Acute pain management is a challenging area encountered by inpatient clinicians every day. While patient care is increasingly complex and costly in this realm, the availability of applicable specialists is waning. This narrative review seeks to support diverse hospital-based healthcare providers in refining and updating their acute pain management knowledge base through clinical pearls and point-of-care resources. Practical guidance is provided for the design and adjustment of inpatient multimodal analgesic regimens, including conventional and burgeoning non-opioid and opioid therapies. The importance of customized care plans for patients with preexisting opioid tolerance, chronic pain, or opioid use disorder is emphasized, and current recommendations for inpatient management of associated chronic therapies are discussed. References to best available guidelines and literature are offered for further exploration. Improved clinician attention and more developed skill sets related to acute pain management could significantly benefit hospitalized patient outcomes and healthcare resource utilization. Acute pain plagues the majority of hospitalized patients at some point during their clinical course . DespiteUncontrolled pain worsens patient outcomes and healthcare costs—adopt a consistent, systematic, and holistic approach to acute pain management.Not every patient is an ideal candidate for every medication, but every patient in pain is a candidate for multimodal analgesia optimization.There are tremendous benefits to employing anti-inflammatories and few good reasons to withhold them in the management of acute pain.Gabapentinoids have a complex risk/benefit ratio and decision-making should be nuanced.Low-dose ketamine is a powerful analgesic even in opioid-tolerant patients and is generally well-tolerated with appropriate institutional protocols.Empiric opioid regimens should include consideration of optimal agent selection, dosing, route of administration, and supportive therapies.Pain regimens should be evaluated and adjusted at least daily through multidimensional pain assessments to optimize efficacy and safety endpoints.Patients with opioid tolerance, chronic pain, and/or opioid use disorder require higher opioid doses and more supportive therapies.When used for acute pain, patient-specific plans for opioid tapering and harm reduction should be developed and supported across the care continuum.Methadone and buprenorphine should almost always be continued throughout acute pain episodes, but naltrexone must be stopped.We therefore underscore acute pain management as a necessary function of inpatient providers and seek to support diverse practitioners in updating or refining applicable knowledge and skills in this realm. Herein, the interprofessional collaborators have provided our “top ten” recommended clinical pearls for acute pain management, with each including discussion, key considerations, and visual summaries of recommendations. Our specific aim is to provide a focused narrative review and actionable point-of-care resource for acute pain management to inpatient providers. The ten clinical pearls discussed herein include:The reader is referred elsewhere for comprehensive reviews and applied examples of foundational analgesia resources, including equianalgesic opioid calculations and types of pain  ,12.While pain management may frequently be associated with patient perception surveys in the minds of hospital-based practitioners, its importance to patient outcomes extends far beyond satisfaction with care delivery. Uncontrolled acute pain triggers a complex neurohormonal cascade that is toxic to nearly every organ system, as evidenced by increased rates of renal and gastrointestinal dysfunction, infection, cardiopulmonary and thrombotic complications, impaired wound healing, adverse psychological effects, and poorer functional recovery and quality of life ,4,13. FuWhile opioids have an important role in acute (and chronic) pain management, they carry many toxicity risks, including pruritus, nausea and vomiting, constipation, ileus, urinary retention, delirium, sedation and respiratory depression. Such opioid-related adverse events (ORAEs) confer longer lengths of stay and significant economic burden ,16. OpioIndiscriminate opioid prescribing has been criticized as fueling the modern U.S. opioid epidemic, yet many hospitalized patients report inadequate pain relief, signaling both overreliance on opioids and suboptimal pain management strategies by healthcare providers ,19,20,21KEY CONSIDERATION: Opioid avoidance in acute painful conditions is an ill-advised goal as it is neither patient-centered nor evidenced-based; rather, multimodal analgesia and opioid stewardship should integrate into a patient-specific, data-driven approach to inpatient acute pain management.We therefore recommend all hospital-based healthcare providers adopt a consistent, systematic, and holistic approach to acute pain management as a daily patient care function, as depicted in not “as needed”), nonopioid analgesics of different classes in addition to any opioid therapies.The most effective strategy for improving pain control and decreasing adverse medication events in this setting is multimodal analgesia. Such “rational polypharmacy” maximizes benefit while decreasing risk by combining lower doses of multiple medications with complementary mechanisms of action ,25,26,27This recommendation should not be conflated with one of prescribing all available nonopioids to patients indiscriminately or in an overly protocolized manner, however. For example, acetaminophen should form the backbone of most acute pain regimens due to its unique mechanism and wide safety margin, even in patients with chronic liver disease . While iKEY CONSIDERATION: Multimodal analgesia should be balanced and thoughtful, utilizing complementary mechanisms to improve pain control with less high-risk drug exposure, as opposed to overmedicating without regard to cumulative risks or patient-specific factors.AcetaminophenAnti-inflammatoriesNeuropathic agents- gabapentinoids, serotonin reuptake inhibitors, anticonvulsantsCorticosteroidsNMDA antagonistsCentral alpha adrenergic agonistsSystemic anesthetics Topical agents- lidocaine, anti-inflammatories, capsaicinRegional anesthetic modalities in concert with a specialist pain service Physical therapy, cognitive/behavioral therapies, thermotherapies, and other nonpharmacologic modalitiesMultimodal analgesia should utilize modest to moderate doses of however-many agents are sensible for the particular patient at that time, within the scope of available institutional protocols to ensure safe and meaningful use. Providers should consider pain features, risk factors for adverse events, drug interactions, and the global clinical status of the patient when initiating and adjusting multimodal analgesic regimens. At the institutional level, interprofessional stakeholders should proactively engage to create order sets or “pain management menus” that provide