The primary aim of this study was to examine whether markers of cell damage and of the psycho-neuroendocrino-inflammatory/immune (PNI) system could be associated in patients with stable coronary artery disease (CAD) on the next day following percutaneous coronary intervention (PCI).Blood samples of 23 patients , were collected immediately before (pre-PCI), immediately after (post-PCI), and on the day following PCI (1d-PCI). Lactoferrin, LL-37 and interleukin-6 (IL-6) were assayed in plasma, in addition to cortisol and chromogranin A (CgA), as well as CK, ASAT and ALAT. Total and differential leukocyte counts were also analysed., (p<0.05), and cortisol (p<0.01). On the first day after PCI, highly significant associations were found of ASAT with IL-6 and neutrophil count , and of CgA with neutrophil count and monocyte count ; furthermore, cortisol was also associated with neutrophil count .At all the three time points, the monocyte fractions, the monocyte-to-lymphocyte and the neutrophil-to-lymphocyte ratios and CgA levels were elevated. We detected significant peri-procedural changes in the plasma levels of our PNI markers: IL-6 (p<0.05), lactoferrin, LL-37 (both: p <0.0001), CgAThe findings suggest that myocardial damage could correlate not only with an inflammatory reaction but, via neutrophil count, also with increased level of stress in stable CAD after PCI. Furthermore, 1d-PCI neutrophil count may serve as an easy-to-obtain integrative PNI measure in stable CAD. Mainly due to advanced interventional cardiology and improved prevention, the mortality rate of coronary artery disease (CAD) decreased considerably in the last decade. Still, myocardial infarction probably remains a major obstacle to human longevity also in the coming decade . AccordiNeutrophils and monocytes/macrophages have fundamental roles not only in the process of coronary atherosclerosis but in the post-PCI inflammatory events and in reperfusion wound healing responses as well –4, 6–7. Stenting was reported to trigger rapid neutrophil activation with the release of oxygen radicals and granule contents (e.g. lactoferrin) in stable and unstable angina –16. A spCreatine kinase level and the percentage of MB isoenzyme (CK-MB) are widely accepted markers for the diagnosis of peri-procedural necrosis . Post-PCIncreasing evidence has indicated that psychosocial stress and depressive symptoms are major risk factors for the development and prognosis of CAD –25. In aDespite the evidence that psychological stress and inflammation could be linked to CAD and PCI, and that peri-procedural myocardial injury is partially due to secondary inflammation, the associations of inflammatory factors and cell damage markers with neuroendocrine stress markers following elective PCI remain largely unexplored. Therefore, our primary goal was to investigate possible correlations involving cell damage markers and some markers of the psycho-neuroendocrino-inflammatory (PNI) system on the next day after stenting in stable angina. Additionally, we followed the peri-procedural changes of the tested PNI markers.18 men and five women  with stable coronary heart disease participated in our study. Patients were eligible if they had characteristic symptoms of stable angina with positive stress test; the main inclusion criterion was significant stenosis of at least two major coronary arteries shown by coronary angiography (significant diameter stenosis >50%) to avoid unifocal coronary atherosclerosis. Exclusion criteria were: bypass surgery, cardiogenic shock, acute inflammations or any immunological disorders, malignancy, immunosuppressive/anti-inflammatory medication (except low-dose aspirin), recent major trauma/surgery, impaired renal and liver function, and drug/alcohol abuse. Our patients did not have myopathies, muscle damage, neuromuscular diseases, neurological disorders, nor electrolyte  problems.2), eight patients (35%) had diabetes mellitus (type 2), and two (9%) were current smokers.We used the New York Heart Association functional classification to estimate the severity of heart failure . There were three (13%), four (17%), and five (22%) patients in NYHA groups I, II, and III, respectively, whereas the rest of the patients did not have heart failure. The patients were on average overweight  and 20 (87%) patients, respectively. In addition, 16 (70%) patients were administered proton pump inhibitors and nine (39%) had sedatives; none of them took antidepressants. Clopidogrel was given before and after PCI; all patients were administered heparin (70–100 IU/kg) during PCI.Our patients granted informed written consent before their entry into the study. The study protocol was approved previously by the Medical Ethics Committee at the Medical Faculty of University of Szeged . The investigation conformed to the principles of the Declaration of Helsinki.Blood samples were collected immediately before PCI (pre-PCI), immediately after PCI (post-PCI), and on the next day following PCI between 7 to 9 h a.m. (1d-PCI).  Blood cell counts were quantified by standard protocols using an automatic cell counter. In order to assess peri-procedural myocardial cell damage, CK, ASAT and ALAT were assayed in 1d-PCI samples. ALAT and ASAT enzyme activities were measured using commercially available kits  on Roche Modular P800 analyzer. CK and CK-MB activities were determined by kits from Human GmbH  on Roche Modular P800 analyzer. The upper limit of reference values for the tested enzymes were 170 U/l  and 195 U/l  in case of CK, /CK-MB ratio: 6%/, whereas 31 U/l (both genders) for ASAT and ALAT.  Since no reference interval was available for the MLR, we used the ratio of the median values of the monocyte and lymphocyte reference ranges (counts) as reference: 0.19. Similarly, we defined the NLR as the median values of the neutrophil and lymphocyte reference ranges (counts) as reference: 1.86.For the assessment of plasma levels of chromogranin A (CgA), cortisol, lactoferrin, LL-37 and IL-6, blood samples were collected into cooled (4°C) EDTA-Vacutainer tubes, and plasma aliquots were stored at -80°C until analyses. CgA concentrations were determined by a radioimmunoassay kit , reference range: 23–153 ng/ml. Cortisol was assayed by radioimmunoassay , reference range: 160–620 nmol/l.;, reference maximum: 8.7 pg/ml.Lactoferrin concentration was measured by our „in-house” ELISA kit, developed according to Antonsen . In brieFirst, we calculated the necessary sample size to obtain clinically meaningful correlation coefficients of r ≥0.6 between variables of interest with a power of 80% and with 5% level of significance. Although the power analysis resulted in a sample size of n = 19, we enrolled 24 patients to compensate for potential dropouts. Since one patient was transferred to a county hospital before PCI, 23 participants were involved in our study. According to Cohen, the effect size of correlation coefficients (r) with value of 0.5 is large; thus, regardless of p-values, it is unlikely, that correlation coefficients ≥0.6 reflect false associations, even in case of multiple tests . To examTo investigate associations among stress-related, inflammatory—and cell damage parameters, we applied partial correlational analysis with power analysis using R package ppcor; adjustments for sex, age, BMI and diabetes were carried out. As CgA levels are affected by proton pump inhibitor intake and the NYHA stadium of the patient, these factors were also used as controlling parameters for the partial correlational analyses of this variable –35. As b, but significant changes were detected in the total leukocyte count . Normal values were observed for neutrophil counts and fractions, and for lymphocyte counts at all time points.From the data of We were interested in monitoring the changes in the plasma concentrations of the PNI markers following PCI. The concentrations of all the tested markers were altered directly after stenting with the exception of IL-6 Figs . We obseWe were primarily interested in examining if, and to what extent the inflammatory process triggered by stenting could be associated with myocardial damage or with neuroendocrine stress response on the next day following intervention. The characteristic cell damage markers were assayed only on the next day after stenting; all values were in the normal ranges: ASAT: 29.80 ± 27.33 U/l (n = 20), ALAT: 27.38 ± 13.01 U/l (n = 21), and CK: 145.62 ± 172.64 U/l (n = 21). Only one patient was diagnosed with acute myocardial infarction .Concerning the correlations of the cell damage markers with the inflammatory markers, the associations of ASAT with neutrophil count (ng) and with IL-6 proved to be the most significant ones with high effect sizes . , a major inflammatory marker (IL-6) and neutrophil count, and an another one, which involves a marker of neuroendocrine-sympathetic activity (CgA), monocyte count and neutrophil count; these two networks are apparently linked by neutrophil count.We observed elevated values of monocyte fractions, monocyte-to-lymphocyte (MLR) and neutrophil-to-lymphocyte ratios (NLR) both in the pre-PCI and post-PCI, and also in the 1d-PCI samples that probably indicates an inflammatory state. The increased level of initial MLR could reflect a severe atherosclerotic state, since the reciprocals of the pre- (and post-) PCI MLR values (1/0.33 = 3.03) are close to the critical lymphocyte-to-monocyte ratio of 3.1 that has been found in patients with high risk of severe atherosclerotic limb ischemia, which is frequently associated with CAD . The sliAll the tested inflammatory markers changed significantly during/after PCI, supporting the development of an inflammatory reaction initiated by the intervention. As lactoferrin is a specific and reliable activation marker of neutrophils, the significant increase of its post-PCI plasma level over the maximal reference value reflects substantial neutrophil activation triggered by the intervention . Given tLL-37, the only human cathelicidin, is regarded as a novel inflammatory marker . It has IL-6 is a multifunctional cytokine with a central role in inflammation, it is proposed as a major factor linking stress/depression and CAD , 30, 45.The increased plasma cortisol in the post-PCI samples presumably indicates a transient stress response initiated by the intervention that was downregulated by next day.  EnhancOn the first day after stenting, we found highly significant and strong correlations of ASAT with neutrophil count or IL-6, that could indicate an association of myocardial cell damage with inflammatory reaction. Lately, it became increasingly evident that stenting could frequently lead to ischemic myocardial cell damage, that is primarily caused by side branch occlusion or distal embolization by the disrupted plaques; this can bring about a secondary inflammatory response–6. On thThe strong correlation of CK with ASAT supports the usefulness of the latter one as a cell damage marker after stenting in stable CAD. Although CK also showed correlations with neutrophil count and IL-6, surprisingly, these were less strong and significant associations than in the case of ASAT.