quant TL . Statistical analysis of Western blot data was performed using the non-parametric median test or Mann–Whitney Cell proliferation and invasiveness were evaluated with the xCELLigence Real-Time Cell Analyzer System (Roche) that monitors cell events by measuring electrical impedance, as previously reported . To measet al. [n = 796). The TCGA miRNA profiles for primary BCs were obtained from TCGA data portal . The data from the Buffa's study [n = 210) were obtained from the GEO repository (GSE22216). All miRNA profiles were quantile normalized. The association between continuous miRNA expression and survival times was carried out using univariable Cox regression. The Kaplan–Meier survival curves with the cases divided by miRNA expression (median cut) were constructed for survival times up to 120 months and the log-rank test was used to assess their significance. The analyses were performed using BRB-Array Tools-R/BioConductor (version 4.4) and SPSS (version 21). No samples were excluded from the cohorts.The data from the METABRIC study of Dvinge et al. , accessi's study  .


StarTrack, which attributes a specific and unique color-code to single neural precursors, allowing all their progeny to be tracked. We used integrable fluorescent reporters driven by a ubiquitous promoter in PiggyBac-based vectors to achieve inheritable and stable clonal cell labeling. In addition, coupling this to an inducible Cre-LoxP system avoids the expression of non-integrated reporters. To assess the utility of this system, we first analyzed images of combinatorial expression of fluorescent reporters in transfected cells and their progeny. We also validated the efficiency of the UbC-StarTrack to trace cell lineages through in vivo, in vitro and ex vivo strategies. Finally, progenitors located in the lateral ventricles were targeted at embryonic or postnatal stages to determine the diversity of neurons and glia they produce, and their clonal relationships. In this way we demonstrate that UbC-StarTrack can be used to identify all the progeny of a single cell and that it can be employed in a wide range of contexts.Clonal cell analysis defines the potential of single cells and the diversity they can produce. To achieve this, we have developed a novel adaptation of the genetic tracing strategy, UbC- In the brain, groups of clonally related cells are responsible for forming all the adult neural circuits. Thus, clonal analys1is of single cells is a powerful means to understand how neural cells acquire their identity and functional differences. There is currently much controversy regarding the commitment and heterogeneity of neural progenitors. Classical theory considered the radial glia as the main neural stem cell capable to generating all neural cell typesStarTrack strategy in order to study clonally related astroglial cells that are derived from single progenitors using the hGFAP promoter15StarTrack, to perform clonal analysis and tracing of neural cell types derived from single progenitors. Unlike other lineage approaches, our ubiquitous lineage-tracing method avoids the expression of non-integrated copies of electroporated plasmids, taking advantage of the Cre-lox strategy to achieve unequivocal labeling of the entire progeny of single progenitors. Indeed, the strategy behind this method represents an exciting tool for other areas in the field of biomedicine, avoiding the use of genetically engineered mouse models.Retroviral vectors carrying a single reporter gene were able to show that radial glia cells are indeed a common progenitor for both glial and neuronal cells59101213StarTrack is based on the combination of six different fluorescent reporter proteins (XFPs) cloned into integrable, floxed and ubiquitous constructs : mT-Sapphire, a UV-excitable monomeric GFP mutant XFP with a large stokes shift; mCerulean, the brightest monomeric fluorescent cyan protein; EGFP, a weak dimer enhanced green fluorescent protein; YFP, weak dimer yellow fluorescent protein widely used; Kusabira Orange (mKO), the brightest orange monomer; mCherry, a photostable red monomer. The appropriate spectral separation of these fluorophores allows the independent confocal acquisition of photostable XFPs with minimal overlap  of CreERT2 in the presence and absence of hyPBase. In addition, a ubiquitous EGFP plasmid (UbC-EGFPflox) flanked by LoxP sites and a ubiquitous non-floxed and non-integrable mCherry encoding plasmid (UbC-mCherry) were co-electroporated. The UbC-mCherry plasmid was used as a control vector given that it is not integrated into the genome by hyPBase and CreERT2 does not inhibit it. After IUE of UbC-EGFPflox, UbC-mCherry and CreERT2 alone, without hyPBase, the plasmids remained as episomal copies. As such, when Tx was not administered, green and red fluorescence was evident in neurons but not in glial cells .To variants  a mixturvariants . Fluoresytoplasm . By cont nucleus , contribStarTrack construct that expressed a XFP was electroporated into HEK cells independently, to determine the minimal spectrum separation with the confocal acquisition parameters employed. Their spectra led to the ordering of the fluorophores as mT-Sapphire, mCerulean, EGFP, YFP, mKO and mCherry, and they were successfully excited with five different confocal laser lines: 405, 456, 488, 514 and 561 nm. To avoid spectral overlap, emission wavelengths were chosen as close as possible to the peak spectrum, with a range of 10 nm for each XFP. When acquired individually, the electroporated XFPs gave a fluorescent signal in the channel designated to each particular fluorophore  of embryonic brain slices. After 48h, labeled cells were located within the SVZ, and clonal cells were dispersed throughout the lower cortical layers  or negative (<20) signal for each fluorophore . The ordor-codes . Besidesor-codes . As hyPBor-codes . Single StarTrack mixtures were performed in vivo at both at embryonic and adult stages . This produced a unique color code for each parental cell and its descendants, allowing neurons and/or different glial cells from