decision-support and cohesive operationalizing of a variety of multimodal modalities ,22. ThesThere are multiple non-pharmacological therapies that can complement medication therapies. Three examples of complementary therapies with increasing evidence and uptake into inpatient practice include massage therapy, acupuncture, and music therapy. Massage therapy has been vigorously studied in regard to cancer pain, but the tenets can be applied to acute pain management. Therapeutic massage may increase blood and lymphatic circulation, decrease inflammation and edema, relax muscles, increase dopamine and serotonin levels, and reduce levels of anxiety, depression, anger and fear ,32. AcupNon-steroidal anti-inflammatory drugs (NSAIDs), including selective COX-2 enzyme inhibitors like celecoxib, exert a potent analgesic effect in acute painful states that outperforms that of opioids when compared directly in randomized, double-blind trials . This isKEY CONSIDERATION: NSAIDs target an important source of pain instead of just interfering with the perception of pain, making them among the most effective and important analgesics available in acute pain management.While many dogmatic safety concerns and perceptions exist with NSAIDs, most of these are no longer supported by published evidence and/or do not preclude the short-term use of NSAIDs to treat acute pain in hospitalized patients . ImportaGabapentinoids  are some of the most widely prescribed medications in the United States especially in the perioperative setting, where opioid sparing regimens as part of enhanced recovery after surgery (ERAS) protocols have become the standard of care . The incWhen considering a gabapentinoid for your patient, it is important to understand their pharmacology and metabolism. Gabapentinoids exert their analgesic effect by binding to the alpha-2-delta subunit of N-type voltage-gated calcium channels in the central nervous system, decreasing the influx of calcium through the channels, and subsequently reducing the outflow of excitatory neurotransmitters to mitigate neuropathic pain ,76. The KEY CONSIDERATION: Gabapentanioids are heavily renally eliminated and require dose adjustment in altered kidney function. Patients undergoing dialysis who are apt to miss appointments or who are unable to tolerate a complete hemodialysis session are at risk for drug accumulation and adverse events.The side effect risks of gabapentinoids were likely underrecognized in initial studies supporting their use in enhanced recovery protocols. Because of binding to the alpha-2-delta subunit of N-type calcium channels, which are widely expressed in the hippocampus and cerebellum, gabapentinoids cause dizziness, balance disorders, ataxia, visual disturbances, sedation, somnolence, and cognitive impairment. Gabapentinoids in combination with other central nervous system (CNS) depressants, such as opioids, have an increased risk of noninvasive ventilation and naloxone use in the perioperative setting . In the KEY CONSIDERATION: Gabapentinoids are narcotics with dose-dependent adverse effects, which may include respiratory depression, especially with concomitant CNS depressants or in high-risk patients.We therefore recommend, when considering a gabapentinoid as part of a multimodal analgesic regimen, careful assessment of patient risk factors for respiratory depression and cumulative exposure to CNS depressants. A “low and slow” approach is also essential to safe and successful use, as adverse event risks are dose-dependent and modified by tolerance. We recommend initiating gabapentin at no more than 300–600 mg per day in gabapentinoid-naïve patients, given in divided doses , with further downward adjustment in patients of advanced age or renal impairment. Hold parameters for the aforementioned adverse events should also be incorporated into medication orders as appropriate.Ketamine is an NMDA antagonist with a host of other pharmacologic properties, resulting in a wide range of effects across its dose–response curve. Ketamine’s complex pharmacology has led to its exploration in a wide variety of therapeutic and psychotropic uses across multiple decades, though its misuse or suboptimal prescribing can lead to significant adverse events. While it has traditionally been used in higher dose ranges  for general anesthesia or tranquilization in acute agitation, ketamine exerts a powerful analgesic effect at low doses  without conferring respiratory depression, making it an attractive pain management modality ,83,84,85KEY CONSIDERATION: Among available nonopioid analgesics, ketamine has the strongest evidence supporting improved pain control and opioid-sparing properties in patients with preexisting opioid tolerance. It may also interrupt the pathological processes of central sensitization and opioid-induced hyperalgesia, conferring benefit in severe or difficult-to-treat pain.Low-dose ketamine, also referred to as analgesic-dose or subdissociative ketamine, has been found to be safe and effective for acute pain in inpatient populations. Randomized controlled trials in the emergency department and postoperative wards have suggested analgesic effectiveness and tolerability comparable to morphine, in addition to opioid-sparing effects ,91,92. PKEY CONSIDERATION: Collaborative, evidence-based institutional protocols and prescriber/staff education are essential to the safe use of analgesic-dose ketamine in hospitalized patients. With appropriate provider knowledge  and standardized procedures, low-dose ketamine can be safely employed for acute pain management by general practitioners on general medical/surgical inpatient units.We therefore recommend that hospital providers familiarize themselves with available guidelines for using low-dose ketamine for acute pain , and conWhen prescribing opioids for acute pain, clinicians will need to consider several factors including the pain type, its severity and location, the speed of analgesic effect necessary for the situation, the analgesic duration of action, ease of use, resources available in the institution, patient preferences/adherence, and cost. The clinician will need to assess not only for the preferred opioid agent, but which route of administration would be the most suitable, what would be the best dosage for that route, and if there are unique limitations and/or contraindications for that patient ,102.When selecting an empiric opioid regimen for patients without prior exposure to opioids, there are “generally preferred” agents that are more likely to be safe and successful in practice, as described in the framework offered in Empiric opioid dosing for acute pain should be stratified based on the patient’s degree of pain or expected pain, preexisting opioid tolerance, and risk for opioid-related adverse events (ORAEs). Opioid tolerance vs. naïvety has historically been considered a binary classification, often demarcated at opioid exposure of ≥60 mg of oral morphine equivalent dose per day for at least 7 days ,114. OurWe therefore recommend