Polycystic liver disease (PLD) is a genetic disorder in which patients suffer from progressive development of multiple (>10) hepatic cysts. Most patients remain asymptomatic during the course of their disease. However, a minority of PLD patients suffer from symptoms caused by hepatomegaly leading to serious limitations in daily life. Untreated symptomatic PLD patients score significantly worse on health-related quality of life (HRQoL) compared to age and gender-matched populations. Currently, liver transplantation is the only curative treatment for PLD. The main goal of other available therapies is to strive for symptomatic relief and improvement of HRQoL by suppressing disease progression. In this review, we summarize the effect of PLD treatment on patient-reported outcome measures with a distinction between HRQoL and symptom severity. At present there is heterogeneity in application of questionnaires and no questionnaire is available that measures both HRQoL and PLD symptom severity. Therefore, we recommend the combination of a validated PLD-specific symptom severity questionnaire and a general HRQoL questionnaire to evaluate treatment success as a minimal core set. However, the specific choice of questionnaires depends on treatment choice and/or research question. These questionnaires may serve as a biomarker of treatment response, failure, and adverse events. Polycystic liver disease (PLD) is a genetic disorder in which patients suffer from progressive development of multiple >10) hepatic cysts. The genetic origin of PLD can be divided in 2 etiologies: autosomal dominant polycystic kidney disease (ADPKD) with presence of multiple cysts in kidneys and liver, and autosomal dominant polycystic liver disease (ADPLD) where cysts are confined to the liver. Patients affected by PLD are predominantly female (>80%)0 hepaticThe natural disease course of PLD remains unknown. Although cyst burden may increase, liver function is generally preserved and most patients remain asymptomatic during the course of the disease. A minority of PLD patients suffer from symptoms caused by hepatomegaly or strategically located cysts. Although this group is small, these symptoms can lead to serious limitations in daily life when patients experience a high symptom burden. Commonly reported symptoms include abdominal distention, pain, early satiety, weight loss, nausea, vomiting, dyspnea, and restriction in range of motion. In contrast, specific biliary symptoms such as jaundice 3%) or cholangitis (1%) are rare.% or cholPLD-specific symptom patient-reported outcome measures (PROMs) have been developed which differentiate between PLD patients and those from the general population depending on PLD-related symptoms.Currently, liver transplantation is the only curative treatment option for PLD. The main goal of other available therapies is to achieve symptomatic relief and improvement of HRQoL by suppressing progression of disease. However, these therapies all possess an unique efficacy/safety balance that may come with technical success yet compromise HRQoL. Many studies have taken reduction of (height-adjusted) total liver volume ((h)TLV) or total cyst volume (TCV) as the primary outcome measures to determine treatment success. These surrogate endpoints might be less meaningful to patients compared with symptom burden or HRQoL depending on the type of treatment. For example, a previous study demonstrated that a larger hTLV correlated with lower SF-36 scores and a higher symptom severity.This raises the question whether liver or cyst volume alone is the optimal outcome measure to determine treatment efficacy or that this should be combined with PROMs that evaluate treatment impact using the patient’s HRQoL and symptom severity. In this article we will review the current literature on HRQoL in PLD, with special emphasis on the effects of currently available therapies on these PROMs.PubMed was searched for terms related to PLD, HRQoL, and the different treatments discussed in this article, on February 28, 2022. Additional  articles were identified through literature snowballing. Studies were included when they were written in English and addressed PLD treatment in combination with either HRQoL or symptom severity. Supplementary file 1, Supplemental Digital Content 1, http://links.lww.com/JCG/A886 contains an overview of the search strategy.Many factors can affect a patient’s HRQoL, including disease-specific symptoms. The most objective measures currently available to measure (factors of) self-rated HRQoL are PROMs. In total 16 articles were identified, yielding 10 different questionnaires which were queried at more than 10 different time moments. These findings are summarized in Table Six studies used a gastrointestinal (GI) symptom questionnaire, which was originally designed to capture early side effects associated with oral iron supplementation,Both PLD-Q and POLCA can be used to provide insight into PLD-related symptoms and to monitor symptom severity before and after treatment. However, it is important to note that these questionnaires are not developed to measure treatment side effects, complications, or overall HRQoL. Furthermore, both questionnaires lack a treatment threshold that can be used in clinical practice.The EQ-VAS is a frequently used tool in healthcare to obtain a quick global estimation of the patient’s self-rated overall HRQoL. The EQ-VAS is a visual analog scale and allows patients to rate their general health from 0 to 100. The EQ-VAS is fast and can be applied without context and the construct validity is satisfactory. However, the test-retest reliability and responsiveness of EQ-VAS is limited, and it does not provide qualitative information on symptoms the patient experiences. Using the EQ-5D a more comprehensive insight can be obtained, this questionnaire includes 5 health levels  and includes the EQ-VAS.For a more detailed assessment of HRQoL, the SF-36 is most frequently used in PLD research. The instrument uses a recall period of 4 weeks and can distinguish 8 different domains of HRQoL: physical functioning, social functioning, role limitations due to physical or emotional problems, mental health, energy, pain, and general health. These 8 domains can be combined into two endpoints: a PCS and a MCS score. The SF-12 is a shortened version of the SF-36 and only yields results for the summarizing PCS and MCS scores.The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core-30 (EORTC QLQ-C30) is a more elaborate questionnaire developed to assess general disease symptoms and HRQoL in cancer patients and has been used in PLD.Lastly, in previous studies for PLD patients the Eastern Cooperative Oncology Group performance status (ECOG-PS) has been used for PLD patients.Table Most patients with PLD are asymptomatic and the disease is frequently discovered as an incidental finding on radiologic imaging for other purposes. In PLD patients with no or only mild symptoms a conservative approach is appropriate. Female patients should be discouraged from taking exogenous estrogens as estrogen exposure is associated with liver growth. Routine surveillance is not recommended in PLD patients unless aggravation of symptoms occur.Multiple treatment therapies have been developed for symptomatic PLD patients with a decreased HRQoL. Treatment choice depends on symptoms, liver phenotype, and patient or center preferences.Aspiration sclerotherapy (AS) is recommended in PLD patients with a dominant or strategically located symptomatic cyst. After the cyst fluid is aspirated, a sclerosing agent is infused that aims to destroy the inner epithelial lining to prevent cyst recurrence. Median volume reduction of AS ranges between 76% and 100%At present no studies have been identified that investigate the effect of AS on HRQoL. The effect of this procedure on symptom severity has been discussed in a systematic review. Symptomatic relief was achieved in 72% of PLD patients, which is remarkably lower compared with the patients with solitary cysts 94%.Together these studies demonstrate that AS effectively reduces symptom severity, with only mild postprocedural complications. The assumption that symptom reduction after AS also results in HRQoL improvement cannot be substantiated sufficiently at present with the available literature.Cyst fenestration can be performed in patients who experience symptoms from multiple large cysts. Cyst fenestration is usually performed with a laparoscopic approach and combines internal aspiration with laparoscopic deroofing of one or more liver cysts.A recent literature review included 1314 patients, who underwent fenestration for symptomatic hepatic cysts. A subgroup analysis showed that postoperative complications, including ascites, arterial or venous bleeding, and biliary leakage, were more frequent in PLD patients (10.8% vs. 29.3%).P=0.01) and postprandial fullness (P=0.02) was significant. Furthermore, a correlation between liver volume and pain was found.However, 1 study used the gastrointestinal questionnaire to assess symptoms 4 weeks before and after cyst fenestration in 12 PLD patients. Reporting of all symptoms decreased, but only the decrease of abdominal distension , these numbers are based almost exclusively on cohort studies without PROMS and merely clinical follow-up data. Only 2 studies used validated questionnaires to assess symptoms and performance status before and after surgery.The first is a retrospective study that analyzed 29 patients who underwent partial hepatectomy combined with extensive cyst fenestration (>30). Patients’ HRQoL were classified using the ECOG-PS. Most patients (84%) were classified as category 1  at baseline. A postoperative liver volume reduction of 52.8% was achieved and after a mean follow-up of 70.8 ± 65 months 84% of the patients were completely asymptomatic. Overall morbidity (Clavien-Dindo grade ≥ II) rate was 41.4% and mortality was reported in 4 patients (13.8%). ECOG-PS normalized or improved in 21 patients (84%), stabilized in 3 patients (12%), and worsened in 1 patient (4%).Another prospective study included 17 PLD patients suffering from severe PLD with a median liver volume of 4781 mL (IQR: 3303–6228).Although hepatic resection seems an effective volume-reducing therapy that improves HRQoL, most studies are conducted with small patient numbers and are conducted in expertise centers which precludes extrapolability to other centers. Despite the cost of a high morbidity and mortality associated with PHCF, patients with a severe symptom burden and suitable liver phenotype could be treated with this procedure.PLD is a rare indication for liver transplantation (LT) as liver function remains preserved even in the most severe cases. LT is reserved for patients with severe