single-fate restricted, bi-and tri-potent precursors to be identified.We developed UbC-The enormous cell heterogeneity of the adult brain will not be totally understood without specific studies to analyze all the progeny of single cells. Transcriptome analyses196StarTrack included a reporter gene. Here we employed a hyperactive version of the mPBase (hyPBase) that integrates a variable number of transposable copies into the genome at a 10-fold higher transposition rate (9 copies on average), without compromising genomic integrity2612-StarTrack constructs resolved both the issues related to integration and the inhibition of non-integrated constructs in order to produce a reliable clonal signal in all the progeny, irrespective of the lineage or time of electroporation. To analyze early-generated cell lineages, the tamoxifen administration should be performed 1dpe to accurate label all the cells from single progenitors with the same color-code.We previously used the PiggyBac system for long-term clonal tracking of the glial lineage in different brain regions2223StarTrack and UbC-H2B·StarTrack mixtures ascends to more than four thousand. In fact, UbC-StarTrack is the clonal method with the widest range of XFPs, expanding the clonal combinations to allow accurate identification of sibling neural cells. While other approaches used Red-Green-Blue reporters13StarTrack potential, we estimated the XFP combinations produced in cells from different IUE brains. In terms of individual fluorophore expression, mKO, YFP or Cerulean could be detected in 30–40% of the cells analyzed, indicating they are useful XFPs for the purpose. The less frequent XFPs were relevant as the clones that expressed these reporters are reliable because of their lower probability. Conversely, prevalent XFPs like EGFP or mT-Sapphire are less informative to define cell clones but they are necessary to increase the number of possible combinations and they contribute to defining less probable combinations. According to the data obtained from more than 6,000 cells, some fluorophore combinations accurately define clones and others, as single-fluorophore expression, do not.Regarding the evaluation of the clonal markers, the theoretical number of possible combinations after the co-electroporation of both UbC-StarTrack: fluorophore combination, fluorophore location within the cell and fluorophore intensity.Given the high levels of DNA transposition of the PiggyBac transposaseStarTrack IUE: clonal cell dispersion and the number of cells per clone. Regarding clonal dispersion, sibling neurons were more scattered than glial cells since neurons often migrate to their final position far from their site of birthStarTrack tracked large oligodendrocyte clones that were recognizable by their morphology and due to their position in the adult corpus callosum. Thus, the distribution of clonally related cells and their cell number was related to their cell lineage.To achieve the study of all neural cells we used UbC promoter, which drives consistent expression in different cell types35StarTrack is also a versatile method that is finely tuned and adapted to a broad number of specific variables regarding cell fate tracking. Indeed, we generated 18 ubiquitous, bright and stable XFPs, which can be readily combined with other expression vectors. Moreover, this approach could be adapted to analyze any cell lineage by using appropriate promoters in order to simultaneously track different populations. Moreover, genetically engineered mouse models can be combined with the UbC-StarTrack constructs, providing additional information. Indeed, in transgenic mice containing any XFP the loss of a specific fluorescent UbC-StarTrack construct will provide significant information. Our floxed constructs are suitable to track clonal related lineages, even in Tx inducible transgenic mice lines. In fact, using such transgenic mice, Tx administration will activate the conditional gene while the non-integrated UbC-StarTrack constructs will be inhibited, overcoming the need for the CreERT2 plasmid electroporation. Moreover, in vitro or ex vivo clonal analysis using a wide range of primary cultures could be performed.Together, these data demonstrate the use of this approach to efficiently track the entire progeny of specific single progenitors. Besides being an efficient method for lineage analysis, UbC-StarTrack will undoubtedly be a useful tool to understand the fundamental events in cancer biology, serving to address the lineage heterogeneity within primary tumors or metastatic niches, to track cancer stem cells and their progeny, and to study the precursor-progeny relationships within tumorigenic cells. Another important area, in which our method could add important information is that of neural disease, providing information as to how cell clonality may be involved in the recovery from injuryFinally, UbC-Wild type C57BL/6 mice from the Cajal Institute animal facility were treated according to the European Union guidelines on the use and welfare of experimental animals (2010/63/EU) and those of the Spanish Ministry of Agriculture (RD 1201/2005 and L 32/2007). Performed experimental approaches were approved by the Cajal Institute, CSIC Animal Experimentation Ethics Committees and the Community of Madrid (Ref.PROEX 44/14). The day of detection of the vaginal plug was defined as the first embryonic day (E0) and the day of birth as postnatal day 0 (P0).Wild type C57BL/6 mice from the Cajal Institute animal facility were treated according to the European Union guidelines on the use and welfare of experimental animals (2010/63/EU) and those of the Spanish Ministry of Agriculture (RD 1201/2005 and L 32/2007). The CSIC Bioethical Committee approved all the procedures used here. The day of detection of the vaginal plug was defined as the first embryonic day (E0) and the day of birth as postnatal day 0 (P0).CloneJET PCR Cloning Kit (Fermentas) and cohesive restriction enzymes (Fermentas) in all cases. The Rapid DNA Ligation Kit (FERMENTAS) was used for ligations and the