-/- mice, elevated sympathetic activity could directly lead to monocytosis via release of hematopoietic cells from the bone marrow (mediated by norepinephrine and beta3-adrenergic receptors) that is followed by increased monocytopoiesis in the spleen and by monocyte delivery to the blood stream [On the basis of the surprisingly strong correlation of CgA with neutrophil count or monocyte count, it is reasonable to suppose an association of the activity of the neuroendocrine-sympathoadrenal activity with the appearance of neutrophil granulocytes and monocytes in the circulation. According to investigations in Apoed stream , 49. Thed stream . We showd stream . We found stream .Since neutrophil count is shared between the two major association networks, hence, this raises the possibility that increased neuroendocrine-sympathetic activity (marked by CgA) could be involved in the peri-procedural myocardial damage by stimulating neutrophil release in patients with stable CAD. Our results indicate that increased cortisol might also be involved in this stress response. Leukocyte mobilization in stress is part of an ancient adaptation mechanism by which the body gets prepared to a possible attack or injury via releasing cells of the innate immune system to the blood, from where they can easily gain access to the site of acute challenge. Although neutrophils are indispensable in the elimination of foreign cells and cell debris, their uncontrolled, excessive activity could be noxious for the healthy cells nearby. A systematic review highlighted the importance of neutrophils in adverse clinical outcomes in acute coronary syndromes and/or after PCI or coronary artery bypass surgery .While sympathetic activation may be a key factor in the release of neutrophils and in enhanced IL-6 secretion, (probably through beta-adrenergic receptor mechanism), increased vagal tone and heightened parasympathetic activity (e.g. by relaxation or vagus stimulation) could result in anti-inflammatory effects in CAD . This isThere are some limitations to be mentioned. First, the exact mechanisms underlying the correlations could not be clarified in this clinical study. Second, no further adjustments could be carried out in the multivariate analysis in addition to sex, age, BMI and diabetes (plus proton pump inhibitor intake and the NYHA stadium in case of CgA), in order to avoid model overfitting. Third, the sample size was small; however, the emerged, highly significant correlations had large effect sizes and thus support the validity of our results. Fourth, only one patient appeared to suffer peri-procedural myocardial infarction, thus it remains unclear, whether our findings could also be valid in a sample of patients with severe peri-procedural myocardial damage remains unclear. Fifth, we included patients with multivessel disease and we assumed that coronary artery stenosis of at least 50% of the vessel diameter in at least two coronary arteries reflects extensive atherosclerosis of the coronary system, although we did not use the sophisticated Syntax or Gensini scores.In conclusion, our results suggest that activity of the stress-related sympathetic-neuroendocrine systems could correlate with neutrophil count that is associated with plasma levels of a major inflammatory cytokine (IL-6) and a cell damage marker (ASAT) in patients with stable CAD on the next day following PCI. Therefore, although not tested, our findings implicate the potential advantage of parasympathetic neuroimmunomodulation in the reduction of myocardial injury during/after stenting. Furthermore, neutrophil count may serve as an inexpensive integrative measure of the activated PNI network and the procedural cell damage after PCI in stable CAD. Further investigations are required to elucidate the details of PCI-related myocardial damage from PNI aspects and to elaborate PCI techniques with the least injuring consequences.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Community health workers can adequately provide DMPA-SC directly or train women on self-injection. Community health workers can adequately provide DMPA-SC directly or train women on self-injection. To assess the supply- and demand-side factors influencing continued use of the injectable contraceptive subcutaneous depot medroxyprogesterone acetate (DMPA-SC).We conducted a 12-month randomized controlled trial in Malawi to measure DMPA-SC continuation rates. A total of 731 women presenting to clinic-based providers (CBPs) at 6 Ministry of Health clinics or to community health workers (CHWs) in rural communities were randomized to receive DMPA-SC administered by a provider or be trained to self-inject DMPA-SC. Data collectors contacted women after the reinjection window at 3, 6, and 9 months to collect data on discontinuation and women's experiences. Twelve months after enrollment or at early discontinuation, women had their final interview, including pregnancy testing. We compared continuation, pregnancy, and safety by whether DMPA-SC or self-injection training was provided by CHWs versus CBPs. We also conducted an exploratory analysis assessing the association between women's sociodemographic factors and the risk for discontinuation using stratified Cox proportional hazards models.P<.001). The risk for discontinuation was also different among health facility catchment sites (P<.001). No other assessed sociodemographic factors were found to significantly influence the risk for discontinuation.The type of provider did not seem to influence continuation, pregnancy, or safety. As reported previously, women in the self-injection group were significantly less likely to discontinue the method compared with women in the provider-administered group (hazard ratio, 0.43; Public-sector CHWs can safely and effectively provide DMPA-SC and train women to self-inject DMPA-SC in low-resource settings. DMPA-SC continuation did not seem to be influenced by the type of provider, whether CBP or CHW, or women's sociodemographic characteristics. DMPA-SC is steadily gaining popularity among family planning users and providers in sub-Saharan Africa as an easy-to-use and accessible contraceptive option. Studies in Senegal and Uganda found that family planning providers preferred the subcutaneous version over the intramuscular formation—providers indicated that DMPA-SC was easier and faster to administer, would decrease stock-outs , and would be less painful and therefore preferable for women.A growing body of evidence shows that self-injection of DMPA-SC is feasible and acceptable and that CHWs can safely provide it.–,–,,,The recent trial in Malawi demonstrated that public-sector family planning providers, including CHWs, can safely provide DMPA-SC and train women to self-inject. However, little is known about whether and how outcomes—including continuation, adverse events, side effects, and pregnancy—vary by supply-side factors such as the type of family planning provider (CHW or CBP) who provides DMPA-SC or self-injection training to women. CBPs in the trial were government nurses and midwives with more health care training than the CHWs, which may influence the quality of services provided. Furthermore, the knowledge base is nascent regarding demand-side factors such as the sociodemographic characteristics of women that may influence 12-month DMPA-SC continuation, especially for self-administered DMPA-SC. The numerous studies that have been conducted to assess determinants of continuation of DMPA-IM indicate that women who receive complete and accurate information on possible side effects are more likely to continue using DMPA,It is unclear how factors influencing continuation of IM and SC formulations will differ, especially when DMPA-SC is self-administered. Two nonrandomized cohort studies in Burkina Faso and Uganda found no difference in continuation rates between DMAP-SC and DMPA-IM when both were administered by CBPs, but findings showed that increased age and partners' acceptance of family planning increased DMPA continuation in Burkina Faso .Given the limited research on factors affecting continued use of DMPA-SC, especially for self-injected DMPA-SC, the aim of this article is to assess the influence of selected supply- and demand-side factors on continued use of DMPA-SC among Malawian women enrolled in a year-long randomized controlled trial. These data can be used to inform task-sharing decisions and optimize service delivery in Malawi and other low-resource settings.–We used data collected as part of a randomized controlled trial we conducted to compare continuation rates between women who self-inject DMPA-SC and women who receive the same product from a provider. The trial was conducted from September 2015 to February 2017 in 6 Ministry of Health clinics and surrounding communities in rural Mangochi District, Malawi. During the trial, CBPs and CHWs randomized 731 women seeking family planning services to either receive DMPA-SC administered by the provider or be trained to self-inject DMPA-SC. Eligible participants were ages 18 to 40 years, in self-reported good general health, able to understand and willing to sign an informed consent document, willing to give contact information for follow-up, willing to have follow-up visits or interviews, willing to be randomized to the self-injection arm or provider-administered injection group, not pregnant according to WHO guidelines, and able to meet eligibility criteria for receiving DMPA per WHO medical eligibility criteria.Our primary outcome was DMPA-SC discontinuation. Women were considered discontinuers if they did not report receiving an injection within the allowable window of time . Given that reinjection provides 3 months of protection, participants without a DMPA-SC injection within the window or who were lost to follow-up were considered to have discontinued 3 months after the previous injection. Those who had not discontinued by 12 months were censored at 12 months, when the study ended.We collected data on adverse events, side effects, and pregnancies occurring throughout the 12-month follow-up. In this article, we report outcomes comparing women assisted by CBPs or CHWs. The analysis of safety data included only participants who successfully received or administered a DMPA-SC injection after randomization.We estimated Kaplan-Meier cumulative probabilities of contraceptive continuation coverage  by provider type and compared the distribution of continuation between these groups using a log-rank test stratified by site using a .05 significance level for a 2-sided comparison. We present these results separately by the original randomization group  since the primary results demonstrated a large treatment effect on continuation.We also assessed factors