symptoms leading to limitations in daily life or untreatable complications, eg, severe malnutrition or portal hypertension with ascites.An observational study, including 21 LT and 15 CKLT patients, studied HRQoL using the SF-36 and self-designed disease-specific questionnaires. Within the first 2 months 5 patients died. Of the remaining 28 patients (78%) who answered 91% felt better and 9% felt worse. Symptoms such as loss of appetite and vomiting, most likely resulting from enlarged liver and kidneys, significantly improved. After LT patients, SF-36 scores were not different compared with the general population.Despite considerable periprocedural morbidity and mortality, LT appears to greatly improve HRQoL in selected PLD patients. However, these findings need to be corroborated in larger cohorts.The pathophysiology underlying cyst formation in ADPKD and ADPLD has not been fully elucidated. Various intracellular pathways are involved, leading to increased fluid secretion and cell proliferation in cholangiocytes and subsequently hepatic cyst formation. These pathways serve as a target for medical interventions. An overview of the (pre)clinical tested medical intervention is given in Figure Somatostatin analogs (SAs) are the only medical treatment for PLD patients that alter the natural progression of the disease. The SAs octreotide, lanreotide and pasireotide halt hepatic cystogenesis through binding to the somatostatin receptor in cholangiocytes. Several randomized controlled trials proved that SAs effectively reduce TLV in PLD.P=0.06).P=0.044), but not in the MCS (P=0.253).A recent meta-analysis studied the effect of SAs on HRQoL with the SF-36 questionnaire. No significant improvement was observed in the patients receiving SAs. Only the SF-36 PCS showed a marginal, nonsignificant improvement in favor of SA . Effects of this type of drug were reported in 2 important publications: a cohort study and a randomized controlled trial.Another study compared everolimus and octreotide combination therapy with octreotide monotherapy, yet showed no difference in effect on TLV.2+ agonist in hepatocytes and cholangiocytes and could restore intracellular Ca2+ homeostasis and halt hepatic cystogenesis. In a randomized controlled trial, UDCA proved ineffective in reducing TLV in PLD.Ursodeoxycholic acid (UDCA) is a CaVasopressin-2-receptor (V2R) is involved in Ca2+-signaling pathway and inhibition of this receptor could theoretically halt hepatic cystogenesis. In a recent study the effect of tolvaptan in PLD patients was studied, but the external validity of this study is limited owing to nonrandomized retrospective design of the study. The effect of tolvaptan on TLV growth rate was not different from the control group.A minority of PLD patients suffer from symptoms resulting from massive hepatomegaly. However, these symptoms may seriously impact HRQoL. In this review we discussed the effects of different treatment modalities on HRQoL and PLD-related symptoms. AS, cyst fenestration, and PHCF effectively reduce pain and symptom severity. A substantiated opinion about their impact on HRQoL cannot be provided, given the low-level of available evidence and the lack of consistency when PROMs were applied in studies. Despite limited availability of robust evidence, LT shows a significant improvement on HRQoL with the SF-36 questionnaire. The effects of medical treatments on HRQoL are not yet fully elucidated. A beneficial effect on HRQoL was found in some studies, while others demonstrate a negative effect of treatment-associated side effects. The volume-reducing effects of medical treatment can also be too modest or too slow compared with surgical therapies to detect tangible changes in HRQol.The effects of PLD treatment are expected to be similar to other diseases defined by mass-related effects . Unfortunately, no evidence is available that discusses HRQoL or symptom severity in liver adenomas or gastrointestinal neuroendocrine tumors. Two studies demonstrated a positive effect of surgical treatment on HRQoL scores in giant hemangioma patients,Most studies discussed in this review used TLV or cyst volume reduction as their primary outcome, while the end goal of treatment is to reduce symptom burden. Development and validation of PLD-specific PROMs allowed the use of these instruments as appropriate primary outcome measures to determine treatment impact in PLD. Despite this progress, there are still a number of issues that need to be addressed before use of PROMs as a bonafide primary outcome (1) general HRQoL versus symptom specific questionnaires, (2) differences in application of questionnaires, and (3) determination of PROM thresholds for treatment guidanceThroughout this review we have made an important distinction between PROMs aimed at HRQoL and PROMs aimed at symptom severity. HRQoL questionnaires measure the physical, mental and social functioning related to an individual’s health. Symptom severity questionnaires address the disease-specific symptoms which could affect HRQoL. PLD therapies should be aimed at reduction of PLD-specific symptoms, thereby ideally also improving HRQol. However, one should keep in mind that symptom severity questionnaires are not validated to measure treatment-associated side effects, which might underestimate a potential negative impact of PLD therapies on overall HRQoL. Currently, no questionnaire is available that can measure both HRQoL and symptom severity in PLD patients. For this reason, we recommend using a combination of both types of questionnaires. As discussed in this review, several validated questionnaires are appropriate to determine symptoms severity and HRQoL, each of which has its (dis)advantages and applicability depends on your research question.Another difficulty in assessing the effect of PLD treatment using PROMS are the differences in how questionnaires are applied. Different questionnaires and different lengths of follow-up periods are used to measure symptoms or HrQol in the studies that address this issue which complicates a comparison of therapy outcomes. We advise baseline measurements before each treatment. The administration frequency of a PROM during medical treatment or after radiologic and surgical treatment should be done in a standardized manner, considering associated side effects, and the length of treatment and individual treatment outcome measures. By frequently administering the questionnaires, recurrence of symptoms can be detected. Treatment specific side effects and complications should also be monitored during checkups. Table Previous studies discussed that treatment initiation is based on symptoms and not liver phenotype. This opens up the possibility to use PROMs in the clinical setting to determine which patients should start or discontinue a certain treatment. To minimize variation between centers and physicians, PROM thresholds for therapies should be determined. Patients with a score exceeding this threshold would be eligible for treatment or inclusion in studies. Liver phenotype can aid in the determination of treatment type. Adequate selection of patients can maximize treatment efficacy and reduce the frequency of unwanted and needless complications. Although at present questionnaires have no thresholds to determine treatment initiation, our recommendation is to administer PROMs when monitoring symptomatic PLD. This provides insight in the patient’s natural disease progression and allows for an objective method to capture dynamics in symptom presentation.In conclusion, validated questionnaires can aid physicians when integrated in daily practice in a uniform and consistent approach. The next step is to determine clinically relevant PROM thresholds. Especially disease-specific PROMS could assist physicians to (1) determine when treatment is indicated, (2) evaluate treatment efficacy more objectively, and (3) swiftly identify symptom recurrence after treatment in PLD patients.www.jcge.com.Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website,


An elderly man with prostate cancer and a deep vein thrombosis (DVT) of the lower extremity diagnosed 12 days ago on apixaban presented with a new-onset palpable rash on both of his legs. Extensive laboratory workup was largely unremarkable, except for multiple skin punch biopsies revealing deposition of immunoglobulin A (IgA) in the superficial blood vessels with infiltration of leukocytes, concerning for a small vessel vasculitis. Given the temporal association along with the negative workup, the rash was attributed to apixaban, and the anticoagulation regimen was switched to dabigatran. At a 2-week follow-up visit, the patient was asymptomatic and tolerating dabigatran without any adverse events. The 2012 Chapel Hill Consensus Conference (CHCC) defines cutaneous small vessel vasculitis as a single-organ vasculitis that involves small arteries in the skin.  We reporA 72-year-old man with prostate cancer on androgen deprivation therapy (monthly leuprolide injections) and likely a provoked deep vein thrombosis (DVT) of the left lower extremity (diagnosed 12 days ago) secondary to the cancer presented to the emergency department with complaints of a rash on his bilateral lower legs which he noticed 1 week ago. He described the rash as red, nonpruritic, mildly painful, and raised. The rash started near his ankles and spread rapidly up his legs to his buttocks. He denied any fever, joint pain, abdominal pain, or changes in urinary habits. He denied any recent travel or experiencing this type of rash before.His other past medical history included hypertension on losartan, type 2 diabetes on insulin, and chronic kidney disease. His only new medication was apixaban 5mg twice daily that he was taking for 12 days for the recently diagnosed DVT. He had completed a seven day course of 10mg of apixaban twice daily and was taking 5mg twice daily on the day of presentation. He had never taken any anticoagulant prior to this and had no known drug allergies or other recent medication changes.On examination, his vitals and systemic exam were unremarkable. Skin exam revealed diffuse palpable tender nonblanching erythematous patches extending from the dorsum of both feet bilaterally to the thigh with few scattered lesions on the buttocks. A fundoscopic eye examination was unremarkable, and there were no other lesions noted on his body. An extensive laboratory workup was performed  revealed perivascular infiltration of neutrophils within vessels in the upper dermis with leukocytoclastia, or neutrophilic debris. A direct immunofluorescence study showed intense granular vessel wall staining with IgA consistent with leukocytoclastic vasculitis.Apixaban was discontinued on the day of admission, and he was anticoagulated with low-molecular-weight-heparin (lovenox). His other home medications including insulin were continued. His pain was managed with acetaminophen and moisturizer was applied to his legs bilaterally. Upon discharge, he was transitioned from lovenox to dabigatran. At his 2-week follow-up visit, he had minimal cutaneous involvement, but no discomfort. At 1-month follow-up, the skin lesions had greatly resolved with near complete resolution.rd patients have active intestinal disease at the time of diagnosis.  Wh Wh15] WhThe literature related to apixaban-induced LCV reveals cases with comparable clinical presentations, histopathologic findings, and a relatively parallel clinical course with nearly identical outcomes. The onset of cutaneous symptoms has been previously documented in 10–28 days after initiating therapy with apixaban.  Our patiThe treatment for LCV is mainly supportive, directed towards the underlying cause, and involves cessation of the offending agent. Steroids can be considered if the disease is severe, as reported in a patient who had complete resolution of symptoms after a 5-week oral prednisone course.  In the pA special mention is the possible cross reactivity of direct oral anticoagulants (DOACs). Anis and Jandreau reported the persistence of rash in a protein C deficient patient when switching from apixaban to rivaroxaban; which eventually subsided within 24 hours of starting warfarin.  Similarl