resulting products were transformed into E. coli JM107 bacteria using the “TransformAid” Bacterial Transformation Kit (Fermentas).Oligonucleotides were obtained from SIGMA-ALDRICH, and the PCR products obtained with these were cloned using StarTrack, we employed a PiggyBac plasmid encoding the enhanced green fluorescent protein (EGFP) under the control of ubiquitous human Ubiquitin C (UbC) gene promoter, pPB-UbC-EGFP . Initially, a cloning vector containing the LoxP sites (34 bp) and a multiple cloning site (MCS) was generated. The resulting plasmid, pPB-UbC-MCSflox was used successively to clone the XFPs: mT-Sapphire, mCerulean, yellow fluorescent protein (YFP), EGFP, monomeric Kusabira Orange (mKO), and mCherry. The XFPs were amplified by PCR from the pPB-GFAP-XFPs StarTrack constructs , and the floxed and integrable plasmids expressing six different XFPs were developed, named UbC-StarTrack. The sequence encoding histone h2B (H2B) was amplified by PCR using the pPB-GFAP-XFPs StarTrack constructs as a template and they were fused to each fluorescent protein in the UbC-StarTrack constructs: UbC-H2B·StarTrack. Moreover, the Tau protein was amplified from the P3-IRES-tauEGFP plasmid (Adgene #15643) and fused to the different XFPs, generating the UbC-TAU·StarTrack constructs. All the constructs were sequenced to assess the efficiency of cloning.To generate UbC-T2 plasmid was a kind gift from Connie Cepko (Addgene #14797). A non-integrable plasmid encoding mCherry under the UbC promoter (UbC-mCherry) was used as a control of electroporation  was kindly provided by Prof. Bradley, while the pCAG-CreERin vitro expression of the constructs generated. Briefly, 1.5–2·105 cells per well were cultured on polyornithine-plated 6-well plates maintained at 37 °C in an atmosphere of 5% CO2, and in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 0.5% penicillin/streptomycin (Invitrogen). Transfection was performed either with the calcium phosphate method or with the commercial transfection reagent TurboFect (Life Technologies). In both cases, a total amount of 3 μgr from the DNA mixture was administrated to each well. The expression of the newly generated constructs was assessed 1 day after transfection. To long-term clonal experiments, cells were passaged every 3–4 days the confluent cultures using 0.05% Trypsin-EDTA (Gibco).HEK293 cells were cultured to validate the StarTrack plasmid mixture was then injected into the SVZ with a borosilicate glass filament. To avoid direct contact of the electrode with the slice, the membranes containing the slices were placed between a protective agarose support, the electrodes were placed in position and then, 2 pulses  were delivered to pass the reporters into the cells. After electroporation, membranes containing brain slices were cultured 1 hour in MEM medium (Life Technologies) and then were placed in Neurobasal medium (LifeTechnologies) for long-term cultures. Slices were analyzed after 48–72 h incubation at 37 °C in an atmosphere of 5% CO2 in Neurobasal.After embedding E14 brains in low melting point agarose dissolved in 1X KREBS buffer, 300 μm slices were placed on polycarbonate membranes (Whatman) placed in KREBS medium. UbC-et al.IUE was performed as described previously  was dissolved in corn oil (Sigma-Aldrich) at a concentration of 20 mg/ml and a single dose of 5 mg/40 gr body weight was administered. When analyzing embryos, Tx was administrated to pregnant females by intraperitoneal injection 24 h post IUE, whereas to analyze adult mice it was administered at perinatal stages.Mice were analyzed at embryonic, postnatal and adult stages. When embryos were used for analysis, the dams were sacrificed, the embryos were extracted from uterine horns and they were then decapitated. Postnatal mice were anesthetized by hypothermia (P1 to P6) or intraperitoneal injection of sodium pentobarbital , their brain was fixed by transcardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer and then post-fixed overnight in the same fixative. Coronal/sagittal serial vibratome sections (50–100 μm thick) were then obtained.Fluorescent labeling was visualized under an epifluorescence microscope  with the appropriate filter cubes (Semrock): UV-2A (FF01-334/40-25) Cerulean (FF01-405/10), GFP (FF01-473/10), YFP (FF01-520/15), mKO (FF01-540/15), mCherry (FF01-590/20) and Cy5 (FF02-628/40-25). Images were acquired on a Leica TCS-SP5 confocal microscope, capturing the different XFPs in separate channels. The wavelength of excitation (Ex) and emission (Em) for each XFP were : mT-Sapphire , mCerulean , EGFP , YFP , mKO , mCherry , and Alexa 633 . Confocal laser lines were in-between 25–40% in all cases and the maximum projection images were created using confocal (LASAF Leica) and NIH-ImageJ software.The imaging data was analyzed with a custom macro integrated into ImageJ (NIH). The critical step in the macro analysis was to appropriately select the labeled cells for which the threshold for every image in each confocal channel was adjusted in order to create positive cell selection for each XFP. Background subtraction was employed before the threshold selection by applying a smooth filter to improve cell signal selection and a binary image was then created to generate the selection. A watershed filter was applied after the binary image to separate contiguous tagged cells and to analyze them as individual points. Once the selection had been performed, minimal fluorescence intensity was considered as a positive value. Subsequently, the data were automatically organized in a table indicating the maximal and minimal XFP intensity value for each labeled cell and its specific color code represented in the potential clonally related cells. Further analysis was performed to determine sibling cells based on the fluorescence intensity ranges and the location of the fluorophore (nucleus or cytoplasm).How to cite this article: Figueres-Oñate, M. et al. UbC-StarTrack, a clonal method to target the entire progeny of individual progenitors. Sci. Rep.6, 33896; doi: 10.1038/srep33896 (2016).