that could potentially influence DMPA-SC discontinuation using Cox proportional hazards models with each of the following covariates: treatment group , site , woman's age, marital status, whether she works outside the home, parity, education, religion, previous experience with contraceptives and injectable contraceptives, and whether a CBP or CHW provided DMPA-SC or self-injection training at enrollment.We assessed each covariate separately and planned to include all covariates found significant at the .05 level in the univariate models in a multivariable model. Except for when we analyzed the effect of site specifically, site was used as a stratification variable in the models as consistent with the randomization scheme. Hazard ratios for discontinuation and 95% CI were provided for each covariate modeled.The study protocol was reviewed and approved by the Protection of Human Subjects Committee at FHI 360, Durham, NC, USA, as well as the College of Medicine Research and Ethics Committee, University of Malawi. All study staff completed training on research ethics, the protocol, and informed consent administration. All trial participants provided their informed consent to participate. The trial was registered with ClinicalTrials.gov (NCT02293694).Participants' sociodemographic characteristics are shown in P=.77). Though the continuation rates were much lower in the provider-administered group , we did not find a significant difference between women who received DMPA-SC from a CBP [0.48  and those who received the method from a CHW [0.44 ] (P=.78). The incidence rate of discontinuation for those who received self-injection training from a CHW was 9 per 100 injection cycles  compared with 6 per 100 injection cycles  among those who were trained by a CBP. For the provider-administered group, the incidence rate for those who received DMPA-SC from a CHW was 21 per 100 injection cycles  compared with 19 per 100 injection cycles  among those who received the method from a CBP.Cumulative probabilities of continuation and 95% CI for each quarter by type of provider at enrollment and treatment group are presented in There was no significant difference in continuation rates between women who received DMPA-SC self-injection training from clinic-based providers versus CHWs.P=.49) or for those in the provider-administered group (P=.26). The most common reason for discontinuing was due to missing the reinjection window  and the provider-administered group (P>.99) were not statistically significant.Data from pregnancy tests were incomplete due to refusals, loss to follow-up, and data collectors neglecting to administer a pregnancy test as planned (pregnancy status was unknown for 12% in the self-administered group and 21% in the provider-administered group). Among 612 women tested, 8 pregnancies were identified; 1 with a conception date prior to enrollment and 7 during follow-up. Of the 7 pregnancies, 3 occurred in the self-injection group (1 among CBP clients and 2 among CHW clients) and 4 in the provider-administered group (1 among CBP clients and 3 among CHW clients). Differences observed by type of provider within the self-injection group (P=.21), 6 months—15% vs. 18% (P=.74), and 9 months—12% vs. 14% (P=.70). Similarly, there were no statistically significant differences between those who received DMPA-SC from a CBP compared with a CHW: 3 months—34% vs. 31% (P=.61), 6 months—23% vs. 22% (P>.99), and 9 months—15% vs. 19% (P=.69). Among women who reported side effects, the majority across all groups reported little to no effect on daily life.The percentage of continuing women who experienced side effects decreased over time across all groups . The difThe percentage of women experiencing side effects were not significantly different by provider type.P=.73 for the differences in proportion of women experiencing adverse events). Twenty-eight related or possibly related adverse events were reported by 17 women in the provider-administered group (data not shown). Nine of these events were reported by 7 women who received DMPA-SC from CBPs and 19 of these events were reported by 10 women who received DMPA-SC from CHWs; these differences were not statistically significant (P=.28 for the differences in proportion of women experiencing adverse events). Furthermore, there were no significant differences between the groups in the types of adverse events reported. There were 5 serious adverse events reported during the trial by 4 different women. Two events related to DMPA-SC  were reported by the same woman in the provider-administered group who was enrolled by a CHW and resolved without sequalae. The other serious adverse events, including 1 death (suspected liver cirrhosis), were unrelated to DMPA-SC.Twenty related or possibly related adverse events were reported by 10 women in the self-administration group (data not shown). Nine of these events were reported by 3 women who received self-injection training by CBPs and 11 of these events were reported by 7 women who received training by CHWs. These differences by type of provider were not statistically significant (P<.001). Risk for discontinuation was also different among clinics (P<.001).The results of the Cox model are presented in Self-injectors were significantly less likely to discontinue DMPA-SC than those who received it from providers.Contraceptive continuation is important for reducing unintended pregnancies. This is one of the first studies to explore factors that affect continued use of DMPA-SC through 12 months, including self-administered DMPA-SC. A retrospective study of 2015–2016 Demographic and Health Survey data in Malawi found a 12-month discontinuation rate of 41% for injectable contraceptive users.We did not find evidence that the type of provider influenced the risk of discontinuing, pregnancy, or safety, which suggests that CHWs—not only CBPs—can provide DMPA-SC or training on self-injection in low-resource settings without hampering continuation. Wait times at health facilities are often long and health facilities are often overcrowded and understaffed. Permitting CHWs to train women on DMPA-SC self-injection in community settings would enable women to circumvent the long lines and alleviate some of the work load at these health facilities. CHWs are based in rural and low-income areas where there is often high unmet family planning need, and they are more likely to remain in their communities once trained.Permitting CHWs to train women on DMPA-SC self-injection could increase access to contraception and alleviate the work load of other providers.,,,Of the factors explored, treatment group and health facility catchment site were the only factors that significantly influenced the risk of discontinuation, with self-injection leading to a reduced risk of discontinuation compared with provider administration. Consistent with previous studies of DMPA-IM,Education levels did not affect women's ability to self-inject.,Our findings are consistent with findings from nonrandomized prospective cohort studies in Senegal and Uganda, which observed that clients who self-injected DMPA-SC had a lower risk of discontinuing relative to clients who received DMPA-IM from CBPs.Our results are also similar to a study of provider-administered DMPA-SC in urban Nigeria that found no differences in continuation at 3 months according to the place women received DMPA-SC.One limitation of our study is that women's reported outcomes may have been influenced by social desirability bias. Another challenge we faced was missing data for the pregnancy outcome. Given this, our pregnancy data should not be used for estimating the DMPA-SC failure rate. The study was also not designed to assess whether women with different characteristics or being assisted by various types of providers had different risks of discontinuation; therefore, the sample size for some of the comparisons may be too small to be conclusive. Furthermore, these are non-randomized comparisons and may be affected by selection biases. Lastly, there are numerous other variables and combinations of variables that we did not explore but which may influence continuation.Although we did not find any of the sociodemographic factors associated with DMPA-SC discontinuation to help us target future efforts, the differences observed across sites may indicate the presence of other underlying factors that would be interesting to explore in future studies. For example, providers' management of clients who would like to continue using injectables but arrived late for their scheduled reinjections  has been documented to vary and to directly affect clients' continued use of contraception.


TP53 deletion/mutation or unmutated IGHV genes, though the clinical responses are rarely complete. More recently, chemo-free combinations of venetoclax+rituximab, venetoclax+obinutuzumab or ibrutinib+venetoclax have been shown capable of inducing undetectable MRD in the bone marrow, opening the way to protocols exploring a MRD-based duration of treatment, aiming at disease eradication. Thus, beside a durable disease control desirable particularly for older patients and/or for those with comorbidities, a MRD-negative complete remission is becoming a realistic prospect for CLL patients in an attempt to obtain a long-lasting eradication and possibly cure of the disease. Here we discuss the standardized and innovative technical approaches for MRD detection in CLL, the clinical impact of MRD monitoring in chemoimmunotherapy and chemo-free trials and the future clinical implications of MRD monitoring in CLL patients outside of clinical trials.In chronic lymphocytic leukemia (CLL), there is a growing interest for minimal residual disease (MRD) monitoring, due to the availability of drug combinations capable of unprecedented complete clinical responses. The standardized and most commonly applied methods to assess MRD in CLL are based on flow cytometry (FCM) and, to a lesser extent, real-time quantitative PCR (RQ-PCR) with allele-specific oligonucleotide (ASO) primers of immunoglobulin heavy chain genes (IgH). Promising results are being obtained using droplet digital PCR (ddPCR) and next generation sequencing (NGS)-based approaches, with some advantages and a potential higher sensitivity compared to the standardized methodologies. Plasma cell-free DNA can also be explored as a more precise measure of residual disease from all different compartments, including the lymph nodes. From a clinical point of view, CLL MRD quantification has proven an independent prognostic marker of progression-free survival (PFS) and overall survival (OS) after chemoimmunotherapy as well as after allogeneic transplantation. In the era of mechanism-driven drugs, the paradigms of CLL treatment are being revolutionized, challenging the use of chemoimmunotherapy even in first-line. The continuous administration of ibrutinib single agent has led to prolonged PFS and OS in