We found a significant and heterogeneous effect of immune dysfunction on the brains of both male and female mice. However, by imaging the whole brain and using Bayesian hierarchical modelling, we were able to identify patterns within the heterogeneous phenotype. Certain structures—such as the corpus callosum, midbrain, and thalamus—were more likely to be affected by immune dysfunction. A notable brain–behaviour relationship was identified with neuroanatomy endophenotypes across mouse models clustering according to anxiety-like behaviour phenotypes reported in literature, such as altered volume in brains regions associated with promoting fear response . Interestingly, genes with preferential spatial expression in the most commonly affected regions are also associated with multiple sclerosis and other immune-mediated diseases. In total, our data suggest that the immune system modulates anxiety behaviour through well-established brain networks.Extensive evidence supports the role of the immune system in modulating brain function and behaviour. However, past studies have revealed striking heterogeneity in behavioural phenotypes produced from immune system dysfunction. Using magnetic resonance imaging, we studied the neuroanatomical differences among 11 distinct genetically modified mouse lines ( The immune system plays an important role in brain–behaviour interactions. During pregnancy, for example, maternal illness increases the risk of nervous system disorders in offspring –5 and maWhile the role of specific immune components is an area of active investigation, existing studies generally examine brain regions or immune components in isolation, thereby sacrificing the ability to observe broader patterns that may explain the observed heterogeneity. Structural magnetic resonance imaging (MRI) is a useful methodology for studying relationships between genetics, brain regions, and behaviour with several studies demonstrating a strong link between the volume of brain structures and organism behaviour , 22. As Thus, here we used whole-brain MRI to explore the effect of immune system mutations on the brains of male and female mice. We imaged the brains of 11 genetically engineered mouse lines, each deficient in different elements of the immune system, using a standard pipeline for specimen preparation, data acquisition, and image registration. Brain structure was highly susceptible to immune dysfunction and we found regional variations in this susceptibility. The most susceptible regions tended to express genes associated with immune-mediated diseases. We also identified that strains with similar anxiety-behavioural phenotypes displayed analogous neuroanatomical effects. Lastly, we identified candidate brain regions and networks that may be involved in modulating the anxiety behaviours seen in mouse models of immune dysfunction.ad libitum water and diet#2918 . All breeding colonies were maintained using homozygous mutants with two exceptions: Cxcr2 colony was maintained using heterozygous mutants due to the fragility of homozygotes [The study consisted of 371 mice  Animal Care Committee (AUP 17-0175H) in accordance with recommendations of the Canadian Council on Animal Care, the requirements under the Animals for Research Act, RSO 1980, and the TCP Committee Policies and Guidelines. As subjects were assigned to groups based on sex and strain, experimenters were not blinded to subject and subjects were not randomised.A multi-channel 7.0-T MRI scanner with a 40-cm diameter bore  was used to acquire 40-micron-isotropic images of 16 brains concurrently  paramet using a Post hoc simulations to assess power were also performed  . Post hod) distribution.Structure volumes were standardised using Z-score. An anatomical hierarchy  was used5 iterations). Similar results were seen after excluding the Cxcr2 strain and when using a bootstrap-based networking procedure [η2) distribution. Hellinger distance, η2, and d statistics are illustrated in Supplementary Fig. The posterior also provided the distribution of neuroanatomy phenotype as beta-values for each structure and strain (referenced to wild-type). Hellinger distance was used to assess the dissimilarity of neuroanatomy phenotype between each pair of strains. Strains were annotated with anxiety phenotype based on existing literature  constituted the ROI for this analysis. Preferential expression for each gene was evaluated using fold-change: defined as mean expression in ROI divided by mean expression in the brain.Using previously published methods , 44 and k-means with k = 4 determined by elbow-method [For disease enrichment and Gene Ontology (GO) enrichment analysis, all genes were part of the background set, while the target set was the top 4000 genes with the highest preferential expression. Similar results were seen with target sets of the top 3000 and 5000 genes. For disease enrichment analysis, mouse genes were annotated with human diseases using two databases: NCBI  , axon ensheathment , and myelination . Using the DisGeNet [p < 10−8, FDR < 10−4), Alzheimer’s disease , and multiple sclerosis (MS) .We sought to link the neuroanatomical findings with immune dysfunction by comparison with genome-wide gene expression patterns from the ABI database . For thiDisGeNet  and NCBIDisGeNet  databaseAs there is strong evidence that MS is an immune-mediated disease , we deciUsing neuroanatomical imaging with ex vivo MRI, we confirm heterogeneous and widespread effects of the immune system on the brain using a selection of immune-related genetically engineered mouse strains. To identify patterns underlying this heterogeneity, we studied neuroanatomical phenotypes on three scales: analysing each strain individually, analysing groups of strains unified by a common anxiety-like behavioural phenotype, and aggregating across all strains. When analysing strains individually, we found results consistent with other studies. For example, we did not find altered morphology of the dorsal hippocampus in Rag1 mutants, which is consistent with histology studies . We founAnalysis of the heterogeneous structural findings for association with behavioural phenotypes revealed important insights and suggests that neuroanatomical endophenotypes cluster significantly with anxiety-like behavioural phenotypes. Previous use of mouse models has revealed several neural circuits associated with producing anxiety behaviours , 61, butImmune dysfunction plays an important role in many neurological disorders, but the impact of the immune system is difficult to study because the aetiology is highly polygenic. For example, MS is associated with over 1000 genetic risk variants , but manThere are limitations in our work that are important to discuss. Our study focuses solely on static loss-of-function mutations in isolated immune system components, which is a simplification used throughout the existing literature –16. HoweIn summary, we imaged the neuroanatomy of 11 different mouse mutants, each deficient in particular components of the immune system. By using a consistent high-throughput protocol for data acquisition and analysis, we characterised the diverse effects of immune dysregulation on the brain and identified patterns underlying this heterogeneity. The observed neuroanatomical differences in the mouse models of immune dysfunction are clustered by their anxiety phenotype, recapitulating known brain networks implicated in modulating anxious behaviours. Exploratory analysis of gene expression shows that the brain regions that are most affected by immune dysfunction have a preferential spatial expression of genes associated with MS and other immune-mediated diseases.Supplementary FiguresSupplementary Tables


Colorectal cancer (CRC) starts as a localized tumor and becomes a systemic disease with fatal consequences. However, clonal dynamics during progression are not well understood. Here, we present various techniques to model the different stages of CRC progression. Using genetically engineered mouse models (GEMMs) or organoid transplantation, localized tumors as well as liver metastases with characteristic intra-tumor heterogeneity were generated. The optical barcoding of transplanted organoids revealed niche-dependent clonal selection, implying that distinct niche factors control clonal outgrowth.Colorectal cancer (CRC) is among the deadliest cancers worldwide, with metastasis being the main cause of patient mortality. During CRC progression the complex tumor ecosystem changes in its composition at virtually every stage. However, clonal dynamics and associated niche-dependencies at these stages are unknown. Hence, it is of importance to utilize models that faithfully recapitulate human CRC to define its clonal dynamics. We used an optical barcoding approach in mouse-derived organoids (MDOs) that revealed niche-dependent clonal selection. Our findings highlight that clonal selection is controlled by a site-specific niche, which critically contributes to cancer heterogeneity and has implications for therapeutic intervention. With a continuously rising incidence and more than 900,000 associated deaths worldwide per year, CRC is one of the main contributors to cancer related fatality . In womeBraf and Alk5 (encoding for TgfbrI). Resulting tumors were mainly located to the right-sided colon and showed similar to