Nine dogs with stage-2 HSA underwent 18FDG-PET-CT following splenectomy and prior to commencement of chemotherapy. Routine staging  was performed prior to 18FDG-PET-CT in all dogs. When abnormalities not identified on routine tests were noted on 18FDG-PET-CT, owners were given the option to repeat a PET-CT following treatment with eBAT. A PET-CT scan was repeated on Day 21 in three dogs. Abnormalities not observed on conventional staging tools, and most consistent with malignant disease based on location, appearance, and outcome, were detected in two dogs and included a right atrial mass and a hepatic nodule, respectively. These lesions were larger and had higher metabolic activity on the second scans. 18FDG-PET-CT has potential to provide important prognostic information and influence treatment recommendations for dogs with stage-2 HSA. Additional studies will be needed to precisely define the value of this imaging tool for staging and therapy monitoring in dogs with this and other cancers.Positron Emission Tomography-Computed Tomography (PET-CT) is routinely used for staging and monitoring of human cancer patients and is becoming increasingly available in veterinary medicine. In this study, 18-fluorodeoxyglucose ( Malignant cells rely on anaerobic glycolysis for energy consumption. Anaerobic glycolysis is a relatively inefficient process, resulting in a strongly increased demand for glucose in cancer cells, which can be visualized with FDG. Following administration, FDG extravasates into the interstitium, where it is internalized via facilitative glucose transporters (GLUT), which are present on all cell membranes and especially enhanced on tumor cell membranes. FDG is then phosphorylated into FDG-6-phosphate, which, unlike glucose, is trapped inside the cell and cannot enter the glycolysis pathway [Positron Emission Tomography-Computed Tomography (PET-CT) is commonly used in human cancer patients to non-invasively diagnose, prognosticate, evaluate therapy-induced changes in tumor metabolism, and monitor response to antineoplastic therapy –3. The cquipment –7. 18FDG18FDG-PET-CT provides information otherwise not obtainable with conventional diagnostic tools, potentially improving the quality of staging and treatment monitoring [7 Increased uptake of FDG has been demonstrated in canine lymphoma [18FDG-PET-CT as a staging and monitoring tool in dogs with hemangiosarcoma (HSA).A recent study evaluating the effectiveness of this image-guiding method in dogs with various naturally-occurring malignancies showed that nitoring . This stlymphoma ,10, canilymphoma  and varilymphoma ,7. Howev18FDG-PET-CT to diagnose systemic disease [18FDG-PET-CT to detect potential cancerous lesions in dogs with stage-2 splenic HSA following splenectomy, as compared to conventional radiography and ultrasonography. We hypothesized that 18FDG-PET-CT would allow for identification of potentially malignant lesions that were otherwise undetectable by routine imaging modalities.HSA is a malignant neoplasm of blood vessel forming cells. It is highly metastatic and invariably fatal in dogs, resembling human angiosarcoma. It occurs primarily in the spleen, followed by right atrium, skin and subcutis, and liver . Metasta disease . We thusDogs that received PET-CT scans were undergoing diagnosis and treatment for HSA at the Veterinary Medical Center (VMC), University of Minnesota (UMN) according to a clinical trial investigating safety and efficacy of eBAT administered following surgery (splenectomy) and prior to five cycles of doxorubicin chemotherapy starting on Day 21 .All imaging studies were conducted with approval and under the oversight of the UMN Institutional Animal Care and Use Committee (IACUC Protocols 1110A06186 and 1507-32804A). Inclusion criteria for PET-CT scanning included: histopathologic diagnosis of splenic HSA obtained following splenectomy; no evidence of gross regional metastasis at surgery, nor regional or distant metastasis from standard diagnostic modalities ; acceptable risk profile for general anesthesia; normal blood glucose level at baseline; and written informed client consent. There were no restrictions based on age, gender, neuter status, or other physical parameters. If abnormalities were noted on the PET-CT that were not visualized on routine staging tests, owners were given the option for their dogs to have a subsequent PET-CT on Day 21 after completion of the experimental eBAT therapy and prior to initiation of doxorubicin.18FDG, with 60-min uptake time prior to PET-CT scanning. Dogs were anesthetized using propofol  for induction (5 mg/kg or to effect) and isoflurane/oxygen mixture via endotracheal tube for maintenance, placed in sternal recumbency, and whole-body CT and PET data (100% of body coverage) were acquired with a standard protocol using Siemens mCt-64 Biograph TrueD HD PET/CT scanner fitted with high resolution crystals (HD PET) . High resolution, diagnostic quality CT was employed for all studies. The scanner was cross calibrated between the dose calibrator and scanner and all clocks were synchronized. The protocol started with pre and post contrast CT images acquired from head to tail using CareDose4D and a kernel of B30f medium smooth with a slice thickness of 1.0 mm and 0.8 mm slice interval. The whole-body emission PET scan of the same region followed at 3 min per bed table position at a resolution of 256 x 256. Images were subsequently reconstructed into 3 mm slice thickness by 3mm slice interval images, CT matrix 512 x 512, slice thickness 1.0 mm, fov = 600 mm. The reconstruction method used was TRUEX (HD-PET) with a gaussian filter with ordered-subset expectation maximization algorithm. Attenuation correction of PET images was performed using the CT data. Contrast medium  was given at a dose of 2.0 ml/kg.PET-CT scans were performed at the Center for Clinical Imaging Research (CCIR), UMN, after splenectomy and before chemotherapy administration. Dogs were fasted for a minimum of 12 hours with water freely available. Pre-medication with diazepam  at 0.1 mg/kg and butorphanol  at 0.2 mg/kg was used to minimize anxiety and muscular activity during the radiopharmaceutical uptake period . Blood gAll images were evaluated by one or more of three board certified veterinary radiologists  using Carestream Vue PACS  and by a Nuclear Medicine and Diagnostic Radiology board-certified medical radiologist (JF) using Siemens Syngo Via MI Oncology Version 3.0 (VB1) workstation software. All scans were inspected for any areas of increased metabolic activity. Abnormalities, if present, were measured on post-contrast CT scans in the sagittal, transverse, and dorsal planes.18FDG uptake was evaluated subjectively, and a representative region of interest (ROI) was drawn with freehand technique over the maximum size of each lesion on pre- and post-contrast images and mean attenuation was calculated in Hounsfield