institutions construct acute pain order sets with various stratified dosing regimens available alongside evidence-based decision support to guide providers in making this determination. The reader is also urged to recognize that the opioid equivalent doses described above are not intended for direct patient care applications such as when changing between opioid agents. The reader is referred to other evidence-based resources when navigating the complexity of converting existing opioid regimens to alternative opioids .The intravenous (IV) route of administration is the fastest route to provide analgesia. Intermittent IV bolus doses as needed (“prn”) are suggested for titration of opioids for severe acute pain since this option provides rapid onset without the uncertainty of medication absorption by other routes. However, the risks and costs of IV agents are higher than their oral counterparts, so IV analgesic regimens should generally be converted to enteral ones as soon as appropriate . This caOral administration of analgesic agents has been the gold standard, being simple, noninvasive, demonstrating good efficacy with high patient acceptability, and similar efficacy as the intravenous route . The subKEY CONSIDERATION: The route of opioid administration should be tailored to patient status and degree of need for rapid onset, with the enteral route being the safest and easiest when possible. The sublingual route of administration also offers a fast onset of action.The subcutaneous route of administration can be chosen if oral, SL, or IV routes are not available or optimal. Examples include combinations of cases of limited venous access, intractable nausea, emesis, dysphagia or bowel obstruction, or if mental status precludes safe oral and SL administration. Intermittent subcutaneous administration can be employed for as-needed opioids, or continuous subcutaneous administration can be pursued in more select cases . Care siKEY CONSIDERATION: The subcutaneous route of administration for opioids is a valued option in unique situations; the intramuscular route of administration should be avoided.When opioids are being utilized, even if only on an “as needed” basis, a prophylactic bowel regimen should be maintained to minimize opioid-induced constipation . OtherwiKEY CONSIDERATION: Always begin a scheduled stimulant bowel regimen when initiating opioid therapy; as-needed laxatives or docusate alone are not sufficient to prevent opioid-induced bowel dysfunction.Opioid exposure, especially in the postoperative period, can also elicit the known ORAEs of nausea and vomiting. This can delay discharge from the hospital or surgical center, delay the return to normal activities of daily living after discharge home, and increase costs of care . We therSeveral validated pain assessment tools are available for use when evaluating pain regimens  122,123123,124. However, analgesic regimens should not be adjusted based on pain scores alone. A consistent systematic approach that assesses pain location, quality, severity, and response to therapy will help ensure a complete picture of the patient’s pain experience . PatientWhen assessing regimen efficacy, a working knowledge of expected time to peak drug effect and duration of drug effect can be helpful in making decisions about medication adjustments. Assessing the patient’s pain control, and adverse effects, at the expected time to peak can help determine if the dose chosen is adequate. If the patient’s pain is not well controlled at the expected time to peak, we should not expect it to be controlled for the remaining expected duration of effect. Expected time to peak and drug duration is dependent upon route of drug administration and formulation .A framework for guiding patient-specific opioid adjustments is offered in KEY CONSIDERATION: Assessing for drug efficacy and adverse effects at the expected time to peak after drug administration is the best way to determine whether the selected dose is right for the patient. Carefully assess the nature of breakthrough pain in order to determine the best approach to regimen adjustments.Before discussing treatment approaches to the opioid tolerant patient, it is important to distinguish between some key terms: tolerance, dependence, and opioid use disorder (OUD). Opioid tolerance and dependence are natural physiologic processes associated with chronic opioid use. Tolerance occurs when repeated exposure to the same dose of opioid results in diminishing effects. Opioid dependence is evidenced by withdrawal syndrome upon abrupt discontinuation, rapid dose de-escalation, or administration of an opioid antagonist . Opioid KEY CONSIDERATION: A relationship of mutual trust and respect is vital to manage the patient’s pain. Chronic pain comes with complex psychological impacts that can inform the patient’s physical pain experience and can affect treatment response.As with opioid-naïve patients, optimization of non-opioid analgesics should still be the goal for opioid-tolerant patients. It is important to take a thorough inventory of the patient’s baseline opioid regimen, as well as adjuvants being used. Assess whether this acute pain episode is an exacerbation of the patient’s chronic pain, or an acute injury or surgery unrelated to the chronic source of pain. Gauge whether this pain episode is expected to change the patient’s pain baseline or if chronic pain is likely to continue despite resolution of the acute episode. When determining a pain regimen, total daily opioid requirements should be taken into account, both around-the-clock and as-needed administrations. Whenever possible, the patient’s home regimen  should be continued. This will help avoid withdrawal symptoms through the iatrogenic creation of a relative opioid deficit and maintain the patient’s baseline level of pain control . HoweverOpioid therapies started for acute pain should arc with the course of acute pain, meaning prescribers should initiate opioids at a thoughtful empiric regimen, titrate to effect and tolerability during acute painful episodes, and then taper the regimen as acute pain resolves. Nonopioid therapies should be maintained throughout this process to optimize analgesia and facilitate opioid weaning ,132,133.KEY CONSIDERATION: Opioid tapering goals and regimens must be patient-specific and prospectively, collaboratively discussed with the patient and other care team members in order to be safe and successful. Pre-discharge patient counseling on the pain management plan and opioid safety are essential.When acute painful episodes can be anticipated, such as those after a scheduled surgery, prospective patient education should be provided including the anticipated type, severity, and duration of pain, in addition to how pain will be managed and the typical extent of opioid exposure . EvidencOpioid tapering strategies after acute painful episodes should be very different for patients without prior opioid exposure than for patients with preexisting opioid tolerance and/or those on chronic pain therapies