patients’ mucinous histopathology . T. TvillinvillinCreER transgene specifically led to recombination in intestinal enterocytes and triggered floxing of loxP flanked Apc and Trp53 (p53). The localized injection of 4-OHT into the mucosa of the colon of villinCreER Apcfl/fl p53fl/fl mice led to rapid tumor development within two weeks , the oncogene Kras (KrasG12D/+), the tumor suppressor Trp53 (p53fl/fl), and the TGF-β pathway (Alk5/Tgfbr1fl/fl) (AKPT) [We also performed intra-colonic injection of the 21 clones into the mucosa as described above to understand the clonal selection in the colon. However, in 12 mice (out of two independent experiments), no tumor establishment was detectable, showing that none of the 21 clones had the ability to grow successfully in the colon. Since parental KPN organoids showed growth in the colon C,D, we s) (AKPT) . Three rHere, we described a versatile tool kit used to establish preclinical CRC models, enabling the generation of CRCs at different stages with high relevance for the human disease. Importantly, the colonoscopy-guided mucosal injection of CRC cells, organoids, virus, or other substances is fast and leads to robust results. Notably, this procedure was performed without invasive surgical manipulation, which significantly improved animal welfare. These methods represent a highly reproducible approach to generate CRC liver metastases. Depending on the type of induction and route, the most relevant sites for primary tumors and metastasis were modeled.Apc and Trp53, showed the rapid establishment and growth of well-differentiated adenomas. Therefore, the colonoscopy-guided injection enables the generation of early-stage adenomas, invasive metastatic carcinomas, as well as different CRC subtypes. The approach described here shows a high engraftment rate with ~80% for intra-colonic injections and 100% in the intrasplenic model. Recent studies suggested the usage of NOD SCID gamma (NSG) mice to increase the success rate of organoid injections [The ability to generate primary- or secondary-site CRC in the correct anatomical location with a high tumor uptake is crucial for cancer research. Here, we described the induction of tumors harboring different genetic backgrounds, thus permitting modeling of relevant CRC subtypes. The colonoscopy-guided injection allows tumor organoids with various genetic alterations to be implanted into immunocompetent or immunodeficient mice. We used AKPT organoids which resemble the classical route of CRC or KPN organoids to model the less characterized serrated route , showingjections ; howeverHuman CRC is believed to be initiated by one mutated cell; nevertheless, the localized injection of 4-OHT will activate the Cre-mediated recombination in multiple crypts. To our knowledge, the targeting of single cells for recombination has not been tested by 4-OHT dilution to date and might be beneficial to study clonal evolution of tumors. Various technologies have been established to model CRC with localized disease. One technique that can be compared to the approach presented here is the generation of a rectal prolapse to inject organoids; notably, this application is feasible without special equipment ,39,40. HFuture technical improvements should enable the modeling of right-sided tumors with needle-guided injections. This might be realized with the use of flexible endoscopy probes and will reduce pan organ recombination effects induced by systemic tamoxifen injections. The local administration of tumor cells, organoids, or virus particles that lead to a single tumor could also be the base for further important developments. The use of specific virus particles harboring the CRISPR/Cas9 system to generate deletions and mutations of CRC driver genes could be extended for genetic screening approaches. More relevant to recapitulate the clinical setting is that a single tumor in the colon is suitable for surgical resection. Indeed, such models would resemble the human treatment regimen and enable the study of metastatic recurrence or tumor cell dormancy.Apc-deficient organoids were not able to form liver metastases, whereas organoids derived from fApcl/fl, KrasG12D/+, and Trp53fl/fl (AKP) mice or engineered organoids resembling the same genetic alterations developed metastases in one third or in one sixth of the mice after 12 or 16 weeks, respectively [Generating metastatic CRC models is crucial for our understanding of late-stage CRC, which is the main cause of CRC mortality. Tail–vein injections to generate lung metastases are established for CRC ; howeverectively . To modeectively ; howeverectively ,23. Sincectively , these sIntra-tumor heterogeneity is believed to be causative for the progression and therapy resistance of CRC ,55, whicTo understand the clonal selection during CRC progression, we employed an optical labelling system that allows longitudinal tracing of individual clones at different sites. Similar to previous studies in mouse models of pancreatic cancer, breast cancer, and squamous cell carcinoma ,57,58, wThe clonal labelling of CRC cell lines demonstrated that the positioning of a clone is more critical for its outgrowth than cell intrinsic signals ,62. ThisThe modeling approaches presented here are straightforward tools to generate CRC at various stages of progression, from adenomas to locally advanced tumors to metastasis. By utilizing these models, we demonstrate that niche-dependent clonal selection of CRC in varying TMEs has potential implication for future therapeutic strategies.


Introduction: Musculoskeletal injury (MSI) contributes to global health burdens. Effective MSI prevention is necessary. MSI risk factor screening tools can be used by employers to identify and mitigate occupational hazards. Rigorous synthesis of the effectiveness of these tools has not taken place. We synthesized literature on effectiveness of MSI risk factor screening tools for reducing injury through informing prevention interventions. Materials and Methods: A literature search of Medline, Embase, Cochrane Library , CINAHL, Scopus and PsycINFO databases was performed. Included studies required an analytic design, used an MSI risk factor screening tool to inform an intervention in a working-age population and reported an outcome of MSI development, injury or compensation/work absence. Data extraction and study quality rating (Downs and Black criteria) were completed. Studies were sub-categorized as having used a single MSI screening tool (single-tool) to inform an injury prevention intervention or involving multiple simultaneous screening tools (multiple-tool). Study outcomes were synthesized when possible. Results: Eighteen articles representing fourteen studies met our inclusion criteria. No high-quality studies were identified (maximum Downs and Black score of 19). Studies did not employ previously validated tools but instead, typically, those purpose-built for a single use. The results were inconsistent both when using tools alone and in combination with other tools. Outcome measure heterogeneity precluded meaningful meta-analysis. Conclusions: There is limited evidence regarding use of MSI risk factor screening tools for preventing injury. Rigorous studies that utilize previously validated tools are needed. Musculoskeletal injuries (MSI) are among the largest contributors to the global burden of pain, disability and work loss . The preThroughout this review, MSI risk factor screening tools are viewed in the context of informing interventions to prevent MSI and its effects. Previous research has reviewed the measurement properties of MSI risk factor screening tools, with varying reliability and validity reported . ReliabiResearch is needed to assess the current scientific literature involving evaluation of the effectiveness of MSI risk factor screening tools for informing injury prevention interventions through a rigorous epidemiological lens. This will provide critically important information regarding whether these tools can successfully be used to prevent MSI and its consequences. Therefore, our research question was: “in working age adults, what is the effectiveness of MSI risk factor screening tools for preventing onset and consequences  of work-related MSI?”This review followed Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines . The revA systematic literature search was carried out by a health sciences librarian (L.D.) in Medline via Ovid (1946–18 March 2021), Embase via Ovid (1974–18 March 2021), Scopus (searched 19 March 2021), CINAHL Plus with Full Text (via EBSCOhost) (1937–19 March 2021), Wiley Cochrane Central Register of Controlled Trials (CENTRAL) (searched 19 March 2021) and APA PsycINFO  databases. Team members collaborated to develop a sensitive search strategy that utilized two approaches: (1) searching by the names of specific tools identified in a preliminary literature review or provided by stakeholders , and (2) searching generically with combinations of subject headings and keywords pertaining to MSI, occupational settings and screening tools. The results of both approaches were limited to quantitative primary research studies only. The grey literature was not searched, which is a change from our protocol. After preliminary searching of the voluminous grey literature, it was determined that this searching would not result in rigorous evaluations, which was the focus of this study. Our definition of MSI was adapted from WorkSafeBC’s definition that encompasses injuries and disorders of muscles, tendons, ligaments, joints and soft tissues (nerve and vascular injury) . For thiA PICOS  framework was used for development of article inclusion criteria. Articles were required to have an English-language title and abstract and were eligible if they assessed:P—a working-age population in a working environment (exclusion of pre-employment screening and studies in military populations);I—applied an MSI risk factor screening tool using individual or workplace-related risk factors to prevent MSI injury and its related consequences ;C—compared to other MSI prevention strategies that were not informed by an MSI risk factor screening tool;O—reported on at least one primary outcome related to MSI development, injury or compensation/insurance claims ;S—utilized an analytic study design .We expected that MSI risk factor screening tools would affect injury outcomes by conditionally applying a specific prevention intervention based on risk levels or scores identified by the screening tool. Tool results inform implementation of a tailored intervention aimed at mitigating any identified risk factors. For example, if the tool identifies excessive physical loading as a risk, the recommendation may be to modify work tasks or activities to reduce risk or