Units (HU). Maximum standardized uptake value (SUVmax) and peak SUV (SUVpeak), defined as the average SUV within a small, fixed-size region of interest (ROIpeak) centered on a high-uptake portion of suspected tumor lesions as well as other abnormalities were calculated [max and SUVpeak of the liver were also calculated for each dog and used as background reference. Contrast enhancement of each lesion was graded as homogeneous, heterogeneous, or ring. Findings from the diagnostic CT portion of the studies were compared to those from the fused PET-CT images.lculated . SUVmax 18FDG-PET-CT scans are summarized in 18FDG-PET-CT scan was 18 days (average 21 ± 8 days). A second 18FDG-PET-CT scan was performed in three dogs following the detection of a right auricle mass (Dog 2), renal changes (Dog 3), and a liver nodule (Dog 6).The demographic characteristics of the nine dogs that had peak and SUVmax for 18FDG uptake in the liver were calculated for each dog (peak was 2.12 ± 0.51 (range 1.36 to 2.75) and the mean reference SUVmax was 2.23 ± 0.56 (range 1.44 to 2.94). The reference SUVpeak and SUVmax values were tightly correlated (R2 = 0.99), but neither value was correlated with the blood glucose concentrations.Reference SUVeach dog . The meapeak and SUVmax are shown in Individual pre-injection glucose concentration values and areas of increased FDG uptake with corresponding SUVWhen analyzing the diagnostic CT portion of the studies, findings included a sclerotic lesion at the right humeral methaphysis with no specific cortical or periosteal reaction or destruction and mild to moderate enhancing regional soft tissues surrounding multiple joints with increased amount of joint fluid in the left elbow joint (Dog 1), a mass associated with the right atrium that was progressive on the second scan (Dog 2), irregular contrast-enhancement along the lateral cortical margin of the left kidney that was no longer observed on the second scan (Dog 3), a focal region of bone lysis surrounding the roots of the left upper incisors (Dog 5), a lesion at the apex of the gall bladder that was progressive on the second scan (Dog 6), and enlarged lymph nodes that corresponded to increased uptake on the PET portion of the studies (Dogs 1–3). The reactivity in the pleural space in Dog 3 and the enhanced muscular activity in the crus of the diaphragm (Dog 4 and 7) noted on the PET studies did not correlate with CT abnormalities.18FDG PET-CT scan in Dog 2 showed increased metabolic activity in the right atrium, auricle, and ventricle. The degree of activity within the left ventricular wall was considered normal whereas the focus of high activity in the right auricular appendage raised suspicion for a malignancy  bone scans in 29 pediatric patients with bone and soft tissue sarcomas showed that PET-CT was able to detect all malignant bone lesions compared to a 99Tc-MDP accuracy of 80%, leading to the conclusion that 18FDG-PET-CT was equivalent or superior as a diagnostic modality [Metastatic dissemination of HSA occurs mainly by the hematogenous pathway. Common sites of involvement include lungs and liver, but other sites, such as bone and central nervous system (CNS) can also be affected in some cases. While pulmonary metastatic disease can be frequently detected by conventional radiography, and abdominal metastases are commonly identified by abdominal ultrasonography, skeletal radiographs or bone scintigraphy to detect bone metastasis and magnetic resonance imaging (MRI) to identify CNS lesions are not routinely conducted before or after splenectomy is performed or the decision to pursue chemotherapy is made by the pet’s owners. Notably, a study comparing modality . In our 18FDG-PET-CT could fill this gap. Even though widespread adoption of this modality will require greater availability and significant cost reductions, these barriers have been largely overcome by other advanced imaging methods, such as ultrasound, computerized tomography, and magnetic resonance imaging over the past two decades. Unlike the more commonly utilized procedure to incorporate low resolution CT studies to PET studies, our 18FDG-PET-CTs consisted of diagnostic quality CT scans that allowed the identification of abnormalities that, for the most part, correlated with the increased uptake on PET. Therefore, it is arguable that CT scans alone could sufficiently improve the diagnostic quality of current staging and monitoring protocols. While this may be true, our data indicate that 18FDG-PET-CT has potential to complement and expand the quality and quantity of information that can be obtained from these currently accepted technologies, and thus help improve the management of veterinary cancer patients. It must be acknowledged that the interpretation of PET-CT studies presents several challenges. One of the main challenges is the variability in FDG uptake by normal tissues. Other factors include FDG uptake related to inflammation or infection, malignant lesions with unusually low avidity for FDG, altered biodistribution of FDG related to hyperglycemia, and bone marrow activation, among others [Because the nature of a splenic mass is often unknown prior to surgery and because the identification of widespread lesions could profoundly influence the decision to proceed with treatment, improved imaging modalities that provide more accurate and complete staging information represent an unmet need in veterinary medicine. g others . While mIn conclusion, PET-CT is a promising tool for staging and monitoring of canine HSA and studies using larger populations are warranted to further evaluate the sensitivity of this imaging modality for staging, to assess therapeutic responses, and to monitor duration of remission. Ideally, necropsy should be a requirement moving forward to correlate the PET-CT characteristics of any possible lesions with histopathology. These studies will be crucial as the availability of PET-CT increases at veterinary institutions and novel targeted therapies are developed.S1 TableAge (years), sex and neuter status, body condition score, presence of hemoabdomen, stage, and time (days) from diagnosis to PET-CT#1 and PET-CT#2 (where applicable) for the nine dogs enrolled in the study are shown.(DOCX)Click here for additional data file.S2 TableAreas of increased FDG uptake on the initial PET-CT scans (PET-CT #1), their presumed diagnosis based on appearance and SUV peak and max are shown for the nine dogs enrolled in the study and the pre-injection glucose concentration values (mg/dl) are listed for each dog.(DOCX)Click here for additional data file.S3 TableAreas of increased FDG uptake on the repeat PET-CT scans (PET-CT #2), their presumed diagnosis based on appearance and SUV peak and max are shown for the three dogs in which scans were repeated, and the pre-injection glucose concentration values (mg/dl) are listed for each dog.(DOCX)Click here for additional data file.