relapsed/refractory and treatment naïve CLL, including those with  IGHV) genes, CD49d and CD38 expression, along with the identification of chromosomal abnormalities  and of recurrent mutations in TP53, ATM, NOTCH1, BIRC3, and SF3B1 genes, have all been recognized as features capable of predicting the outcome of CLL patients (Chronic lymphocytic leukemia (CLL), characterized by the clonal expansion of mature B lymphocytes in the peripheral blood (PB), bone marrow (BM), spleen and lymph nodes , is diagTP53 deletion/mutation and IGHV mutations) and status , but international guidelines are rapidly changing the current paradigms thanks to the efficacy of novel drugs/drug combinations (see below).Patients are treated only when the disease becomes symptomatic and/or progresses to more advanced clinical stages. Effective treatments include combinations of chemotherapy ; bendamustine (B); chlorambucil (Chl) with anti-CD20 monoclonal antibodies (rituximab (R), obinutuzumab  or novel agents such as the B-cell receptor (BCR) inhibitors  or BCL2 inhibitors (venetoclax), introduced in the last 5 years. Treatment choice is currently based on patients' age and comorbidities, disease biology (Unlike in adult and childhood acute lymphoblastic leukemia (ALL), where the assessment of response to therapy by minimal residual disease (MRD) monitoring drives therapeutic choices, MRD analysis in CLL has been only recently introduced.Whilst morphologic response criteria are not sufficiently sensitive to predict outcome after treatment, several studies have demonstrated that patients who achieved a clinical complete remission (CR) as defined in the International Workshop on CLL (iwCLL) response criteria  but with−4) being considered MRD responders. However, the predicted benefit on PFS needs to be confirmed with longer follow-up  (EMA guidelines), with patients who achieve clinical CR and uMRD  and real-time quantitative polymerase chain reaction (RQ-PCR) –28. MoreIn this review, we discuss the standardized and innovative technical approaches for MRD detection in CLL, the clinical impact of MRD monitoring in chemoimmunotherapy and chemo-free trials, and the future clinical implications of MRD monitoring in CLL patients outside of clinical trials.−4).The general principles to be fulfilled for a MRD detection assay are the capability of identifying malignant cells on the basis of a univocal profile that is not shared by their normal counterparts and the reliability and reproducibility of the method, that should be easy to perform and simple to interpret. The two major approaches satisfying these criteria are FCM and allele-specific oligonucleotide (ASO) of immunoglobulin heavy chain (IgH) RQ-PCR. The availability of standardized guidelines both for FCM –26 and An = 117), significantly higher values of MRD were found in the BM compartment compared to PB. In the few discordant paired samples (n = 17), 94% were PB uMRD/BM MRD+  restriction has been the simplest and most commonly used FCM approach for MRD evaluation, virtually applicable to all CLL cases but with a low sensitivity. The introduction over the years of an increasing number of antibodies in the work panel (see below) has allowed a more accurate definition of residual CLL cells. However, in order to save labor work, time, reagents and contain costs, the use of a simple upfront clonality assessment, by CD19/CD5/κ/λ screening, can identify samples with high levels of residual disease, in which a full multicolor MRD analysis is unnecessary .−4 .Beside the choice of the antibody panel, another crucial aspect of FCM monitoring is the number of acquired events to reach the sensitivity of 1 leukemic cell in 10,000 normal cells , intermediate if <1% and ≥.01% or <10−2 and ≥ 10−4  and positive if ≥1% or ≥10−2 .Generally, for a correct MRD estimation, at least 500,000 events need to be acquired for each tube with the identification of at least 20 clustered events displaying a CLL immunophenotype. The presence of MRD is reported as the percentage of pathologic cells within the total leukocyte population. Conventionally, MRD is defined as undetectable if <0.01% or <10The first consensus document by the European Research Initiative on CLL (ERIC) group has identified a standardized method for MRD identification and reporting, based on specific monoclonal antibody combinations and on codified fixed gating strategies, aimed at improving the sensitivity and reducing the inter-operator variability and false-positive rate . This as−4). Moreover, this standardized 4-color FCM assay has proven highly reproducible, being validated in most multi-center randomized clinical trials . We. We−4). −4 (−5) with a single-tube assay. Our group also demonstrated the applicability of a single 8-color combination tube (CD81/CD38/CD20/CD43/CD5/CD45/CD19/CD3) obtaining comparable MRD values with those resulting from the ERIC 4-color panel, bypassing the issue of sample cellularity that represents the main sensitivity limitation of FCM MRD analysis , diversity (D) and joining (J) gene segments rearranged during early lymphoid differentiation. The unique d region . A monocMRD evaluation by RQ-PCR is based on the identification at diagnosis of a patient-specific molecular target on which sequence primers and probes are designed and then used to monitor the disease during clinical follow-up .IGHJ gene, with a fluorescent probe located between the two primers. The other approach combines both highly specific ASO primers together with probes; also the latter, in challenging cases, might be specific for the patient's rearrangement. It has been reported that the use of patient-specific primers and probes represents a more powerful strategy for RQ-PCR analysis in B-cell lymphoproliferative disorders, being independent of the mutational load of the IGHV and IGHJ regions, with evaluable results in more than 90% of cases (−5) (Different strategies for the design of primers and probes have been investigated –54. Brieof cases . It is ises (−5) .We have demonstrated in 185 FCM uMRD samples collected after chemoimmunotherapy that there is a sizable proportion of samples (22.7%) proving positive by ASO IgH RQ-PCR. Patients with FCM MRD-/RQ-PCR MRD+ showed a poor PFS similar to that of FCM MRD+ cases .Challenges related to RQ-PCR-based MRD evaluations are represented by the need of diagnostic material for all MRD evaluations of each patient and the occurrence of the hypermutation process that requires a specific primer/probe design (see above).Overall, RQ-PCR results in MRD monitoring of CLL are interpreted according to the guidelines developed for ALL by the European Study Group for MRD detection in ALL (ESG-MRD ALL) , today kHowever, it has to be underlined that at the single patient's level the kinetics of MRD is more relevant than a single MRD assessment, since the increase of MRD over time and not only its persistence or “fluctuation” is eventually followed by clinical relapse.Despite the high sensitivity and specificity of RQ-PCR approach, as well as its established role for MRD evaluation in CLL, this method has important limitations especially due to the requirement of standard curves generated from dilutions of the tumor-specific target identified at diagnosis, and to the dynamic of intrinsic technical variations not fully eliminable. Moreover, a certain proportion of samples could fall in a window of inadequate quantification i.e., PNQ samples. To overcome these limitations, the ddPCR technique has recently been adopted for MRD measurement in different hematologic malignancies –33. This−4->10−6). This was highly predictive of 12-month disease-free survival compared to cases with a negative IGH-HTS MRD (<10−6)   . OverallMoreover, NGS-based MRD assessment may not only overcome some disadvantages of PCR-based methods but also enable the analysis of the genetic diversity and clonogenic heterogeneity which may contribute to a better understanding of the biology of the disease. Thus, the definition of “monoclonality” of CLL could be challenged by the availability of genome-wide analyses . Since tThe availability of HTS approaches has also expanded the sources of DNA for disease genotyping and monitoring. Plasma circulating tumor DNA (ctDNA) is readily detectable in patients with CLL, providing a unique opportunity for a comprehensive genotyping of the disease from all the involved compartments, including the lymph nodes, and for a non-invasive serial analysis of clonal evolution and possibly MRD investigation –67. In tOther sources of nucleic acids, such as cell free-RNA/microRNA or those derived from exosomes could be explored in the future as biomarkers of residual disease in CLL, as done in other tumors , 70.TP53 mutations under chemoimmunotherapy , ≥10−4 up to <10−2 (intermediate MRD), and ≥10−2 (high MRD). In the German trial, after FCR, PB MRD was undetectable in 63%, intermediate in 24% and high in 13% of patients, corresponding to a PFS of 68.7, 40.5 and 15.4 months, respectively, with a significant difference for all comparisons; PB uMRD and intermediate MRD showed a similar OS, significantly better than high MRD  CLL8 trial, the first-line phase III study randomizing physically fit CLL patients to receive FC or FCR , in whichigh MRD . FCR indhigh MRD  and in 4high MRD .p < 0.001) and a shorter PFS . A yearly blood MRD monitoring performed in 85 patients with uMRD status at EOT, documented the conversion to MRD positive in 38, after a median of 48 mo after EOT. The “MRD relapse” anticipated the clinical progression by a median of 24 months, a potential window for early intervention strategies  of low-risk patients alive and progression-free for 6 or more years from FCR , FCM PB MRD was negative in all. In a long-term follow-up analyses of the above mentioned studies  was negative in all 15 long-term responders with mutated IGHV. Interestingly, MRD at the end of FCR has not the same significance in patients with unmutated vs. mutated IGHV, since unmutated IGHV CLL who achieved uMRD at the EOT showed a significantly shorter PFS than patients with uMRD and mutated IGHV   .These observations proved the long-term persistence of a deep response among low-risk CLL patients after FCR; although representing a minority, this subgroup might still benefit from a conventional chemoimmunotherapy approach even in the era of novel compounds.