have workers undergo a fitness or strengthening program to increase their manual handling abilities. A variety of risk factor/intervention combinations are possible for both physical and mental risks at the workplace. This process is conceptualized in a logic map in We made modifications to our review protocol prior to our analysis. Specifically, to capture all potentially relevant articles, studies did not require a minimum sample size to be included. Additionally, we clarified that eligible study populations must not have been identified as injured prior to study enrolment; thus, eligible outcomes became incident MSI, compensation claims or insurance claims. Following completion of the database search, article titles and abstracts were added to online review manager Covidence  and de-dFull-text articles were independently assessed for eligibility by a smaller subset of the research team. Articles had to be deemed eligible by two team members, and disagreements at full-text stage were resolved by consensus prior to or after consulting the third team member. Articles for which consensus was not reached at the full-text stage were provided to the entire research team for discussion. Additional articles were identified directly for full-text eligibility screen through citation searching of included articles and systematic reviews identified during screening. One article was identified as a subsequent analysis of a study population from an article included at full-text stage and was retrieved for full-text eligibility screening. A standardized spreadsheet was used for data extraction of included articles. One reviewer performed the initial data extraction, with verification by a second reviewer. Extracted article data included study design, study setting and context, participant characteristics, MSI screening tool descriptions and alternative treatments of study arms, outcome measure descriptions and reported outcome results. Effect estimates were presented where possible.Included articles were synthesized depending on their method of MSI risk factor screening tool application. The first category of “single-tool” articles contains studies that, in at least one study arm, applied a single MSI screening tool to inform an intervention in isolation from any other additional screening tools, assessments or interventions. These study designs provide the most direct assessments of MSI screening tool effect. The second category of “multiple-tool” articles applied one or more MSI risk factor screening tools in combination with other assessments and interventions (that may or may not have been informed by the screening tool of interest). For this latter group of articles, it was deemed that the causal effect of any single MSI screening tool use could not be meaningfully isolated from the causal effect of distinctly separate but concurrently applied assessments and associated interventions. Consultation with community partners indicated that prevention interventions within industry contexts are most often pragmatically applied in “multiple-tool” situations. The Downs and Black (D&B) quality assessment checklist was used to assess included article quality . The quaThis review follows principles of best evidence synthesis and incorporates components of Synthesis Without Meta-analysis reporting guidelines, the latter of which is intended to complement PRISMA reporting guidelines ,18. All The initial database search yielded 12,207 results, and 4025 duplicates were removed; 8182 articles were screened for potential eligibility, of which 79 full-text articles were reviewed for inclusion. Percent agreement during abstract screening ranged from 88% to 100%, and all discrepancies were resolved through consensus. Fourteen articles met the inclusion criteria following full-text review and were included for analysis. Citation searching from the included articles, key systematic reviews and incidental related articles yielded 15 articles that were retrieved for full-text analysis. Four articles were retained from this second identification group. In total, 18 articles were included for quality assessment and data extraction. Most articles excluded at the full-text stage did not evaluate the effect of an MSI risk factor screening tool see .Article quality appraisal was applied using the Downs and Black criteria . ArticleFive single-tool articles representing four studies were identified and retained following quality assessment. All five articles were scored as medium-quality, meeting at least half of the methodological criteria ,22,23,24Thirteen multiple-tool articles were identified ,35,36,37Six articles were scored as poor quality and are not characterized in this paper beyond their quality appraisals ,32,33,34No high-quality studies are present in the analysis, and each study utilizes a different MSI risk factor screening tool. Therefore, there is insufficient evidence to determine the effect of any specific MSI risk factor screening tool on any of the previously identified outcome categories.Three medium-quality single-tool studies show either conflicting evidence ,23 or noTwo medium-quality single-tool studies show no change ,21,24 inOne medium-quality study shows no change ,24 in meOne medium-quality study shows conflicting evidence  in measuNo included single-tool studies assessed workplace-associated MSI outcomes following an MSI-risk-factor-screening-tool-guided intervention. Two medium-quality multiple-tool studies show decreases in workplace-associated MSI ,27,28,36No included single-tool studies assessed measures of claims cost following an MSI-risk-factor-screening-tool-guided intervention. One medium-quality multiple-tool study shows decreases in claims cost ,27,28 anNo included single-tool studies assessed measures of self-rated health status following an MSI-risk-factor-screening-tool-guided intervention. One medium-quality multiple-tool study shows no change  in measuThe current evidence is insufficient to characterize the effect of MSI risk factor screening tool use on relevant MSI outcomes when used by themselves. The available evidence demonstrates an inconsistent effect of screening tool use on musculoskeletal discomfort and work absence. When used in combination with other tools and interventions in the context of a broader injury prevention program, there is mixed evidence for the effect of MSI risk factor screening tools on workplace-associated MSI and claims costs. For more certain conclusions on the utility and real-world effectiveness of MSI risk factor screening tools, high-quality randomized controlled trials should be conducted examining the impact of the currently available tools on MSI injury and related outcomes. If used in workplace settings, MSI risk factor screening tools should be one component of a broader MSI risk mitigation strategy.This study utilized rigorous epidemiological data synthesis methods to assess the current state of the scientific literature regarding the effect of using MSI risk factor screening tools to inform injury prevention interventions on important outcomes, such as MSI and related claims and resource utilization. In total, 18 articles representing 14 studies met the article inclusion criteria dictated in the final protocol. Of these 18 articles, only 12 met the minimum quality criteria for retention in the literature synthesis. Of these twelve articles, only five—representing four studies and containing no overlap in screening tools used—used an MSI risk factor screening tool to guide an intervention in a manner that enabled meaningful isolation of the effect of the tool as compared to the effect of other distinctly separate but concurrent tools and interventions. Despite plausible isolation of the effects of these remaining screening tools, outcome measures were too heterogeneous to allow effect size data pooling; rather, the highest level of evidence that could be gleaned from the current literature is, overall, whether screening tools were or were not associated with a positive health effect for specified outcome measure categories.There are numerous supplementary findings from this systematic review. First, none of the named tools from the preliminary database search that were identified as commonly used  were found to have been evaluated rigorously beyond their own validation studies. This literature shows that, instead, MSI risk factor screening tools are, in practice, typically purpose-built or adopted from local occupational health centres. Occupational health and safety professionals designing these novel tools would see minimal examples supporting use of specific screening tools in the literature and instead may base their designs on international standards for biomechanical risk factors, which themselves are not definitively robust . Any docSecond, there is a distinct difference between the characteristics of single-tool and multiple-tool studies, the former group requiring that the effect of a single tool be identifiable. Notably, the selected study sample in multiple-tool studies was more consistently representative of its source population. These studies used a more pragmatic approach to screening and intervention and may better reflect actual practice, where, often, numerous assessment tools and potential interventions are simultaneously introduced in an attempt to improve some aspect of MSI. One conclusion from this finding is that MSI risk factor screening tool use is commonly only one component of a broader MSI risk mitigation strategy. It remains unclear how the effect of MSI screening tools changes with different types of concurrent interventions. This is an additional research avenue made clear from the results of the current systematic review.This study provides, to the authors’ knowledge, the first systematic review specifically assessing the effects of MSI risk factor screening tools in actual work environments for informing MSI prevention programs. The strengths of the study included use of a robust database search strategy created through collaboration with an experienced health sciences librarian, use of up-to-date guidelines on systematic review structure and reporting and involvement of multiple stakeholder groups to provide guidance on practical needs of the occupational health and safety industry. Previous research synthesis has focused instead on the variety of available MSI risk factor screening tools , the effOverall, there is a small quantity of insufficient research and limited evidence regarding use of MSI risk factor screening tools for informing injury prevention interventions. For more certain conclusions on the utility and effectiveness of MSI risk factor screening tools, high-quality research on the currently available tools is necessary.