or MOUD . In opioAlongside opioid tapering plans, hospital providers should consider appropriate harm reduction strategies for patients being discharged on opioid therapies for acute pain. Concomitant narcotics, including benzodiazepines, gabapentinoids, and sedative-hypnotics should be assessed ,152,153.KEY CONSIDERATION: Co-prescribing take-home naloxone is evidenced-based harm reduction and should be widely considered by hospital providers prescribing opioids at discharge, alongside appropriate educational and continuity of care efforts.We therefore recommend hospital providers discharging patients on opioid therapies for acute pain consider patient-specific goals and therapy plans for the post-discharge phases of care, including the intended use and duration of opioid and nonopioid pain therapies, patient and caregiver education, co-prescription of take-home naloxone, and pain management-related follow-up as appropriate.The percentage of hospitalized patients on chronic medication for opioid use disorder (MOUD) is increasing, and hospital providers must therefore understand the fundamental concepts relating to managing pain in this high-risk population . There anot to manage OUD, but rather to reduce the risk of diversion of buprenorphine-containing products, i.e., the crushing and injecting of the dosage form to achieve opioid-induced euphoria. Naloxone undergoes tremendous first-pass hepatic metabolism (approximately 97–98%), resulting in very low oral bioavailability when taken as intended, and can even be co-administered with other opioids in the treatment of opioid induced constipation [Buprenorphine is a partial mu-opioid receptor agonist and kappa-opioid receptor antagonist FDA-approved in 2002 to treat OUD. It is effective for management of cravings due to its high mu receptor affinity and slow release  Buprenortipation  A commonKEY CONSIDERATION: Buprenorphine/naloxone combination products DO NOT have to be changed to buprenorphine-only products during acute painful episodes due to the low bioavailability of naloxone.Methadone is a full mu-opioid receptor agonist and NMDA antagonist approved for the management of OUD in the clinic setting. Doses for management of OUD vary from 60–120 mg per day with some patients requiring a higher total daily dose (TDD) to manage cravings and suppress withdrawal symptoms . Barringn = 22) and methadone (n = 29). The study found no differences in pain scores between the two groups nor no significant differences in patient-controlled analgesia requirements between the two groups or with those patients who did not receive methadone on the day of surgery. However, as found with the previous studies, patients who were not given buprenorphine the day after surgery used significantly more patient-controlled analgesia compared to those who received their dose [While it may seem counterintuitive, there is ample published evidence and experience to support the continuation of methadone and buprenorphine regimens during acute painful episodes, including through surgical encounters. Continuation of MOUD improves pain control, reduces the risk of relapse, and has been shown to decrease as-needed opioid requirements in this patient population . A smalleir dose . Furthereir dose ,172.KEY CONSIDERATION: Methadone and buprenorphine should be continued during acute painful episodes, including surgical encounters, alongside opioid-tolerant doses of as-needed pain medications within multimodal analgesic regimens.Both methadone and buprenorphine have high affinity at the mu opioid receptors, providing suppression of opioid withdrawal symptoms and cravings for 24–48 h, making them ideal therapies for OUD . The durKEY CONSIDERATION: The duration of analgesia from buprenorphine and methadone is shorter than their duration of reducing cravings and withdrawal symptoms- splitting the home dose throughout the day should be considered as part of the multimodal analgesic regimen during periods of acute pain. The patient’s usual dosing regimen can then be resumed once acute pain has subsided and/or at hospital discharge.Naltrexone is a competitive mu-opioid receptor antagonist that blocks the effects of endogenous and exogenous opioids. It is available for the treatment of OUD in a depot intramuscular (IM) injection dosed once monthly . It is aThe primary strategy to manage pain on those patients who are taking naltrexone is to stop naltrexone prior to the planned procedure. For oral naltrexone, therapy should be stopped 72 h prior to the procedure to allow for the medication to be metabolized and to increase the number of available opioid receptors. For patients receiving IM naltrexone, the dose should be held for at least 30 days prior to elective surgery . It is iKEY CONSIDERATION: Naltrexone-containing products must be stopped prior to any scheduled elective procedures to ensure adequate pain management for patients. Patients who have naltrexone interrupted will rapidly lose their tolerance to opioids and should be monitored closely when opioid therapies are needed.These management recommendations for MOUD during acute painful episodes are summarized inTable 5. Additionally, patients with OUD who are in acute pain should be afforded safe and effective multimodal analgesia by hospital providers, including appropriate as-needed opioids dosed for tolerance as discussed previously. Additionally, coordination of care with the patient’s Addiction Medicine team should be standard of care, to the extent possible. The patient should also always be prospectively included when discussing pain management strategies, especially those patients with OUD. For example, the team may, unknowingly, prescribe a specific opioid which may trigger a relapse and is not preferred by the patient based on their previous drug of choice. Holistic care is important for management of pain crises as it allows for better understanding of the patient, their risk factors, and what you, the clinician, are able to do to help meet pain goals.Acute pain management in hospitalized patients can challenge inpatient healthcare providers, but increased familiarity with core concepts and resources can support a consistent and effective approach to daily practice. We hope the pearls and tools offered in this piece help diverse hospital-based prescribers and staff in providing better pain management and improving patient outcomes.