−4 i.e., <1 CLL cell per 10,000 leukocytes), 132 (27.8%) intermediate-MRD  and 252 (53.2%) positive-MRD . Patients with PB uMRD had a median PFS of 56.4 months vs. 23.9 months for intermediate-MRD patients vs. 13.9 months for positive-MRD patients . Median OS was not reached (NR) in the PB undetectable and intermediate MRD categories vs. 60.0 months for PB positive-MRD patients . Thus, MRD retains its prognostic significance even in elderly and less fit CLL patients treated with less intensive regimens. uMRD at EOT was significantly more common in patients receiving G-Chl vs. those receiving R-Chl . At the multivariate analysis, PB MRD (positive vs. undetectable) at EOT independently predicted PFS—along with treatment arm (G-Chl vs. R-Chl), serum thymidine kinase and immunogenetic risk factors—and OS—along with Binet stage C, total CIRS score, serum thymidine kinase and immunogenetic risk factors  on R-Chl or Chl single agent in 781 previously untreated and unfit CLL patients . MRD was factors . UnfortuThe FCR combination, the standard front-line therapy for fit CLL patients without del(17p), was compared in the randomized phase III study (CLL10) to the potentially less toxic combination consisting of BR , in prevIGHV achieved uMRD, but also a high proportion of those with lymphocyte count ≥50 × 109/l, disease bulk ≥5 cm, Binet stage B+C or unmutated IGHV did. Lower uMRD rates were seen in patients harboring 17p or 11q deletions.In the GREEN trial, a non-randomized, open-label phase IIIb study investigating G alone or plus chemotherapy (FC or Chl or B) in TN or R/R CLL patients, a subgroup analysis of 158 TN CLL patients receiving B+G was reported. ORR was 81.0%, with uMRD in 59.5% and 27.8% of patients for PB and BM, respectively . InteresThese clear evidences on the association between MRD status and PFS prompted the application of a meta-regression model to the three German phase III trials , in order to determine whether the chemoimmunotherapy effect on MRD response in PB samples at the EOT can predict the effect of treatment on PFS . This moTP53 deletion/mutation, and confers an advantage in PSF and OS over ofatumumab in R/R CLL patients and over Chl in elderly TN   in the iLLUMINATE trial  plus an anti-CD20 monoclonal antibody to chemoimmunotherapy in the first-line treatment of CLL patients were recently presented. Ibrutinib+rituximab vs. ibrutinib single agent vs. BR in TN elderly CLL patients (Alliance North American Intergroup Study A041202) ; ibrutin, E1912) ; ibrutin, E1912) . In all TE trial . ContrarTE trial .n = 181) with few high-risk TN CLL with TP53 deletion/mutation (n = 27), where the addition of rituximab to ibrutinib therapy did not improve the 3y-PFS (86.9 vs. 86%) or the 3y-OS (89 vs. 92%) compared to ibrutinib single agent and did not significantly increase the ORR (92% in both arms) or CR rate (26 vs. 20.2%) as best responses, despite the faster achievement of CR and lower level of BM MRD evaluated by 4-color FCM .Another concept is the administration of ibrutinib in combination with agents with different mechanisms of action, in order to achieve uMRD and discontinue the treatment. A limited duration of therapy  could avoid the continuous exposure to the drug, the development of mutations conferring resistance, cumulative toxicities and unaffordable costs. The GCLLSG CLL2-XXX phase II trials , enrolliTP53 deletion/mutation . Again, ibrutinib with anti-CD20 monoclonal antibodies does not seem the best agent to achieve uMRD in most patients in order to plan a MRD-based discontinuation.Although the rate of ORR was comparable among the 3 combinations, ibrutinib in combination with G induced higher PB uMRD than ibrutinib+ofatumumab at the end of induction (EOI) (48 vs. 14%), but the best combination resulted venetoclax+G  . These dp < 0.0001)  .IGHV, in order to maximize the quality of response, to reduce the number of chemotherapy cycles and to stop ibrutinib in the case of uMRD  for the treatment of 45 TN CLL patients with mutated  of uMRD . After t of uMRD .The BCL2 inhibitor venetoclax in the first phase 1 dose-escalation study in R/R CLL patients induced an ORR 79% with 20% CR . MRD staTP53 deletion/mutation and NOTCH1 mutation; refractoriness to BCR inhibitors; bulky lymphnodes) and depth of response  were independently associated with the duration of response to venetoclax , it has been shown that uMRD in the PB and BM was achieved in 27 and 16% of cases, respectively, mostly within 2 years of treatment . Both prnetoclax .−4) in 64% and low-MRD (≥10−4- <10−2) in 18% of patients. Whilst 70% and 98% of patients with uMRD at EOT remained in uMRD with no PD, respectively, those with high-MRD (≥10−2) at EOT were a high risk subgroup. In fact, PD at 9.9 m after completion of venetoclax was 79% in patients with high-MRD at EOT, 13% in those with low-MRD and only 2.4% in uMRD patients. Different quantitative levels within the range of MRD positivity have been shown to be at least as important for PFS as the distinction between MRD negativity (below 10−4) and MRD positivity (≥10−2) in the chemoimmunotherapy setting  . VR was  setting , 84. It  setting , but thi−4) have been re-treated with venetoclax after an asymptomatic progression after 25, 29, and 43 months off, and re-attained a PR, followed in one patient by a PD 18 months later  and type of therapy. Residual nodal disease did not. Thus, VR followed by venetoclax monotherapy induced such deep remissions that became the first novel regimen allowing a fixed-duration treatment. Whilst patients with high-MRD are a subgroup at high risk of progression, patients with BM uMRD can maintain long-term responses after the discontinuation of venetoclax therapy post-VR , 117. ThThese results push in the direction of a MRD-driven duration of therapy with venetoclax, with safe interruption in patients with BM uMRD responses, and of a possible MRD-driven early re-introduction of venetoclax in cases who lose the response.Results on the combination of venetoclax + G have been recently released and are impressive.In the phase Ib trial by Flinn et al. , 46 R/R TP53 deletion/mutation), intermediate MRD in 8% and positive in 2%. Treatment was discontinued in about one third of patients under maintenance due to the PB uMRD achievement, with follow-up not yet available.In the phase II CLL2-BAG trial , 31 R/R TP53 deletion/mutation on the PFS is still present. In the venetoclax + G arm, ORR was 84.7% with 49.5% CR/CRi; PB and BM uMRD, assessed by ASO-PCR 3 months after therapy discontinuation were 75.5% and 56.9%, respectively, and were sustained. Patients with clinical CR and PB uMRD were 42.1% and those with clinical CR and BM uMRD were 33.8%.In the phase III CLL14 trial , impressThe newest combination is represented by ibrutinib+venetoclax. The TAP CLARITY trial based on ibrutinib+venetoclax in 54 R/R CLL patients and with a MRD-driven stop therapy strategy, exceeded the primary endpoint to indicate promising efficacy. The ORR was 94%, CR/CRi 54%. At m +14, PB MRD4 was 57% (28/49) and BM MRD4 39% (19/49)  and 69% at m +18 .A phase I trial explored the combination of venetoclax, ibrutinib and G in 12 R/R patients, with the aim of maximizing the efficacy and try a fixed duration treatment, with promising results in terms of uMRD in both PB and BM .Which is the most effective anti-CD20 monoclonal antibody in combination with venetoclax and whether the addition of an anti-CD20 monoclonal antibody to venetoclax+ibrutinib combination is needed remain two open questions .TP53 deletion/mutation.Results from a number of first-line clinical trials are awaited. The UK NCRI FLAIR CLL10 trial for fit TN CLL with no del(17p) has been further extended to enroll 1516 CLL patients in order to compare FCR vs. ibrutinib+R vs. ibrutinib single agent vs. ibrutinib+venetoclax (duration of therapy defined by MRD). The GCLLSG CLL13 compares chemoimmunotherapy to venetoclax+R vs. venetoclax+G vs. venetoclax+ibrutinib+G in TN fit CLL patients without TP53 deletion/mutation and R/R disease, so much that the indications for SCT in CLL have dramatically changed , MRD and clinical response has contributed to demonstrate the existence and the protective effect of graft versus leukemia (GVL) against CLL (p < 0.0001)  . The ana 0.0001) , 125, thThese concepts will be relevant in the era of CAR-T cell immunotherapy. Indeed, CAR-T cells cleared the BM from CLL cells in most of 24 treated cases, as documented by negative FCM in 17/21 (88%). Twelve of these patients underwent deep IGH sequencing, and 7 (58%) had no malignant IGH sequences detected in the BM. The achievement of molecular responses in CLL patients who responded by iwCLL criteria was associated with a 100% PFS and OS (median 6.6 months follow-up) after CAR-T cell immunotherapy –129.MRD is a powerful prognostic tool to predict the outcome of CLL patients in the context of several clinical trials and is now an accepted surrogate marker to assess treatment efficacy in randomized trials before clinical endpoints can be evaluated. Despite these evidences, no MRD-driven decision making in CLL patients is currently applied in the clinical practice according to the most recent iwCLL guidelines, with the only exception represented by the pre-emptive immunotherapy after SCT.Moreover, although the achievement of uMRD in CLL is the desirable goal to maximize the length of PFS and the way to pursue the eradication of the disease, it should be kept in mind that this is not necessarily true for the entire CLL patients' population. In the era of several novel compounds that have changed the therapeutic scenario both in R/R and TN CLL patients, the achievement of a durable disease control rather than a MRD-negative remission with ibrutinib may be more desirable especially for older patients and/or for those with comorbidities, in order to spare toxicity. In all other patients, i.e., young, fit and/or high-risk patients, venetoclax-based combinations aiming at achieving BM uMRD are the most promising way to significantly impact on the subsequent relapse risk and to provide a long-term disease remission, if not cure.Several are the potential clinical implications of MRD in guiding CLL patients' treatment, such that MRD is likely to move to the clinical practice in the near future for a personalized management of CLL patients. However, the validity of the MRD-driven approaches mentioned below is still an area of uncertainty that needs dedicated studies to be conclusively defined.First, MRD could be useful to decide when to stop treatment. There are some evidences about the use of early uMRD to guide the reduction of the number of administered FCR cycles, an approach that could spare overtreatment/toxicity in FCR treated patients, nowadays limited to those with low-risk genetics. MRD as a tool to limit the duration of chemo-free combinations is under evaluation, with a relevant impact in terms of compliance, clonal selection/resistance, toxicity, and costs.Second, in the near future, the longitudinal monitoring of MRD will also allow to study the kinetics of the disease: the increase/reappearance of MRD by itself could guide the re-challenge of therapy after treatment discontinuation.BTK, PLCg2, BCL2 mutations), could guide the switching to a non cross-resistant agent or combinations of agents, in order to anticipate the clinical relapse.Third, the subsequent screening of emerging mutations conferring resistance to a given drug  of the Fondazione Italiana Linfomi, though no other collaboration, with one of the authors IG.