F-statistic for each interval of the temporal kinematic waveform was compared to the critical value using a variance equality test to identify significant differences in the waveform. Comparative tests between two types of gait terminations found that subjects may exhibit greater kinematics variance in most lower limb joints during UGT caused by unexpected stimulation . Significant greater variances during PGT were exhibited only in the MPJ sagittal and frontal planes at the early stimulus delay phase (4-15% and 1-15%). This recorded dataset of temporal kinematic changes caused by unexpected stimuli during gait termination is essential for interpreting lower limb biomechanical function and injury prediction in relation to UGT. Given the complexity of the gait termination task, which involves both internal and external variability, the variance equality test can be used as a valuable method to compare temporal differences in the variability of biomechanical variables.The efficacy of the variance equality test in steady-state gait analysis is well documented; however, temporal information on where differences in variability occur during gait subtasks, especially during gait termination caused by unexpected stimulation, is poorly understood. Therefore, the purpose of the current study was to further verify the efficacy of the waveform-level variance equality test in gait subtasks by comparing temporal kinematical variability between planned gait termination (PGT) and unplanned gait termination (UGT) caused by unexpected stimulation. Thirty-two asymptomatic male subjects were recruited to participate in the study. A Vicon motion capture system was utilized to measure lower extremity kinematics during gait termination tasks with and without unexpected stimulation conditions. The  Gait is the essential behavioral characteristic for quadrupeds represented by humans to move on solid substrates. Since this process involves a complex interplay of the nervous, musculoskeletal, and cardiovascular systems, any disorder of body function may affect gait performance . By quanSimilar to physiological signals, most of the measured values in gait analysis  are not constants but fluctuate over time, varying from one stride to another . Due to Gait variability measures have been considered better predictors of decreased mobility than absolute gait measures  , 10–12. Conventional gait tasks, such as walking and running, are continuous from initiation to termination, with the essential requirement to complete body movement while maintaining stability –17. The However, few studies estimate the variability of biomechanical variables during the two types mentioned above of gait termination. In particular, there is a lack of measurements that provide temporal information about where variability differences occur. Meanwhile, more information was required to verify further the efficacy of the waveform-level variance equality test in gait subtasks represented by gait termination. Given the research interest in temporal information on gait variability, the current study aimed to investigate temporal differences in group variance along the lower extremity kinematics waveform during PGT and UGT caused by unexpected stimulation through a waveform-level variance equality test. Combined with the importance of hip/knee extension and flexion strategies in stabilizing and performing stopping tasks during UGT , we hypo2. The inclusion criteria were (i) adult male with dominant right leg; (ii) no musculoskeletal disorders that could potentially affect gait performance; (iii) no prior history of lower limb injuries or surgeries were reported in the first half of the year preceding data collection. Before the experiment, all subjects understood the study aim, requirements, and protocol and were thoroughly familiar with the procedures of experimental data collection. The Ethics Committee from the University (RAGH20201218) approved the study, and a written consent form was obtained from all individuals before participation.Thirty-two participants were recruited to participate in this study, with ages of 23.63 ± 1.39 years, heights of 177.38 ± 5. 49 cm, weights of 67.97 ± 6.66 kg, and BMIs of 21.59 ± 1.70 kg/mEach subject was instructed to perform gait termination trials randomly under two conditions: PGT and UGT, in the motion capture laboratory with normal indoor temperature and light. The gait trial protocol was consistent with the previously reported experimental procedure , 26. BefDuring the PGT trials, subjects were asked to walk barefoot along a 20 m walkway surrounded by eight infrared cameras. The gait activity was stopped when the participants reached a specific area within the walkway. Termination was established once the subject brought their feet together. For the UGT trials, subjects were not told beforehand which walkway area to stop in. Instead, they were given a termination signal during walking. The staff sent the termination instruction to the subjects by randomly ringing a bell. Participants needed to stop once they received the auditory signal delivered when their dominant foot stepped onto the walkway UGT area. To minimize the influences of anticipated sensory cues, twenty-five per cent of trials involved the termination signal, but the other seventy-five per cent did not. Each subject was required to provide six successful gait trial datasets, including three PGT and three UGT trials.Kinematic data of the dominant lower extremity (right limb) joints was unilaterally measured via a three-dimensional motion capture system  at a frequency of 200 Hz. Twenty-five reflective markers with a diameter of 9.0 mm were placed in each subject wearing tight-fitting pants to define the anatomical coordinate system and the center of the lower limb joint, including the hip, knee, ankle, and metatarsophalangeal joint. Based on a previous study , the pelExperimental data of each gait termination trail during the stance phase were processed and analyzed in Visual 3D software  from C3D files created by Vicon Nexus Software . A second-order low-pass Butterworth filter filtered the trajectory of reflective markers with a cut-off frequency of 10 Hz . The joiF-test to compare group variances across an entire waveform [Temporal differences in group variance along the lower extremity kinematics waveform during PGT and UGT caused by unexpected stimulation were investigated in this study. Due to the one-dimensional time-varying characteristic of the lower limb kinematics data, statistical analysis applied a waveform-level variance equality test proposed by Kowalski et al. . Based owaveform , 29.F-statistic was calculated by dividing the group with the bigger variance by the group with the smaller variance, forcing the F-test into a right-tailed test [F-critical was calculated as an inverse survival function of the F distribution with alpha (0.05), degrees of freedom, the number of discrete field nodes, and the field smoothness as inputs by the one-dimensional random field theory [The led test . F-critid theory , 30. AllKnee angle curves and waveform variances in three motion planes during gait terminations are reported in MPJ angle curves and waveform variances in sagittal, frontal, and transverse planes during two types of gait terminations are reported in Investigating the temporal kinematic differences between the two gait terminations might provide further insight into the biomechanical mechanisms of stopping gait induced by unplanned stimuli. The primary objective of this study was to explore temporal differences in group variance along the kinematics waveform of lower extremity joints during PGT and UGT caused by unexpected stimulation through the waveform-level variance equality test. In general, our hypotheses were partially correct, as some subjects exhibited greater kinematic variances in most lower limb joints  during UGT caused by unexpected stimulation . In contrast, significantly greater variances during PGT have been exhibited only in the MPJ sagittal and frontal planes at the early stimulus delay phase.Previous findings , 31 suggAs a multisegmental system, the movements of the hip, knee, and ankle joints are linked with the lower extremity kinetic chain , 34. In Considering that gait termination may be challenging for the elderly and patients with balance disorders –22, 36, While acknowledging the results of this study, some limitations should be considered. Firstly, this study only compared the variability in the kinematics of lower limb joints and did not analyze the intrinsic kinematics or even the joint kinetics. Secondly, in the present study, all subjects were healthy young males, which resulted from the motivation to alleviate gender- and age-related differences in locomotion function. Lastly, the validity of the waveform-level variance equality test may not be fully verified by the results alone.The sources of variability in gait measurement can be divided between the individual internal and environmental external . Gait te


What are the brain network features of apathy in late-life depression, and how are they associated with treatment outcomes?In this secondary analysis of a 12-week nonrandomized single-group trial of escitalopram titrated to 20 mg/d, compared with 20 older adults with depression without apathy, 20 older adults with depression and apathy showed distinct alterations in network connectivity among circuits that support goal-directed behavior. Apathy-related variability in functional connectivity was associated with poor antidepressant response and persistent cognitive dysfunction after escitalopram treatment.This study suggests that, among older adults with depression, distinct network abnormalities may be associated with apathy and poor response to first-line pharmacotherapy and may serve as promising targets for novel interventions. Apathy is prevalent among individuals with late-life depression and is associated with poor response to pharmacotherapy, including chronicity and disability. Elucidating brain networks associated with apathy and poor treatment outcomes can inform intervention development.To assess the brain network features of apathy among individuals with late-life depression and identify brain network abnormalities associated with poor antidepressant response.This secondary analysis of a single-group, open-label nonrandomized clinical trial of escitalopram conducted at an outpatient geriatric psychiatry clinic enrolled 40 adults aged 59 to 85 years with major depressive disorder from July 1, 2012, to July 31, 2019.After a 2-week washout period, participants received escitalopram titrated to a target of 