As sites enriched in lipid transfer proteins (LTPs) and small solute transporters, MCSs confer the “non-vesicular” direct exchange of lipids and small solutes  How do the structural attributes of MCS proteins regulate lipid metabolism? 2) How does bulk lipid trafficking—in particular of phospholipids—work, and how is this regulated? 3) What growth conditions or genetic programs control the metabolic changes conferred by MCSs? And given the links between MCSs and disease pathology: 4) Do MCSs provide pharmacological targets for therapies combating the ever-increasing list of MCS-related disorders?In this Research Topic we focus on functional aspects regulated through MCSs. These functional aspects impact inter-organelle communication, organelle structure, organelle inheritance and biogenesis, lipid and protein traffic, but also encompass cellular processes such as autophagy, membrane stress responses, cell death and ageing. Our knowledge about MCSs has exponentially increased throughout the past few years during which we have come to appreciate the importance of MCSs as platforms for general subcellular organization that dynamically coordinate multi-organelle processes. This research topic includes six review articles and one original research article that highlight recent advances in the field of MCS research, and describe the complex functional connections between lipid trafficking, metabolite channeling, autophagy, cell death and quiescence.Choudhary and Schneiter summarize the current knowledge on ER-lipid droplet (LD) contacts, but also LD contacts with mitochondria and peroxisomes. In particular, this article discusses the function of specific LD-contact site components that generate the unique membrane interface that drives LD formation, lipogenesis and lipolysis. Emphasizing the cellular need to coordinate cellular lipid homeostasis between organelles, the authors also consider the roles of membrane LD-mitochondria and LD-peroxisome tethers, which support unique metabolic chemistries that are spatially segregated with cells. Because cellular bioenergetics and metabolism depends on LD interactions with these organelle membranes, the contribution of LD contacts on health and disease is also discussed.In the review article “A Unique Junctional Interface at Contact Sites Between the Endoplasmic Reticulum and Lipid Droplets” Choudhary and Schneiter focus on LD biogenesis, Renne and Hariri concentrate on LD contacts with other organelles and their roles in fatty acid (FA) metabolism and trafficking. Their review article, “Lipid Droplet-Organelle Contact Sites as Hubs for Fatty Acid Metabolism, Trafficking, and Metabolic Channeling,” describe mechanisms by which LD contacts impact FA metabolism, including catabolism, synthesis, desaturation, elongation, and incorporation of FAs into complex lipids. In particular, the authors give insight into the importance of LD–ER contacts for metabolic channeling of FAs into LDs, which is an important requirement for lipid storage. They further discuss the function of LD contacts with peroxisomes and mitochondria for FA-beta-oxidation, and the roles of LD-vacuole contact sites for FA storage and mobilization.Whereas Mechanisms of Non-Vesicular Exchange of Lipids at Membrane Contact Sites: Of Shuttles, Tunnels and, Funnels,” Pascal Egea describes mechanistic features of lipid transfer proteins (LTPs) at the heart of MCSs. Based on structural and functional properties, LTP mechanisms are categorized into 1) diffusion, 2) sliding and 3) bridging-based. These mechanisms are discussed in depth as illustrated by the structural attributes of Ups1/Mdm35, ERMES, and other LTPs.In the review article “Amit Joshi. Although these peroxisomal MCS include those with LDs as reviewed by Choudhary and Schneiter and Renne and Hariri, this article further extends to peroxisomal contacts with the ER, mitochondria, vacuole and plasma membrane. The author not only describes the structural features of these peroxisomal MCSs but also discusses functional aspects of peroxisomal MCSs important during processes such as peroxisome biogenesis.A particular focus on peroxisomal MCSs is presented in the mini-review “Peroxisomal Membrane Contact Sites in Yeasts” by Lenoir et al. also contribute to this special issue with their review article “Transport Pathways That Contribute to the Cellular Distribution of Phosphatidylserine”. The authors give a comprehensive overview of the current understanding on phosphatidylserine (PS) biosynthesis and the maintenance of its distribution in cellular membranes, including lateral distribution within bilayers. They discuss the intracellular trafficking of PS between membranes by different LTPs at ER contact sites with mitochondria, the plasma membrane, and with the autophagy isolation membrane. PS exchange between the two layers of the same membrane by flippases, and scramblases is also discussed.Struyfs et al. takes a different viewpoint on lipid membranes. Here the authors focus on the membrane interaction of antimicrobial peptides (AMPs), in particular antimycotic peptides. Because fungal infections are on the rise worldwide, this article provides a fresh perspective on the growing knowledge on lipid membranes, as well as MCSs and their regulation, that can be exploited for medical and pharmacological purposes. In particular, the authors describe AMP structures and mechanisms allowing for specific fungal membrane interactions linked to programmed cell death, autophagy, mitochondrial dysfunction and MCSs.The review article “Membrane-Interacting Antifungal Peptides” by Peselj et al. report on the role of lipids in the regulation of quiescence. They investigate the storage and consumption of neutral lipids upon glucose or phosphate starvation. The authors find that upon glucose exhaustion, LDs were degraded by lipophagy. Upon nitrogen exhaustion, however, cells exhibit a nucleus-vacuole junction expansion and accumulate LDs on the vacuole surface. Significantly, the formation of steryl esters and their hydrolysis and mobilization from LDs is critical for survival of phosphate starvation but not during glucose starvation. They conclude that the mechanism by which neutral lipid homeostasis adapts to both support quiescence and to exit from quiescence is specifically dependent on the limiting nutrient.In the original research article “Sterol metabolism differentially contributes to maintenance and exit of quiescence” Altogether, this compilation delivers a focused picture of yeast MCSs and their impact on lipid homeostasis and other functional contributions to cell ageing, cell death, and autophagy.