Purpose: Angiogenesis in liver cancers can be characterized by hepatic functional perfusion imaging (FPI) on the basis of dynamic contrast-enhanced ultrasound (DCEUS). However, accuracy is limited by breathing motion which results in out-of-plane image artifacts. Current hepatic FPI studies do not correct for these artifacts and lack the evaluation of correction accuracy. Thus, a hepatic DCEUS-based dual-phase multi-parametric FPI (DM-FPI) scheme using a derivative principal component analysis (PCA) respiratory gating is proposed to overcome these limitations.Materials and Methods: By considering severe 3D out-of-plane respiratory motions, the proposed scheme's accuracy was verified with in vitro DCEUS experiments in a flow model mimicking a hepatic vein. The feasibility was further demonstrated by considering in vivo DCEUS measurements in normal rabbit livers, and hepatic cavernous hemangioma and hepatocellular carcinoma in patients. After respiratory kinetics was extracted through PCA of DCEUS sequences under free-breathing condition, dual-phase respiratory gating microbubble kinetics was identified by using a derivative PCA zero-crossing dual-phase detection, respectively. Six dual-phase hemodynamic parameters were estimated from the dual-phase microbubble kinetics and DM-FPI was then reconstructed via color-coding to quantify 2.5D angiogenic hemodynamic distribution for live tumors.Results: Compared with no respiratory gating, the mean square error of respiratory gating DM-FPI decreased by 1893.9 ± 965.4 (p < 0.05), and mean noise coefficients decreased by 17.5 ± 7.1 (p < 0.05), whereas correlation coefficients improved by 0.4 ± 0.2 (p < 0.01). DM-FPI observably removed severe respiratory motion artifacts on PFI and markedly enhanced the accuracy and robustness both in vitro and in vivo.Conclusions: DM-FPI precisely characterized and distinguished the heterogeneous angiogenic hemodynamics about perfusion volume, blood flow and flow rate within two anatomical sections in the normal liver, and in benign and malignant hepatic tumors. DCEUS-based DM-FPI scheme might be a useful tool to help clinicians diagnose and provide suitable therapies for liver tumors. Liver cancer is respectively the second and sixth major causes of death in developing and developed countries In comparison with several perfusion imaging techniques based on computerized tomography and nuclear magnetic spin imaging, DCEUS is a more low-cost, real-time, and non-irradiating technique for the easy bedside use Among quantitative DCEUS techniques, functional perfusion imaging (FPI) based on bolus detected kinetics was proposed to quantify detailed hemodynamics in angiogenesis Conventional FPI depicts and quantifies the 2D hemodynamics in angiogenesis at only one anatomical section without 3D perfusion information To weaken the influence of respiration on abdominal DCEUS, breath-holding is still used for short examinations. However, it is infeasible for long perfusion imaging , especially for patients with cardiopulmonary diseases To overcome limitations of abovementioned compensation strategies, semiautomatic and fully automatic respiratory gating methods employing machine learning algorithms have been developed in recent years. Using independent component analysis, a posteriori respiratory gating DCEUS approach was proposed based on an assumption of independence between microbubble kinetics and breathing motion In vitro and in vivo abdominal DCEUS-based FPI of a single respiratory phase was reconstructed by considering slight out-of-plane motion in vivo respiratory gating FPI is unknown because of the uncontrolled DCEUS without respiratory motion and the unknown ground truth of in vivo respiratory curves in vitro and in vivo hepatic perfusion examinations under the condition of extreme out-of-plane respiratory motion. A list of abbreviations used in this study is given in Table in vitro perfusion experiments (n = 20) to validate the accuracy of the proposed respiratory gating DM-FPI scheme. Table 5 bubbles/mL; SonoVue, Bracco, Milan, Italy), which were made of phospholipid encapsulated sulfur hexafluoride, was injected via an infusion outfit into the flow phantom. The transducer's 3D rotary motion was regulated by an electronic control system  to simulate out-of-plane breathing motion and to result in an extreme 3D periodic rotational motion in DCEUS loops. Thus, DCEUS loops exhibited an serious deformation of the mimicked hepatic vein with a simulated respiratory frequency of 0.27 Hz in vitro DCEUS loops with respiratory motion artifacts. Moreover, in vitro static DCEUS loops without breathing motion artifacts at fixed end-of-expiration (EOE) and end-of-inspiration (EOI) phases were respectively recorded as ground truths to assess the respiratory gating accuracy in the proposed DM-FPI scheme.An ultrasound scanner  utilizing a linear transducer was applied for ents n = 0 to valiin vivo hepatic (n = 14) perfusion experiments to validate the feasibility of the proposed respiratory gating DM-FPI scheme. Experimental acquisitions included 8 healthy rabbits, 6 patients with diagnosed symptoms of hepatic cavernous hemangioma  and hepatocellular carcinoma . Patients  were 45 to 69 years old. As performed in the previous studies An ultrasound platform  using a convex array was utilized for 8 bubbles/mL) was intravenously injected 8 bubbles/mL) was also intravenously injected and then immediately washed with 0.9% normal saline in vivo DCEUS loops with respiratory motion artifacts were also recorded using the same pulse-inversion contrast harmonic imaging mode. All in vitro and in vivo DCEUS sequences covered the major perfusion phases including pre-contrast, arriving, rising, filling, and complete or part falling phases of microbubbles.After the rabbits were anesthetized with a 3% concentration of mebumalnatrium, a bolus of SonoVue diluent  The alteration in  p is the component order, represents the i-th vector of principal component in  is the spatial weighting vector of  and is quantified by using the maximal eigenvectors from the covariance p is larger than 4, the remanent component in the variance is smaller than 1.7% in p is 4 in this study  corresponds to the respiratory kinetic curve , which can be estimated as:where were first removed by smoothing to obtain . Zero-crossing dual-phase detection was applied on the first-order derivative curve of  to alternately identify the two extreme phases of EOI  were then extracted pixel-by-pixel RG denoising to remove the clutter originated from the thermal noise and speckle effect RG was guaranteed.As shown in Fig. RG for each group data. Two time parameters are wash-in time (WIT) and wash-out time (WOT), which are proportional to the blood flow; two intensity parameters are peak value (PV) and area under curve (AUC), which are related to the local perfusion volume; and two ratio parameters are wash-in rate (WIR) and wash-out-rate (WOR), which are in direct proportion to flow rate As depicted in Fig. In vitro and in vivo FPIs without respiratory gating (FPInoRG) were reconstructed from TICs without respiratory gating (TICnoRG) to compare with respiratory gating FPI (FPIRG) of the same perfusion parameter. TICnoRG was acquired from the same DCEUS loops used for TICRG and FPIRG but without respiratory gating. Moreover, without breathing motion disturbances, in vitro static TIC (TICstatic) and FPI (FPIstatic) were considered as the control groups to assess the accuracy of the proposed respiratory gating DM-FPI scheme. TICstatic was extracted from the ROI at the same location as the in vitro static DCEUS sequences. Additionally, FPIRG, FPInoRG, and FPIstatic of the same hemodynamic parameters were adjusted to the same dynamic ranges to demonstrate the performances of the DM-FPI scheme RG was evaluated by utilizing the SCR RG and TICstatic, as defined by the following equations The respiratory gating veracity of TICP(f) is the TIC power spectral density. cf and sf are the cut off and sample frequencies of TIC, respectively. MSE between TICnoRG and TICstatic was also calculated to compare with the MSE between TICRG and TICstatic.where RG and FPIstatic and the mean correlation coefficients (R) between FPIRG and FPIstatic in the entire perfusion region, as defined by the following equations The accuracy of respiratory gating DM-FPI was quantified by utilizing the MSE between FPIRG and Pstatic are the probability densities in FPIRG and FPIstatic of one perfusion parameter, respectively, K is the number of FPI, and N is the points of PRG and Pstatic. The MSE and correlation R between FPInoRG and FPIstatic were also computed to compare with the MSE and R between FPIRG and FPIstatic.where Pin vivo hepatic TICs and DM-FPI were evaluated using the SCR of TICs and mean noise coefficients (MNC) of FPI in the entire perfusion region, as defined by the following equations:The respiratory gating feasibility in j is the red, green, and blue components  in the color map of FPI. is the mean value of FPIj.where FPIp-value < 0.01 was taken for highly significant that was marked by two asterisks in Figures and Tables. A p-value < 0.05 was regarded as statistically significant which were marked by one asterisk in Figures and Tables. Since three types of six FPIs were reconstructed at single EOE or EOI phase for each group of data, the analysis databases of in vitro, in vivo normal, HCH, and HCC data were 40, 16, 6, and 6 for each PFI type at single phase, respectively; the corresponding databases were 240, 96, 36, and 36 for of all types of dual-phase FPIs, respectively. All data processing was executed utilizing MATLAB . Machine learning toolbox of MATLAB was used to extract the respiratory curves, which included the packages of non-negative matrix factorization, independent component analysis, and PCA.Mean values of perfusion results and their standard deviations were calculated and further evaluated using a t-test. Herein, a in vitro and in vivo hepatic respiratory kinetic curves, which acquired from DCEUS loops in contrast mode and fundamental mode, respectively. The order of principal component vector in  is related to reduction dimension  well matched the respiratory motion frequency estimated from the manual frame selection in respiratory gating methods, which were further verified in the cross-validations using non-negative matrix factorization and independent component analysis  was 0.27 Hz in keeping with the simulated ground truth and the results in  were also consistent with the simulated kinetic phases. Therefore, the respiratory kinetics