20 mg/d for 12 weeks.Baseline and posttreatment magnetic resonance imaging (MRI), clinical, and cognitive assessments were conducted. Functional MRI was used to map group differences in resting state functional connectivity (rsFC) of the salience network, and diffusion MRI connectometry was performed to evaluate pathway-level disruptions in structural connectivity. The Apathy Evaluation Scale was used to quantify apathy, and the Hamilton Depression Rating Scale (HAM-D) was used to quantify the primary outcome of depression severity.z score >2.7; Bonferroni-corrected threshold of P < .0125). Compared with participants without apathy, those with apathy had lower structural connectivity in the splenium, cingulum, and fronto-occipital fasciculus . Twenty-seven participants completed escitalopram treatment; 16 (59%) achieved remission (HAM-D score <10). Lower insula-DLPFC/midcingulate cortex rsFC was associated with less symptomatic improvement (HAM-D % change)  and a higher likelihood of nonremission  after treatment and, in regression models, was a mediator of the association between baseline apathy and persistence of depression. Lower dorsal anterior cingulate-DLPFC/paracentral rsFC was associated with residual cognitive difficulties on measures of attention  and executive function .Forty participants  with depression were included; 20 participants (50%) also had apathy. Relative to nonapathetic participants with depression, those with depression and apathy had lower rsFC of salience network seeds with the dorsolateral prefrontal cortex (DLPFC), premotor cortex, midcingulate cortex, and paracentral lobule and greater rsFC with the lateral temporal cortex and temporal pole (This study suggests that disturbances in connectivity between the salience network and other large-scale networks that support goal-directed behavior may give rise to apathy and may be associated with poor response of late-life depression to antidepressant pharmacotherapy. These network disturbances may serve as targets for novel interventions.NCT01728194ClinicalTrials.gov Identifier:  This secondary analysis of a nonrandomized clinical trial assesses the brain network features of apathy among individuals with late-life depression and identifies brain network abnormalities associated with poor antidepressant response. One-third to one-half of patients with LLD also have apathy,3 a persistent and disabling syndrome of impaired motivation characterized by reduced goal-directed behavior, emotional blunting, and cognitive weaknesses.4 Apathy worsens clinical outcomes in LLD. Apathy results in social isolation, poor self-care, and sedentary behavior, with broad health consequences.10 Relative to older adults with depression without apathy, those with comorbid apathy are far more likely to withdraw from medical care and have a more severe clinical course, higher caregiver burden, and functional decline.7Late-life depression (LLD) is a leading cause of disability11 Apathy is associated with poor response of depression to selective serotonin reuptake inhibitors (SSRIs), the first-line pharmacotherapy for LLD, and efficacious nonpharmacologic treatment alternatives for this patient population are scarce.14 Despite the high prevalence and clinical impact of apathy among individuals with depression, little is known about its optimal treatment and, more broadly, about the brain-based mechanisms of apathy.Remission rates of LLD are especially low among those with prominent apathy.16 The SN, which includes the insula and dorsal anterior cingulate cortex (dACC), attributes motivational value to a stimulus. The SN dynamically coordinates the activity of other large-scale networks, including the executive control network and default mode network (DMN), to guide purposeful behavior.18 The few magnetic resonance imaging (MRI) studies of apathy in depression report reduced volume in structures of the SN and preliminary evidence for disruption in functional connectivity among the SN, DMN, and executive control network.20 Abnormalities within the SN may also predispose older adults with depression to poor response to traditional pharmacotherapies.3 However, the network mechanisms linking apathy to poor antidepressant response in LLD are not well understood.An emerging hypothesis suggests that apathy and its impact on treatment response in LLD may reflect compromise of the salience network (SN) and its large-scale network connections.21 Connectometry is less susceptible to crossing fibers and partial volume effects and offers greater localization of microstructural abnormalities compared with conventional tract-based analysis25 but, to our knowledge, has not yet been applied to the study of apathy. Moreover, connectometry can be used alongside resting state imaging to identify network properties of apathy in major depression.Identifying network abnormalities in apathy is crucial for understanding its behavioral manifestation in individuals with LLD and for developing new strategies of treatment in clinical practice. Connectometry is a novel approach to diffusion MRI analysis that quantifies the local connectome of white matter pathways.Our primary objective was to evaluate functional connectivity and structural network disturbances associated with apathy in older adults with LLD. Informed by our conceptual framework, we focused on functional connectivity of the SN and hypothesized alterations in connectivity among key nodes of the SN and other core circuits that modulate goal-directed behavior  among individuals with depression and apathy. We further applied connectometry to identify pathway-level disruptions in structural connectivity and hypothesized that compromise of frontoparietal and frontolimbic pathways would be associated with apathy in LLD. Our secondary objective was to evaluate whether apathy-related network abnormalities were associated with antidepressant response after 12 weeks of pharmacotherapy with the SSRI escitalopram. Therefore, we assessed whether pretreatment variability in connectivity associated with apathy was associated with mood and cognitive outcomes after antidepressant treatment.Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) (DSM-IV) criteria for major depressive disorder without psychotic features. Participants were recruited for a single-group, open-label escitalopram treatment trial (NCT01728194) through advertisements and clinical referrals. Participants provided written informed consent approved by the Weill Cornell Medicine and Nathan Kline Institute institutional review boards. Exclusion criteria were current or lifetime history of any Axis I psychiatric disorder other than major depressive disorder or comorbid generalized anxiety disorder, high suicide risk, history of electroconvulsive therapy, ongoing treatment with medications associated with depression (eMethods in TREND) reporting guideline for nonrandomized clinical trials.26The study, conducted at an outpatient geriatric psychiatry clinic from July 1, 2012, to July 31, 2019, included participants aged 59 to 85 years who met DSM-IV diagnosis was assigned by a study clinician based on the Structured Clinical Interview for DSM–Revised,27 and inclusion required a Hamilton Depression Rating Scale (HAM-D) score of 18 or more .28 Mild cognitive impairment was assessed using the Petersen criteria29 and determined using a previously described classification procedure.30 Although the primary outcome for the main clinical trial was the Montgomery-Asberg Depression Rating Scale score, several items capture core features of apathy .32 Therefore, the HAM-D score was chosen as the primary depression outcome in the secondary analysis presented here.A 35 a psychometrically validated instrument consisting of 18 items rated on a Likert scale.37 Total scores range from 18 to 72, with higher scores corresponding to more severe symptoms. An examination of the psychometric properties of the Apathy Evaluation Scale found that cutoff values greater than 40.5 and 37.533 had similar sensitivity (88%), although a higher cutoff value had better specificity.34 Accordingly, an Apathy Evaluation Scale score of more than 40.5 was considered a priori to represent clinically significant apathy.35Apathy was evaluated using the clinician-rated version of the Apathy Evaluation Scale,38 and Trail Making Test Part B − A) because they are commonly impaired among individuals with LLD and deficits often persist after antidepressant treatment.40A focused neuropsychological assessment conducted at baseline and after treatment included measures of processing speed, attention, memory, language, and executive functioning. This study focused on measures of attention  and executive function  to account for the number of seeds analyzed (n = 4).A hypothesis-driven seed-to-whole-brain approach was used to test for group differences in resting state functional connectivity (rsFC) of the SN between participants with depression and apathy and those with depression without apathy. The left and right insula and dACC—core SN hubs—were used as a priori seed regions generated from the Human Connectome Project–derived Glasser Parcellation Atlas.21 was applied to derive the correlational tractography showing an association between quantitative anisotropy and apathy.50 Connectometry measures the degree of connectivity between adjacent voxels based on the density of diffusing spins and probes group differences within subcomponents of white matter pathways.21 The tensor metrics were calculated, and quantitative anisotropy was extracted as the local connectome fingerprint.51 A t score threshold of 2.5 was assigned and tracked using a deterministic fiber tracking algorithm,52 and connectometry was performed across the whole brain, excluding the cerebellum.54 To estimate the false discovery rate (FDR), 10 000 randomized permutations were applied to the group label to obtain the null distribution of the track length. An FDR threshold of less than .05 was used to correct for multiple comparisons.52Diffusion connectometryt test, the Mann-Whitney test, and the χ2 test were used to identify individual demographic and clinical variables that distinguished participants with depression without apathy from those with depression and apathy. β Weights and quantitative anisotropy values were extracted from significant clusters and fascicles that differed between groups in rsFC and connectometry analyses, respectively, to evaluate associations with clinical outcomes. Change in mood (HAM-D score) was the primary outcome, and change in attention and executive function served as secondary outcomes. Linear and logistic regression models were generated