The logopenic variant of primary progressive aphasia is characterized by early deficits in language production and phonological short-term memory, attributed to left-lateralized temporoparietal, inferior parietal and posterior temporal neurodegeneration. Despite patients primarily complaining of language difficulties, emerging evidence points to performance deficits in non-linguistic domains. Temporoparietal cortex, and functional brain networks anchored to this region, are implicated as putative neural substrates of non-linguistic cognitive deficits in logopenic variant primary progressive aphasia, suggesting that degeneration of a shared set of brain regions may result in co-occurring linguistic and non-linguistic dysfunction early in the disease course. Here, we provide a Review aimed at broadening the understanding of logopenic variant primary progressive aphasia beyond the lens of an exclusive language disorder. By considering behavioural and neuroimaging research on non-linguistic dysfunction in logopenic variant primary progressive aphasia, we propose that a significant portion of multidimensional cognitive features can be explained by degeneration of temporal/inferior parietal cortices and connected regions. Drawing on insights from normative cognitive neuroscience, we propose that these regions underpin a combination of domain-general and domain-selective cognitive processes, whose disruption results in multifaceted cognitive deficits including aphasia. This account explains the common emergence of linguistic and non-linguistic cognitive difficulties in logopenic variant primary progressive aphasia, and predicts phenotypic diversification associated with progression of pathology in posterior neocortex. et al. integrate advances from cognitive neuroscience and neuropsychology to propose a role for left temporoparietal degeneration in underpinning emergent linguistic and non-linguistic cognitive deficits in logopenic variant primary progressive aphasia.In a new clinico-anatomical model, Ramanan  Key to this resurgence was a seminal case series by Mesulam3 describing progressive aphasia with left perisylvian atrophy and slow rates of disease progression. Marked general cognitive decline emerged only later in the disease course.4 Subsequent classifications of PPA have expanded the phenotype to include patients with motor-speech planning, grammatical and semantic memory deficits, identifying three main clinical variants: a ‘non-fluent/agrammatic’ variant (nfvPPA) characterized by motor-speech dysfunction and/or agrammatism stemming from primary fronto-insular degeneration;5 a ‘semantic’ variant  presenting with degradation of conceptual knowledge due to anterior temporal degeneration;6 and a third ‘logopenic’ variant (lvPPA)7 that forms the focus of the current Review. Clinically, lvPPA patients display slowed, disrupted spontaneous language production marked by word-finding pauses, anomia, phonological errors and poor length-dependent repetition of sentences, amid relatively preserved grammatical processing, motor speech (unlike nfvPPA) and semantic knowledge (unlike svPPA).8 Comprehension of long sentences is also affected and, in part, could arise from poor phonological short-term memory.8–10 This constellation of language difficulties emerges from primary degeneration of the left-lateralized posterior temporal gyri, temporoparietal junction and inferior parietal lobes (TPJ/IPL) and their interstices.11 Recent advances in neuroimaging of disease progression in PPA syndromes conceptualize them as network-level disorders, in which neurodegeneration starts in a syndrome-specific susceptible epicentre and then spreads in a predictable, constrained manner along structurally/functionally connected brain regions.13 Accordingly, with disease progression, atrophy in lvPPA encroaches into regions in the left hemisphere that are functionally coupled with TPJ/IPL (via language and Default Mode networks), followed by regions in the right hemisphere.14–19Primary progressive aphasia (PPA) refers to a group of neurodegenerative disorders affecting language functions in early stages, due to degeneration of a distributed, largely left-lateralized language network.3 When non-linguistic cognitive functions are tested in depth, most lvPPA cases appear to exhibit moderate-to-marked levels of non-linguistic cognitive dysfunction.20 Others find that the severity of non-linguistic difficulties in lvPPA may parallel the severity of aphasia and emerge relatively independent of disease severity.21 This issue is complicated by overlaps in both language and non-linguistic cognitive performance between lvPPA, amnestic Alzheimer’s disease and posterior cortical atrophy: three clinical entities often sharing common pathology.16 Piecing together the diagnostic and mechanistic puzzle of why lvPPA presents with linguistic and non-linguistic cognitive difficulties is vital to ensure accurate diagnosis, characterization and prognostication.To date, the lens of lvPPA clinical investigations has largely focused on language profiles, mainly because, by definition, the main presenting clinical complaint is that of language disturbances. However, a growing number of studies pose an important diagnostic challenge: that numerous lvPPA patients display concurrent, non-linguistic cognitive deficits on neuropsychological testing. Of note, even in Mesulam’s early PPA cases who probably had the logopenic variant, calculation difficulties were noted during clinical presentation.In this paper, we propose a new clinico-anatomical framework that encompasses the ‘multidimensional’ deficits in lvPPA. The combination of core language and variable non-linguistic deficits may arise from shared neurophysiological mechanisms, and the nature and localization of neurocognitive processes supported by the left TPJ/IPL region—the epicentre of atrophy in the syndrome. The contribution of this region and functionally connected areas to language disruption in lvPPA has received significant attention, but its role in cognitive processes beyond language in this syndrome has been relatively overlooked. By integrating insights from contemporary cognitive neuroscience and neuropsychology, we review the evidence of domain-general and domain-selective mechanistic roles for these regions. By then reversing these inferences, we can consider the phenotypic outcome of left TPJ/IPL degeneration to lvPPA, updating our understanding of why some patients present with a relatively pure aphasic profile while others display additional non-linguistic dysfunction.To be clear from the outset, our goal of this Review is not to rewrite the current diagnostic criteria for lvPPA, but rather to explain phenotypic diversification in the syndrome with implications to its accurate diagnosis and timely introduction of multidomain symptomatic treatments.22–25 The impact of TPJ/IPL pathology in lvPPA may further cause disruptions in cognitive processing related to up/downstream regions, such as the hippocampus and right IPL, and connected functional networks, compounding cognitive dysfunction in the syndrome. The current proposal does not exclude contributions of other brain regions to cognitive dysfunction in lvPPA; but in this Review, we focus on TPJ/IPL due to its complex cognitive roles and early involvement in this syndrome. A detailed review of left TPJ/IPL functionality is beyond the scope of our Review26–29; however, by considering key themes regarding its neurocognitive organization, we can provide testable neural hypotheses explaining the multifaceted cognitive presentation of lvPPA.We propose that patterns of verbal and nonverbal cognitive difficulties observed early in the lvPPA disease course are a direct reflection of (i) the cognitive complexity of TPJ/IPL; and (ii) the amount and distribution of neuropathology, atrophy and/or hypometabolism in TPJ/IPL and structurally/functionally connected regions and networks.30 Functional neuroimaging suggests that the multidomain involvement of TPJ/IPL can be traced to its role in deploying key