was estimated by using the second principal component in this study. The estimated respiratory kinetics accounted for 20.3% ± 2.9 % of all components when p was 4. In comparison with in vivo respiratory kinetics, in vitro kinetics contained some information on microbubble kinetics, and the envelope was equivalent to TICRG  in vitro and in vivo TICRG enhanced by 3.3 ± 1.7 (p < 0.05) p < 0.05), respectively, when compared with the corresponding TICnoRG.Figure in vitro groups of dual-phase FPIstatic, FPIRG, and FPInoRG of the mimicked hepatic vein were shown in Fig. noRG, inhomogeneous transient variations in blood velocity, perfusion charge, and rate of flow of the mimicked hepatic vein hemodynamics were clearly presented on the time-, intensity-, and ratio-related dual-phase FPIRG, respectively. Differences in the corresponding hemodynamic distributions depicted by dual-phase FPIRG were also clearly observed at the EOI and EOE phases. Dual-phase FPIRG in Figs. static in Figs. In vitro FPIRG was nearly similar to the ground truth. The inhomogeneous hemodynamic mappings at various respiratory phases were overlapped and distorted in FPInoRG because of the absence of respiratory gating. As shown in Fig. RG and FPInoRG at EOE and EOI phases, respectively. Table RG when compared with FPInoRG in Fig. noRG, the MSE of FPIRG decreased significantly by 1893.9 ± 965.4 (p < 0.05) whereas the correlation R improved significantly by 0.4 ± 0.2 (p < 0.01).Three in vivo DCEUS-based dual-phase FPInoRG and FPIRG in the normal liver of a healthy rabbit, and the HCH and HCC of two patients, respectively. Each group of DM-FPIs included one time-related FPI of WIT or WOT, one intensity-related FPI of PV or AUC, and one ratio-related FPI of WIR. Compared with aliasing in FPInoRG caused by severe out-of-plane breathing motion, in vivo hepatic blood velocity, perfusion charge, and rate of flow were clearly depicted by the time-, intensity-, and ratio-related FPIRG, respectively. Anatomical structures of the right and left hepatic veins in the normal liver were distorted in FPInoRG, which were recovered effectively by the dual-phase FPIRG of WOT. The irregular border of the HCH was accurately identified by the dual-phase FPIRG of WIT and WIR, respectively. The heterogeneous HCC angiogenesis was well quantified in the dual-phase FPIRG of AUC and WIR, respectively.Figures in vivo dual-phase FPIRG and FPInoRG. In comparison with FPInoRG, the decrease in MNC of time-, intensity-, and ratio-related dual-phase FPIRG within the normal liver, HCH, and HCC were shown in Table p < 0.05) in FPIRG. These in vitro and in vivo verifications demonstrated that the proposed DM-FPI scheme considerably removed severe out-of-plane artifacts caused by breathing motion on FPI, improved the accuracy in respiratory gating FPI. The proposed method could also characterize and distinguish the heterogeneous hemodynamics of the hepatic angiogenesis within two sections of liver cancer cells at the EOI and EOE phases.Figure in vitro and in vivo TICs and to guarantee the efficient reconstruction of DM-FPI in vitro and in vivo validations and limitation of memory space in the used commercial ultrasound platforms, the frame rate and acquisition time were different in Table This study used DCEUS video loops to reconstruct DM-FPI. In comparison with radiofrequency signal, nonlinear image processing algorithms may influence DCEUS video signal The maximum and minimum thresholds were utilized to detect respiratory phases in the estimated respiratory kinetics in vitro and in vivo respiratory gating or motion compensation methods based on machine learning algorithms p < 0.05) and computation efficiency was quicker by 509.7 times (p < 0.01). Compared with non-negative matrix factorization, MSE of PCA was reduced by 0.3 (p < 0.05) and computation efficiency was improved by 20.8 times (p < 0.05). Thus, in comparison with independent component analysis and non-negative matrix factorization, the accuracy of estimated respiratory kinetics using PCA was improved and computation efficiency was the highest among those machine learning algorithms Previous studies had discussed the performance of in vitro validation of respiratory gating for DCEUS-based DM-FPI aimed to provide information not available on the accuracy of those previous studies in vivo experiments, only Christofides and Renault et al. evaluated the respiratory gating accuracy through ideal simulation models under in-plane and slight out-of-plane motion conditions, respectively in vitro studies validated the accuracy of FPI with respiratory motion compensation at in-plane condition noRG, the decreases in MSE of FPIRG were range from 1189.5 ± 86.71 (p < 0.05) to 1893.9 ± 965.4 (p < 0.05) under the conditions from slight to serious respiratory motion artifacts. But the increases in correlation R of FPIRG were range from 0.2 ± 0.1 (p < 0.05) to 0.4 ± 0.2 (p < 0.01). These quantitative evaluations emphasized the fact that the noise level, misestimation, and various out-of-plane artifacts in FPIs induced by respiratory motion could be considerably decreased by the proposed respiratory gating DM-FPI scheme.The RG clearly characterized the hepatic angiogenesis in HCH and HCC at EOI and EOE phases  zone in the No. 1 region  zones were characterized in the No. 2 region in FPIRG at the EOI phase and in the No. 2 and No. 3 regions in FPIRG at the EOE phase. The heterogeneous angiogenic hemodynamics in HCC and HCH were visualized and quantified using dual-phase FPIRG. Additionally, angiogenesis not only is an effective indicator to diagnose cancer but also becomes one of the principal targets of several antiangiogenic therapies Moreover, it is well-known that the tumor angiogenesis presents heterogeneity because of differences in cancer size, density, and architecture ion Fig. , angiogeSeveral quantitative DCEUS techniques have been developed to directly quantify the microvascular hemodynamics. 1D TIC analysis techniques were proposed to qualitatively indicate perfusion differences within normal and lesion tissues, which were further developed to estimate single Moreover, other FPI approaches on account of nuclear magnetic spin imaging and computerized tomography are also widely used to diagnose and stage tumors and to monitor their response to treatments in vitro and in vivo hepatic perfusion validations under conditions of severe out-of-plane respiratory motions. Compared with approaches without respiratory gating, the proposed DM-FPI technique significantly removed out-of-plane artifacts and parametric misestimation induced by respiratory kinetics and improved the respiratory gating accuracy and robustness. Respiratory gating for DM-FPI could characterize and distinguish heterogeneous angiogenic hemodynamics within two sections in normal livers and in benign and malignant liver tumors. The proposed scheme may assist clinicians to identify benign and malignant hepatic tumors, characterize tumor grading and staging, evaluate therapeutic effectiveness, and guide appropriate therapies for liver cancers.This study proposed a hepatic respiratory gating DM-FPI technique based on DCEUS utilizing derivative PCA. Accuracy and feasibility of the proposed technique were respectively illustrated through


Childhood abuse related posttraumatic stress disorder (CA-PTSD) is associated with a high burden of disease and with treatment response rates that leave room for improvement. One of the treatments for PTSD, prolonged exposure (PE), is effective but has high drop-out rates and remission rates are relatively low. An intensified form of PE (iPE) was associated with good response and low drop-out rates in PTSD and has not yet been tested in a controlled trial in CA-PTSD. Phase-based treatment (PBT), in which PE is preceded by skills training may improve overall outcomes in this population. We will assess the effectiveness and cost-effectiveness of standard PE, iPE and PBT in patients with CA-PTSD.Multi-center randomized controlled trial. Treatment conditions are: prolonged exposure ; intensified PE ; phase-based treatment .N = 150). Assessments in weeks 0, 4, 8, 16, 26 and 52.Primary outcome: Clinician-rated PTSD symptom severity. Secondary outcomes: loss of PTSD diagnosis, self-reported PTSD symptom severity, comorbid symptom severity and quality of life. Moreover, we will examine cost-effectiveness and moderators and mediators of treatment outcome. Target population: adults with CA-PTSD ; retrospectively registered at June 21, 2017 at  Childhood abuse is associated with severe negative long-term consequences. These include health problems, high health care utilization, a high risk of revictimization, low socio-economic well-being and criminal behavior in adulthood –6. ChildIn international guidelines of PTSD, trauma-focused treatment (TFT) is recommended as first treatment option . SubstanN = 10) also suggest that intensive treatment is safe and acceptable, with an 80% remission rate [Some authors , 18, 19 ion rate . Furtherion rate , 25 showOther authors –30 have Further research on the treatment of CA-PTSD is needed because of limitations of existing studies. Firstly, no studies have directly compared TFT with PBT or iTFT , 18, 33.The aim of the current study is to examine the effectiveness of three different treatment strategies for patients with CA-PTSD. We will carry out a randomized controlled trial (RCT) comparing the (cost-)effectiveness and treatment adherence of a well-established form of TFT, prolonged exposure (PE), with two potential improvements of TFT: intensified PE (iPE) and phase-based treatment (PBT). For the iPE group, PE sessions are delivered in 4 weeks (3 sessions per week), PBT consists of Skills Training in Affective and Interpersonal Regulation (STAIR), followed by PE. We expect more PTSD symptom reduction and lower drop-out rates in iPE and PBT than in PE. We also expect that iPE and PBT will be more cost-effective, given that the treatment protocols include fewer (iPE) and shorter (PBT) sessions. We expect that iPE will lead to faster improvement than PE and PBT. Finally, we expect that PBT will be superior to both PE and iPE with respect to improvement in emotion regulation, interpersonal skills and self-esteem. The primary outcome is clinician-rated PTSD symptom severity. Secondary outcomes are loss of PTSD diagnosis, self-reported PTSD symptom severity, treatment adherence, comorbid symptoms severity and cost-effectiveness. Outcomes will be assessed at baseline, after 4, 8 and 16 weeks and at 6 and 12 months follow-up.In line with previous