to evaluate whether group differences in baseline connectivity were associated with symptom change (percentage change in HAM-D score ) and remission of depression (HAM-D score <10),55 adjusting for age and baseline depression severity. Secondary linear regression models assessed the associations between baseline connectivity and change in performance (posttreatment [T2] − baseline [T1]) on measures of attention (Digit Span Forward) and executive functioning (Trail Making Test Part B-A and Stroop Interference), adjusting for age, educational level, baseline depression severity (HAM-D score), and baseline task performance.The 56 to assess whether the association between presence of apathy and antidepressant response was mediated by apathy-associated abnormalities in connectivity. To estimate the indirect effect, bootstrapping (k = 5000 samples) was used to generate 95% CIs. Mediation results are considered significant if the 95% CI for the indirect path does not contain zero.57 Mediation models were fit in PROCESS, version 3.4.157 for SPSS, version 26 (IBM SPSS).In models with significant associations between baseline connectivity and treatment response, exploratory post hoc mediation analyses were performedForty older adults  with major depressive disorder were evaluated, of whom 20 had apathy at the prespecified threshold . Groups did not differ significantly in age, sex, educational level, or baseline depression severity .z score = 2.82; Bonferroni-corrected threshold of P < .0125)  , midcingulate cortex (MCC), and premotor cortex (mean < .0125) . Using tz score = 3.0; Bonferroni-corrected threshold of P < .0125) . There were no group differences in connectivity using the left dACC seed.Participants with depression and apathy showed lower rsFC between the right dACC seed and the right DLPFC, paracentral lobule (PCL), and anterior premotor cortex compared with participants with depression alone   in the splenium of the corpus callosum, cingulum, and left inferior fronto-occipital fasciculus among participants with apathy compared with nonapathetic participants    achieved remission (HAM-D score <10). Adjusting for age and baseline depression severity, lower insula-DLPFC/MCC connectivity at baseline was associated with less reduction in depressive symptoms (percentage change in HAM-D score) (β [ = .001)  and a hiP = .04) .df] = 0.445 [26]; P = .04) and executive function  (Stroop Interference), while greater connectivity was associated with greater improvement in performance after treatment  on measures of attention . There was an association between apathy and insula-DLPFC/MCC connectivity , which was expected as connectivity was defined by its association with apathy in the baseline sample. There was also a significant indirect effect of insula connectivity, suggesting that group differences in insula-DLPFC/MCC connectivity mediated the association between apathy and treatment response . Path coefficients indicate that baseline apathy was associated with lower insula-DLPFC/MCC connectivity which, in turn, was associated with poorer antidepressant response  after treatment  and compromised structural connectivity in a core set of brain circuits. Moreover, apathy-related alterations in rsFC were associated with reduced symptomatic improvement and a lower likelihood of remission of depression, as well as persistent cognitive dysfunction, after 12 weeks of treatment with escitalopram.60 Evidence for this model has been derived largely from studies of neurodegenerative disorders and stroke,61 leaving the mechanisms underlying motivational disturbances among individuals with depression poorly understood.These findings support an emerging model of apathy, which proposes that apathy may arise from dysfunctional interactions among core networks  that support motivated behavior.62 and sends signals to primary and secondary motor cortices to facilitate action initiation.64 Functional disconnection between the SN and DLPFC may indicate reduced synchrony of systems supporting attentional control, working memory, and cognitive flexibility–core processes disrupted in apathy.65 Moreover, altered communication between the SN and motor systems may result in difficulties translating intention into action.66 These observations converge with reports in healthy adults showing associations between disturbances in rsFC between the SN and executive control network and reduced motivation,68 as well as work linking lower rsFC between the dACC and supplementary motor area to apathy among community-dwelling adults.66In a sample of older adults with LLD, those with comorbid apathy showed reduced rsFC between the SN and the DLPFC, a key node of the executive control network, as well as the premotor cortex and PCL. The SN communicates with the executive control network to mobilize the cognitive resources required for goal-directed behavior18 Increased rsFC with the SN may reflect difficulty modulating or disengaging the DMN, affecting the ability to generate cognitive strategies necessary for motivated behavior while ignoring irrelevant or internally directed stimuli. Consistent with this model, failure to suppress the DMN is associated with lapses in attention and decreased cognitive control performance,69 core features associated with apathy.70 These findings also align with a prior preliminary study showing increased rsFC between the insula and nodes of the DMN among participants with depression and apathy relative to healthy older adults.5Participants with apathy and depression also showed higher rsFC between SN seeds and the dorsal medial subsystem of the DMN. In response to salient stimuli, the SN generates control signals that downregulate the DMN in favor of task-positive networks, such as the executive control network.21 Participants with comorbid apathy showed lower structural connectivity in the splenium, left inferior fronto-occipital fasciculus, and cingulum than those without apathy. The cingulum enables communication between the cingulate cortex, parietal, and medial temporal regions,71 and integrity of this pathway has been associated with processes disrupted in apathy,72 including affective processing, decision-making, and behavioral initiation.69 The fronto-occipital fasciculus connects frontal, insular, occipital, and parietal regions,74 and the splenium is a commissural tract that connects the posterior parietal, temporal, and occipital cortices. Efficient transmission of sensory information to the SN is needed to identify salient stimuli and guide goal-directed behavior.62 Microstructural abnormalities in these pathways may reduce interhemispheric communication and the integration of sensory signals with frontal and insular structures.73 Thus, along with frontolimbic fibers, compromise of pathways that facilitate the transmission of sensory signals for salience processing may be associated with the apathy trait.Along with functional network abnormalities, we applied diffusion connectometry to evaluate pathway-level disruptions in structural connectivity associated with apathy.18 leading to difficulties with salience processing, action planning, and behavioral initiation that manifests clinically as apathy.More broadly, these findings collectively suggest that apathy among individuals with LLD may be associated with disturbances in SN modulation of large-scale circuits (DMN and executive control network) to facilitate goal-directed behavior. This may cause a failure of network integration,Select circuit abnormalities associated with apathy at baseline were also associated with individual differences in response to an SSRI treatment trial. Specifically, low pretreatment insula-DLPFC/MCC connectivity was associated with reduced response and remission of depression, while low dACC-DLPFC/PCL connectivity was associated with residual cognitive difficulties on measures of attention and executive function. In exploratory analyses, lower insula-DLPFC/MCC connectivity mediated the association between presence of apathy and poor antidepressant response. Thus, apathy-related variability in rsFC may predispose older adults with depression to persistent mood and cognitive deficits after traditional antidepressant pharmacotherapy. These data suggest that the effectiveness of SSRI treatment in LLD may rely on an intact ability to detect and transmit signals of salience, and highlight SN-executive control network connectivity as a potential therapeutic target for novel interventions.4 will increase confidence in our results. There are also opportunities to optimize our imaging approach. The functional MRI scan was relatively short based on current standards, and a longer multiband acquisition would improve the precision of signal estimation. For cluster-based analyses, we applied a threshold commonly used in clinical trials and studies with modest sample sizes,48 although a more conservative threshold would further reduce the risk of type I error. We applied a hypothesis-driven, seed-to-whole-brain rsFC approach focused on the SN. Future studies evaluating other networks that may be disrupted in apathy, including the reward network,58 will provide further insights into its etiopathogenesis.This study has several limitations. There was no longitudinal follow-up after acute treatment and a relatively limited neuropsychological battery, so we cannot establish the persistence of treatment differences nor the specificity of cognitive associations. Although we attempted to exclude participants with mild cognitive impairment, it is possible that some participants may have had mild cognitive impairment that was not detected by our evaluation process. In addition, the study sample size is relatively small and was reduced at follow-up. As such, the longitudinal findings, including the post hoc mediation analyses, should be considered preliminary. Follow-up studies with larger samples using multiple cognitive measures and a more comprehensive diagnostic assessment of apathyThis secondary analysis of a nonrandomized clinical trial suggests that disturbances in connectivity among a core set of brain networks that support motivated behavior  may give rise to apathy and predispose depressed older adults to poor response to first-line pharmacotherapies. These findings contribute to our understanding of the functional neuroanatomy of motivational disturbances in depression and the network pathways linking apathy to poor clinical outcomes in LLD. Novel interventions that modulate interactions among affected circuits may help to improve clinical outcomes in this distinct subgroup of older adults with depression, for whom few effective treatments exist.