neurocomputational resources to meet demands of different cognitive tasks.31 Three principal features of this region underpin its domain-general and domain-selective functions:Historical cognitive neuroscience and neuropsychology evidence has implicated left TPJ/IPL in multiple, apparently distinct, cognitive tasks. For instance, Gerstmann’s syndrome, emerging from left IPL damage, encompasses both linguistic  and non-linguistic  complaints.34Neuroanatomically, TPJ/IPL are segregated from primary sensory cortices. This absolves them from processing dynamic fluctuations in the immediate sensory environment, and facilitates the processing of multimodal information and diverse cognitive domains.36 TPJ/IPL harbour this functional capacity, whereas TPJ/IPL lesions characteristic of semantic aphasia impair the use of time- and context-appropriate information across semantic and non-semantic domains.38Computational models suggest that repeat processing, integration and buffering of information can promote extraction of time-, space- and context-invariant representations that can be repurposed to different task demands.39Functional specialization within TPJ/IPL is not sharply fractionated, but varies between subregions in a graded manner and is tied closely to structural/functional connectivity patterns of each subregion. This characteristic is seen in the temporal granularity of information being processed here. For example, cognitive tasks processed over shorter time periods  preferentially recruit anterior TPJ/IPL regions  whereas posterior IPL (e.g. angular gyrus) is preferentially recruited in tasks reliant on longer temporal windows, where processing of accumulating information is required .On the basis of these principles, we evaluate evidence for TPJ/IPL in multidomain cognition, with relevance to lvPPA .41 and extraction and maintenance of phonological representations.42 These specific functions, in turn, may be supported by shared neurophysiological mechanisms within TPJ/IPL. For example, the planum temporale acts as an auditory ‘computational hub’, disambiguating incoming auditory information, matching these representations to existing auditory templates (derived from experience), in turn, supporting its transformation into subsequent motor programmes for different cognitive/behavioural domains .43–45 Ventral and posterior portions, including posterior middle temporal regions, are associated with multimodal semantic-executive related processing,46–48 while angular gyrus participates in constructive elements of verbal episodic recollection,49–51 sentential and combinatorial processing52 and buffering sequential information in coherent contexts.54 These functional specializations are corroborated by extant neuropsychological evidence. For example, conduction aphasia, emerging from damage to the inferior supramarginal gyrus and underlying arcuate fasciculus, specifically disrupts speech repetition but spares semantic-executive control.56 Conversely, transcortical sensory aphasia and semantic aphasia, occurring from lesions in the middle/posterior cerebral artery watershed territory affecting posterior middle temporal regions , display poor semantic-executive processing but relatively intact phonological functions.57 Finally, combined damage to the entire left TPJ/IPL, such as in Wernicke’s aphasia, disrupts both phonological and semantic-executive control functions, irrespective of tested modality.58Considering language functions, posterior superior temporal and inferior supramarginal gyri are involved in extraction of spectro-temporal features of sound59 Longer temporal windows in posterior IPL (angular gyrus) facilitate its involvement in transformation and integration of accumulating information, irrespective of its nature or modality.54 For example, during memory recollection and future-oriented thinking: (i) angular gyrus and medial temporal regions show functional coupling; and (ii) angular gyrus activity scales with the vividness, strength and accuracy of retrieved memories, and shows demonstrable sensitivity to integration of multimodal information.61 Accordingly, primary IPL damage, irrespective of aetiology, impairs performance on cognitive endeavours requiring mental simulations, including episodic retrieval and scene construction,62–64 theory of mind and social cognition65 and spatial navigation.66 Angular gyrus is further integral to transformation and retrieval of numerical knowledge,67 explaining dyscalculia following IPL damage in Gerstmann’s syndrome. The integration role of the IPL further extends to motor behaviour, where supramarginal gyrus directs attention to motor sequences68 while supramarginal and angular gyri together aid integration of individual motor goals and their transformation into external motor actions.69 This particular finding implicates the IPL as a neural substrate of ideational apraxia as well as volitional apathy.70 Taken together, the evidence points to a role for the TPJ/IPL in supporting multiple cognitive processes spanning language and beyond.For non-linguistic domains, left IPL emerges at the nexus of multiple non-linguistic cognitive capacities. As per our third principle , the anterior IPL  shows involvement in tasks requiring relatively shorter temporal processing windows, such as directing bottom-up attention to external stimuli and internally generated thought (e.g. episodic recollection).Reverse inference from the cognitive neuroscience and neuropsychology literature on the nature of multidomain cognitive processes of the TPJ/IPL region .75 On this view, the language and anatomical profile of lvPPA most closely resembles conduction aphasia post-stroke,77 although it must be acknowledged that the major PPA syndromes have either little or only partial analogues in vascular aphasiology, in terms of overall linguistic and cognitive performance.78 If and when the underlying pathology encroaches into anterior and ventral temporal cortices, semantic comprehension impairments, such as those in svPPA, are expected.80 Similarly, if the disease spreads into fronto-insular regions and connecting arcuate/superior longitudinal fasciculus, then motor-speech difficulties (resembling those observed in nfvPPA) may become part of the clinical profile.81The core language deficit of lvPPA is in keeping with the role of the posterior superior temporal gyrus/inferior supramarginal gyrus in phonological processing and working memory. In lvPPA, errors of phonology and lexical retrieval during word formation emerge from dysfunction of these specific regions.82–85 Dyscalculia as early as 1 year post disease onset, and emergence of apraxia within 2 years are also reported.86 A recent case report of an lvPPA patient with canonical TPJ/IPL atrophy noted attentional and visuospatial complaints 87—a constellation of complaints typically seen in posterior cortical atrophy following superior/inferior parieto-occipital neurodegeneration. Another case report of a patient meeting lvPPA clinico-radiological criteria evidenced