Verbal working memory (WM) comprises different processes  that are often compromised in brain diseases, but their neural correlates have not yet been examined in childhood and adolescence. To probe WM processes and associated neural correlates in developmental samples, and obtain comparable effects across different ages and populations, we designed an adapted Brown-Peterson task  to implement during functional magnetic resonance imaging (fMRI). In a sample of typically developing children and adolescents (n = 16), aged 8 to 16 years, our paradigm successfully identified distinct patterns of activation for encoding, maintenance, and retrieval. While encoding activated perceptual systems in posterior and ventral visual regions, retrieval activated fronto-parietal regions associated with executive control and attention. We found a different impact of verbal versus visual concurrent processing during WM maintenance: at retrieval, the former condition evoked greater activations in visual cortex, as opposed to selective involvement of language-related areas in left temporal cortex in the latter condition. These results are in accord with WM models, suggesting greater competition for processing resources when retrieval follows within-domain compared with cross-domain interference. This pattern was found regardless of age. Our study provides a novel paradigm to investigate distinct WM brain systems with reliable results across a wide age range in developmental populations, and suitable for participants with different WM capacities. The ability to maintain relevant information in mind in the presence of interference or distracting information is critical for higher cognitive functions required in daily life. Working memory (WM) is the theoretical construct used to refer to this capacity to simultaneously maintain and process information over brief periods of time according to current task goals –3. StudiFrom a developmental point of view, WM capacity develops rapidly over childhood –13. Thisn steps earlier in the sequence . De. Den stee , ha, han stee  interposed between encoding and subsequent retrieval , 30. In To validate this novel paradigm, we applied it in children and adolescents aged 8 to 16 years. We expected that all would successfully complete our adapted Brown-Peterson fMRI paradigm, which tailors task difficulty to each participant according to their WM capacity. We predicted that distinct activation patterns would be elicited by the two concurrent tasks (i.e. within and cross-domain), not only during the maintenance interval, but also during the subsequent retrieval period. Based on Baddeley’s WM model , the natM = 116.2, SD = 10.4) and normal or corrected-to-normal vision and hearing. The study was approved by the Human Research Ethics Committee at the Royal Children’s Hospital. Written informed consent was obtained from the caregivers of the children and adolescents prior to participation.Participants were 16 healthy children and adolescents aged 8 to 16 years , 9 females and 7 males, recruited though advertisements in local schools and staff at the Royal Children’s Hospital. The wide age range of this sample allowed us to examine whether the adapted Brown-Peterson task was suitable for both children and adolescents. No participant had a documented history of a brain lesion, neurological disability or neurodevelopmental disorder such as autism spectrum disorder (ASD) or attention deficit hyperactivity disorder (ADHD). All participants were right-handed as measured by a score between +40 and +100 at the Edinburgh Handedness Inventory , 33, EngParticipants completed an adapted version of the Brown-Peterson paradigm , 30 impl1) Encoding period.Participants were presented with a series of single upper-case letters for further recall displayed sequentially in the middle of the screen at a rate of one letter per second. All consonants of the English alphabet were used as memory items except W, which is three-syllabic. Series of two and three letters were created for within-domain and cross-domain blocks in such a way that each letter appeared with the same frequency in both blocks. Participants were asked to maintain the letters in order of appearance.2) Maintenance delay filled with a concurrent task.During the maintenance delay of 6 seconds, a concurrent task required to process either verbal or non-verbal stimuli involving within- or cross-domain interference respectively.The within-domain concurrent task was a lexical decision task. Two successive letter-strings were presented for 3 seconds each and required simple motor responses . Words were selected from the “Oxford Wordlist”, which is an Australian database of high frequency words in young children’s writing and reading development . Among tThe cross-domain concurrent task was a face decision task. Two successive pictures were presented for 3 seconds each, requiring similar motor responses . Ten males and 10 females faces with a neutral expression were selected from the NimStim database , and con3) Retrieval period.At retrieval, one single upper-case letter was presented along with either one or two place-holders  made of dashes with a question mark. Participants had to decide if the single letter matched the letter that was presented in that serial position during the encoding period by giving a simple motor responses, i.e. press as quickly and as accurately as possible the left-most/green button or the right-most/red one for positive and negative responses respectively. This was done to make sure that participants memorised both item and serial order information.In addition to the active condition, there was a baseline condition (no-concurrent task) in which participants were required to encode a single letter and recognise it after a short empty delay of 1 second. They were instructed to press as quickly and as accurately as possible the left-most/green button if the single letter was the same during encoding and retrieval; or the right-most/red one if it was a different letter.For both the active and baseline conditions, a randomized inter-trial interval of 2000, 2500, or 3000 milliseconds was presented before the next trial. Three types of blocks of 10 trials each were created: two active blocks, one including the within-domain concurrent task and the other including the cross-domain concurrent task, and a third baseline block. The order of presentation of these three blocks was counterbalanced across participants and repeated twice for a total of six blocks of 10 trials. Within each block, half of the probes were positive  and the position of positive and negative probes were randomized within each blocks.Two important challenges of brain imaging studies examining cognitive development are that differences in both participant age and task performance may influence activation patterns. One concern is whether changes in neural activity reflect changes in functional maturation of the central nervous system, independently of behavioural efficiency, or whether they reflect changes in task performance consequent upon increasing age , 38. ForParticipants completed the adapted Brown-Peterson fMRI paradigm. This fMRI paradigm was presented visually during fMRI using E-prime2 . Initially, participants successfully completed a mock MRI scanner training protocol before the MRI. Participants were prepared for the adapted Brown-Peterson paradigm through training initially outside  and then inside