work , we willAs to mediators, moderately strong evidence exists that between-session habitation and change in post-traumatic cognitions mediate the effects of PE, while mixed evidence exists for emotional engagement, inhibition learning and within-session habituation . Mediatohttps://clinicaltrials.gov/ct2/show/NCT03194113.The IMPACT study is a multicenter RCT comparing prolonged exposure (PE) with intensified prolonged exposure (iPE) and phase-based treatment (PBT). Participants will be randomly assigned to the conditions. Figure Participants are recruited at the departments of Psychotrauma of PsyQ Den Haag and PsyQ Rotterdam. Referrals from other treatment centers will also be accepted. After initial screening, potential participants will receive written and oral information about the study. Patients who are interested in participating are invited for the baseline assessment including screening of in- and exclusion criteria and an informed consent procedure. Informed consent will be obtained prior to the assessment.Inclusion criteria of the study are: 1) age 18–65; 2) diagnosis of PTSD as established with the Clinician Administered PTSD Scale , and at least moderate severity of PTSD-symptoms (CAPS ≥26), and with at least one specific memory for a traumatic event; 3) multiple traumata related to childhood sexual and/or physical abuse that occurred before 18 years of age, committed by a primary caretaker or an authority figure as index event; 4) sufficient fluency in Dutch to complete the treatment and research protocols.Exclusion criteria are: 1) involvement in a compensation case or legal procedures concerning admission or stay in The Netherlands; 2) pregnancy; 3) severe non-suicidal self-injury (NSSI) which required hospitalization during the past three months; 4) severe suicidal behavior: a suicide attempt during the past three months or acute suicidal ideations with serious intent to die with a specific plan for suicide and preparatory acts; 5) severe disorder in the use of alcohol or drugs in last three months; 6) cognitive impairment (estimated IQ < 70); 7) changes in psychotropic medication in the two months prior to inclusion; and 8) engagement in any current psychological treatment.Our sample size calculations are based on the intention to detect at least moderate effect size differences (d = .40) among conditions. To detect this effect size difference in PTSD severity with alpha = .05 (2-tailed) and a power of 0.8, 50 participants per condition are needed. We expect some drop-out which will result in a lower power due to missing values. However, we calculated the sample size based on the conservative assumption that the correlation between the baseline and all further post measurements is 0 and the correlation between post measurements is 1, since we do not have a good estimation for the correlation between the outcome measurements yet. Thus, the actual power is expected to be considerably higher than 0.8 due to the multiple measurement design correcting for power loss due to drop-out , 47.Before randomization, patients complete a baseline assessment of the study. In the preparatory session, patients receive detailed information about the treatment and research procedures and about practical considerations, such as availability. Randomization is carried out by an independent researcher from Leiden University who uses a computerized randomization sequence of permutated blocks of six patients, stratified by gender. Patients are regarded as treatment drop-out if they stop therapy prematurely and as measurement drop-out if they refuse or do not show up for follow-up measurements. Early responders are defined by a score below 16 on the PTSD checklist for DSM-5 (PCL-5) for three consecutive weeks with agreement between patient, therapist and supervisor about finishing the therapy early , 49. MeaBefore participation in the trial, master’s level therapists attend a two-day training in prolonged exposure and a two-day training in STAIR. At the end of these trainings, the therapists have to pass an exam with pilot patients, which is graded by the supervisors of the study. During the study, all therapists receive weekly supervision in (i)PE (by AM and RK) and PBT (by MC and IW). All treatment locations offer the three types of treatment and all therapists receive the same amount of supervision and training. Adherence to the treatment protocols will be checked by independent observers, who will rate randomly selected videotaped therapy sessions.Prolonged exposure therapy (PE) is delivered in 16 weekly sessions of 90 min. The treatment manual is based on the PE protocol by Foa, Hembree, & Rothbaum (2007) .Treatment sessions consist of imaginal exposure (repeated recounting of the most anxiety provoking traumatic memories and processing related thoughts and feelings), and exposure in vivo (approaching trauma-related situations). Between sessions, participants listen to audio recordings of the imaginal exposure on a daily basis, and complete in-vivo homework assignments.Intensified prolonged exposure therapy (iPE) involves three weekly sessions of 90 min PE for a period of four weeks , followed by two PE sessions after one and two months . The same protocol is used as in the PE condition with some minor changes for practical considerations. For instance, when two treatment sessions are given on consecutive days patients are instructed to do combined homework of both sessions. After the first 12 sessions, patients are instructed to keep doing imaginal exposure and exposure in vivo homework for the 13th and 14th sessions. For practical considerations, two therapists deliver the iPE sessions alternately.Phase-based therapy (PBT) is delivered in 8 weekly 60 min STAIR sessions , followeIn Table kappa = .83) for the diagnosis of PTSD and a high internal consistency (α = .88) and test-retest reliability (ICC = .78) for the total severity score [PTSD diagnosis and symptom severity are assessed with the Clinical Administered PTSD scale (CAPS-5) . The CAPty score . Responsty score . Remissia = .94) and test-retest reliability (r = .82) [Posttraumatic symptom severity is also measured with the PTSD checklist for DSM-5 (PCL-5). The PCL-5 has a high internal consistency (r = .82) , 59.To measure clinician-rated symptoms that have been proposed to define complex PTSD  we use tComorbid axis-1 disorders (DSM-IV) are measured with the Mini International Neuropsychiatric Interview (MINI) . DepressPersonality disorders are measured with the Structured Clinical Interview for DSM-IV Personality Disorders (SCID-II) .Moreover, anger, negative cognitions, social support and attentional control are measured using self-report questionnaires State-Trait Anger Scale (ZAV) , the PosThe LEC-5 , 78 measPrior, during and immediately after  exposure, Subjective Units of Distress (SUD) ratings are assessed and prior and after exposure harm expectancies are assessed. Treatment credibility of the three therapies will be checked with the adapted Treatment Credibility Scale . AdditioQuality of Life is measured with the EQ-5D-5 L , 87. TheMoreover, cost-effectiveness is determined with the Trimbos/iMTA questionnaire for costs associated with Psychiatric Illness (TiC-P)  which meA classical associative learning paradigm is administered to measure avoidance behaviors. In this task, emotional, anxiety provoking pictures from the International Affective Picture System (IAPS)- set are used as unconditioned stimulus (US), and pictures of an office containing a light, that changes color  as the conditioned stimulus (CS). Participants can avoid the US by pressing a button, but success is dependent on the CS .Data analyses will be based on intention-to-treat analyses. All randomized patients will be included in the analyses. Due to the structured data, we will use multiple imputation of multilevel data which takes the levels within the data into account .Primary and secondary continuous outcome parameters will be analyzed with multilevel mixed models using a repeated measurement design to correct for the dependencies among the observations , 92. DicFor moderation and mediation analyses, regression based approaches will be used with the PROCESS macro in SPSS . For modCompletion of this RCT will provide more knowledge about the relative effectiveness of three treatment strategies for CA-PTSD. We will directly compare the effects of a well-established treatment (prolonged exposure) and two treatment innovations (intensified prolonged exposure and phase-based treatment) in this difficult to treat patient population. Furthermore, cost-effectiveness of the three interventions will be examined. Finally, moderation and mediation analyses will provide more information for whom and under which conditions these treatments are most effective. Ultimately, this might assist clinicians in personalizing treatment indications and optimizing treatment delivery.We expect to include a cultural and socioeconomic diverse sample, since the participating centers are located in large cities. We protect the generalizability of the findings by using few exclusion criteria. The relatively long follow-up measurements of 6 and 12 months will provide insights in the long-term effects of the therapies. Every type of treatment is supervised by expert supervisors of that specific method. Additionally, all therapists are trained and supervised in both PE and PBT. This prevents biases to the internal reliability of the study and is essential for a meaningful interpretation of the results .Limitations of this study are that not all eligible patients will agree to participate in the study which could result in selection bias. Especially the iPE condition could lead to selection bias since it is more demanding in terms of time investment in the first weeks of the treatment. All reasons of patients to decline participation in the study will be carefully monitored to ensure the generalizability of the results and for implementation purposes. Another limitation is that patients have one therapist in PE and PBT, but two alternating therapists in the iPE condition. This may influence the therapeutic alliance and consequently the results of the treatment. We will assess whether therapeutic alliance indeed differs between condition and, if so, whether this has any influence on treatment results.Patients with CA-PTSD have a high burden of disease. Currently, there is no consensus on treatment-guidelines for this patient group. The results of this study may have important implications for the treatment of patients with CA-PTSD.