the scanner before starting fMRI acquisition . All participants demonstrated understanding of the paradigm before being placed in the scanner. The paradigm was projected onto a screen at the foot of the MRI bed, and participants viewed the images from a mirror attached to the head coil. To minimize head motion during scanning, a soft cloth was placed on the child's forehead, then taped to the head tray, and foam pads were inserted around the head. Responses were provided using an MRI compatible response box with four response buttons. The response box was placed centrally on the child’s stomach and responses were provided by pressing the left-most/green button with the left thumb or the right-most/red button with the right thumb, respectively.Separate repeated measures analyses of variance (ANOVA) were conducted on accuracy measures (percent correct) for the concurrent tasks  and the retrieval period with the type of the previous concurrent task (within- or cross-domain) as within-subject factor. Independent-sample t tests were used to explore sex differences in accuracy. Pearson’s correlation was used to study the relationship between age and accuracy. Statistical analyses were performed using SPSS Statistics V22.0 .MRI was performed on a Siemens 3T MAGNETOM Trio scanner  at the Royal Children’s Hospital. The scanner was equipped with the Syngo MR B17 software release, and a 12-channel receive-only head coil was used. T1-weighted MP-RAGE sequence (Magnetization Prepared Rapid Gradient Echo) were obtained using the following parameters: repetition time (TR) = 1900 ms, echo time (TE) = 2.71 ms, inversion time (TI) = 900 ms, flip angle (FA) = 9°, field of view (FoV) = 256mm, voxel size = 0.7 x 0.7 x 0.7 mm. Functional images were acquired using a T2-weighted with a gradient-echo-planar imaging (EPI) sequence with 32 interleaved slices with 5% gap, voxel size = 2.6 x 2.6 x 3 mm, TR = 2400ms, TE = 35ms, FA = 90°, FoV = 240mm.fMRI data were preprocessed and analysed using SPM8  implemented in Matlab R2014a. The images of each subject were corrected for slice acquisition timing, and spatially realigned to eliminate movement artefacts. Head motions were small in any direction : X = 0.04mm, Y = 0.2mm, Z = 0.01mm; Mean translation: X = 0.08mm, Y = 0.11mm, Z = 0.25mm ; Mean rotation : X = 0.004mm, Y = 0.003mm, Z = 0.002mm) and therefore no participant was excluded from further processing [Statistical analyses were performed using a two-step process, taking into account the intra-individual and inter-individual variance . First lAll six movement parameters  were included as covariates of no interest in the model. The individual statistical images from each condition were then entered in a group analysis at the second level using a flexible factorial design, which provides the flexibility to specify the different period of our mixed block and event-related paradigm. In this random-effects model, independence and unequal variance between subjects and conditions were assumed, allowing for violation of sphericity, as implemented in SPM8. Considering a possible impact of gender on brain-activation, we also added this binary variable as a covariate in the flexible factorial design , 42, 43.We performed exploratory analyses to examine age- and retrieval accuracy-related changes in brain activation during the Brown-Peterson fMRI paradigm. The largest and most relevant clusters of activation identified at the group level were used to define functional regions of interest (ROIs) for each of the different conditions using the marsBaR toolbox . Beta vaWe also performed a whole-brain analysis where different active phases were compared , but now including age and retrieval accuracy as covariates of interest in a multiple parametric regression design using SPM8. For these regressions, a significant threshold of p < .001 uncorrected for multiple comparisons with a minimum extent threshold of 20 voxels was used.s = 33, z = .58, p = .565). Concerning retrieval of the active condition, repeated-measures ANOVA showed no effect of type of concurrent task on response accuracy, F  = 1.278, p = .276 . Hence, differences in brain activity patterns at retrieval could not be explained by differences in WM performance.As far as the concurrent tasks were concerned, the percentage of correct responses was 97% (SD = 4.3) for the within-domain  and 98% (SD = 3.5) for the cross-domain (face decision task). For the effect of the type of the concurrent task, assumption of normality was violated, as assessed by inspection of histograms and results of the Shapiro-Wilk test (p = .001). Therefore, related-sample Wilcoxon-signed rank test was used and showed no significant effect of the type of concurrent task , and no significant relationship between age and response accuracy on the concurrent tasks . There was no significant gender difference for any of the measures, ts < 1, ps > .50.There was no significant relationship between age and response accuracy on the retrieval of the active condition whatever the type of the previous concurrent task , was associated with widespread activations bilaterally in the occipital and ventral temporal lobes , as well as in medial frontal areas (supplementary motor area (SMA), middle cingulate gyrus) and precentral gyrus. Smaller activation foci were found in the insula . ConversComparing activations during the within-domain concurrent task , relative to the cross-domain concurrent task (face decision task), revealed differential increases in the right middle frontal gyrus (Brodmann area 10) and medial fusiform cortex only . ConversThe most critical question concerning the WM system in our paradigm is whether the nature of the concurrent task during the maintenance interval may produce different degrees of competition and thus result in different neural substrates during retrieval. We therefore tested for brain regions that would be differentially activated during the retrieval period when following within-domain concurrent task  or when following cross-domain concurrent task (face decision). Greater increases following the within-domain concurrent task were found in visual areas, with large bilateral clusters in occipital cortices . Conversely, greater increases were found after the cross-domain concurrent task in the left middle and superior temporal cortex, overlapping with usual location of phonological processing , 49, pluSeveral functional ROIs were defined for each of the contrast of interest described above  age; and (b) WM retrieval accuracy for the main contrasts of interest as described above . None of these analyses revealed any significant overlap with activations identified by the main contrasts of interest reported in We report and validate an adapted Brown-Peterson fMRI paradigm that probes for the neural correlates of different WM processes, including encoding, maintenance and retrieval, as well as the effect of within- and cross-domain concurrent tasks during maintenance. Results indicate that this paradigm can be performed equally well by children and adolescents of different ages, with reliable results across different levels of performance. To our knowledge, this is the first study to propose a paradigm to delineate distinct patterns of brain activity for the different WM processes in children and adolescents. We provide the first exploratory results on brain activity related to encoding, maintenance, and retrieval WM processes in children and adolescents, and compare verbal WM in the presence of both verbal (within-domain) and visual (cross-domain) concurrent tasks.As expected, our adapted Brown-Peterson paradigm was successfully completed with high accuracy in the MRI scanner by typically developing children as young as 8 years of age, indicating that it is suitable to examine WM processes in children and adolescents from 8 to 16 years of age. It is important to note that task difficulty was adapted to each participant’s WM capacity using a simple procedure , and we found no significant association between age or task performance and brain activation patterns. These findings indicate that our paradigm is well suited to examine brain systems associated with different WM capacities in different age groups. This may be an important advantage when comparing groups with different developmental trajectories, because previous studies show that WM-related activations may increase with age in parallel with changes in performance and improvements in WM capacity , 18.Secondly, our imaging results demonstrate that, while distributed networks in frontal and visual areas activated in the context of the verbal WM paradigm used here (i.e. during the active conditions compared to the baseline), distinct neural substrates were selectively recruited during the encoding and retrieval periods. The verbal encoding period induced stronger activations in posterior and ventral brain regions, with large bilateral increases in occipital, as well as parahippocampal cortices. In contrast, the verbal retrieval period induced stronger activations in more anterior and dorsal regions, in particular in prefrontal and parietal areas, and to a lesser extent in lateral temporal areas.The predominance of activity in visual cortex together with medial temporal lobe  during encoding is consistent with the need to extract discriminative visual information from the to-be-remembered stimuli and store this information into short-term memory. On one hand, ventral occipito-temporal areas differentially engaged during encoding are crucial for perceptual shape analysis, especially for letters with a letter-sensitive activation in these regions , 51. We As expected, predominant activity in frontal and parietal areas during retrieval is consistent with executive control and attentional focusing. The executive control system serves as an attention controller that allocates and coordinates attentional resources for cognitive tasks, such as retrieval of information encoded in working memory , 60. OurOverall, our findings converge with those of van den Brosh and colleagues (2014), who reported a similar posterior and perceptual network during the encoding phase compared to a more anterior and executive network during the recall phase of a Sternberg item recognition paradigm (which did not include a distracting phase) in children and adolescents aged 9 to 19 years. However, these authors did not find any temporal or parahippocampal activations, possibly reflecting differences in the paradigm and material used (digits in their study vs. letters in ours). More generally, our findings of extensive fronto-parietal and visual activity during WM also dovetail with previous neuroimaging studies investigating brain systems associated with verbal WM in children and adolescents, across different kinds of verbal WM paradigms, such as the Steinberg item recognition task using letters , 67, 68 Study hypotheses were supported by results revealing that brain activation patterns differ as a function of the nature of the concurrent task performed during the maintenance interval. Our design allowed us to compare the impact of within-domain  versus cross-domain (face decision task) concurrent task processing during the maintenance period intervening between encoding and retrieval, while information stored in WM itself did not differ. A lexical decision task was expected to produce within-domain interference, as it involved verbal material resembling the to-be-remembered material (i.e. letters), while a face decision task was considered to induce cross-domain interference as it relied on non-verbal visual processes.As predicted, the within-domain and cross-domain concurrent tasks evoked distinct brain activations when compared to each other. Localised and right-sided activations in the right frontal pole (Brodmann area 10) and medial fusiform gyrus were observed during the within-domain/lexical concurrent task, whereas the cross-domain/face concurrent task elicited much more distributed activations in occipital temporal extrastriate areas, but also left parahippocampal gyrus and fronto-parietal regions. These differences could not be attributed to task difficulty  but most likely reflect the different task demands and perhaps different strategies and processes applied during the maintenance interval. Since verbal information had to be held in WM, it might have produced stronger interference and greater conflict in resource allocation during the within-domain/lexical decision task than the cross-domain/face decision task, eventually affording less efficient engagement of task-specific networks in the former condition and hence lower accuracy. The involvement of the right frontal pole (Brodmann area 10), thought to organize an optimal use of cognitive resources and overcome potential impasses , may refCritically, and in keeping with our hypotheses, the two concurrent tasks (within- and cross-domain) elicited distinct patterns of brain activity during the subsequent retrieval phase, despite the fact that identical stimuli were encoded, maintained and retrieved from WM. This indicates that partly different processes mediated retrieval after within- and cross-domain interference, and thus WM retrieval differed according to the nature of the preceding concurrent task. Large bilateral occipital activations were engaged during retrieval after the within-domain/lexical concurrent task, whereas only limited activity was observed in medial occipital cortex in addition to left superior and middle temporal cortex during retrieval after the cross-domain/face concurrent task. Interestingly, the latter cluster in temporal cortex overlapped with regions often reported in phonological tasks and associated with language networks , 49. A pThe current study is not without limitations. The study sample size could be considered relatively small. We note, however, that it is comparable with previous studies exploring neural correlates of WM , 67, 78.Our study provides new insights into WM-related brain activity. We show a greater role of perceptual brain systems for encoding processes, and a fronto-parietal attentional network for retrieval processes. More critically, we show that a concurrent task during maintenance in WM produced distinct activations not only during the concurrent task itself, but also during subsequent retrieval. We conclude that the specific demands of the concurrent task affect the way memory items are maintained in WM, selective verbal interference resulting in greater reliance on visual cortex for retrieval, whereas visual interference leaves verbal systems of maintenance unaffected, hence resulting in the involvement of language-related areas in left temporal cortex for retrieval. These data accord with WM models postulating differentiated cognitive processes, with distinct neural substrates, according to the concurrent material interfering in verbal WM , 76, 77.