deregulated semantic-executive control when using electrical appliances, wherein the patient ‘clearly understood what these appliances are and what they are used for, but was not sure how to use them’.. This profile appears to fulfil standard definitions of ideational apraxia, and is highly reminiscent of semantic control deficits in semantic aphasia ,88–90 where damage to inferior/middle posterior temporal and IPL (angular gyrus) regions results in context-invariant executive deregulation of verbal and nonverbal semantic information.90 In this regard, probing the status of semantic control functions in lvPPA forms an important line of inquiry as it may inform whether deregulated semantic control partly explains the co-occurrence of some non-linguistic symptoms, such as apraxia, in this syndrome.Relative to healthy controls and other PPA variants, neuropsychological investigations of well-characterized lvPPA patients identify deficits in processing speed, executive function, sustained attention, working memory and visuospatial function.91 Accordingly, an additional under-appreciated cognitive complaint in lvPPA is episodic amnesia, closely linked to IPL involvement in the syndrome. By the time of their first clinic appointment, a third of patients report misplacing objects, missing appointments, getting lost and facing difficulty in learning new tasks.93 Both carer- and clinician-indexed reports further attest to daily memory difficulties.94 On objective standardized neuropsychological memory assessment, verbal and nonverbal episodic memory deficits in lvPPA can be prominent to a magnitude comparable to amnesic Alzheimer’s disease and are associated with changes to integrity of the bilateral IPL (angular gyri) and their structural connections to memory processing regions in the medial temporal lobe (e.g. hippocampus).94–96 Episodic amnesia in lvPPA can also extend to remote autobiographical memories encoded decades before the onset of clinical symptoms (e.g. from teenage and early adulthood years), with poor autobiographical recall, irrespective of the temporal epoch of the memory, correlating with angular gyrus involvement in the syndrome.97Given the role of the angular gyrus in episodic memory processing, atrophy/pathological deposition to this region can impair episodic retrieval.98–101 These impairments have been found to be closely associated with dysfunctional working memory,100 further correlating with integrity of the left IPL and parietal cortices.101 Spatial working memory deficits are also prominent in lvPPA, again associated with degeneration of superior parietal cortices and their functional connections with prefrontal brain regions.102–104 Across clinical variants of Alzheimer’s disease (including lvPPA), increased pathological accumulation in TPJ/IPL and parietal cortex strongly correlates with emergent impairments of episodic and semantic memory, plus executive and visuospatial functions.105 In lvPPA particularly, patients displaying greater visuospatial, executive and general cognitive disturbances typically show intensified atrophy to left IPL/superior parietal regions.106 Corroborative evidence also comes from studies adopting the inverse ‘imaging-first’ classification approach, where lvPPA patients with greater left IPL/parietal atrophy/hypometabolism display poorer nonverbal memory, visuospatial and executive performance, in contrast to lvPPA patients with temporal-dominant atrophy/hypometabolism.107 Finally, there is some evidence for behavioural dysfunction in lvPPA, such as increased apathy, anxiety, irritability, agitation and difficulties in emotion detection.108–112 Ultimately, it is unsurprising that a combination of aphasia, poor memory and general cognition, increased apathy and agitation significantly compounds carer burden and caring-related challenges in lvPPA.114 Additional investigations of the inter-dependence between behavioural and mood symptoms, the neural bases of non-linguistic cognitive and behavioural difficulties, and how these changes relate to underlying pathological and genetic changes in lvPPA are required , in the absence of peripheral hearing difficulties.ired see . NeverthBox 1 Links between multidimensional cognitive impairment, underlying Alzheimer’s disease pathology and progranulin gene mutations in lvPPA. Clinicopathological associations are key to achieving accurate prognosis and treatment decisions. Over 80% of lvPPA patients have underlying Alzheimer pathology, mostly noted as amyloid-positive PET scans.115–118 More recently, reports have emerged of cases showing an lvPPA-like clinical picture amid amyloid-negative profiles. In these cases, a common finding is the mutation of the progranulin gene, which is typically associated with frontotemporal lobar degeneration syndromes.119–122 In addition to the classic linguistic profile of lvPPA, many such cases display concurrent difficulties in reading, episodic memory, executive functions and calculation,124 suggesting links between cognitive multidimensionality and underlying neurobiological and genetic factors. In current neurodegenerative dementia clinico-pathological conceptualizations, lvPPA is considered as an atypical clinical variant of Alzheimer’s disease.16 Therefore, an lvPPA patient with an amyloid-positive scan, a prototypical aphasic profile, poor memory, visuospatial and/or executive dysfunction may rule in several different diagnostic labels including mixed PPA, advanced amnesic Alzheimer’s disease and an Alzheimer’s disease clinical variant with visuospatial and/or language features. As the syndrome straddles clinicopathological boundaries of both PPA and Alzheimer’s disease taxonomies, it is sensible to ask whether multidimensional cognitive difficulties in lvPPA are parsimoniously explained by underlying pathological and/or genetic mechanisms.125 On the other hand, accumulating evidence suggests that lvPPA patients with and without amyloid-positive profiles perform comparably on standardized measures of lexical retrieval, speech and language, executive function, processing speed and verbal memory.126–129 Comparable profiles between high- and low-amyloid burden lvPPA patients are also evident on visuospatial and non-verbal memory tasks, even when disease severity and overall language status are accounted for.130 Moreover, data-driven clustering solutions of lvPPA cognitive performance find no significant differences in amyloid burden, demographic and disease duration indices between lvPPA with mild, marked or no co-occurring general cognitive deficits.20 Likewise, when contrasting lvPPA cases with and without progranulin gene mutations, comparable language, cognitive, and neuroanatomical profiles  are notable.120 Due to the rarity of such reports, more work is required on this front. However, most findings suggest that the pathological or genetic profile, by itself, may not be sufficient to explain variations in non-linguistic cognitive difficulties in lvPPA. Rather, as we propose, the key factor in variable cognitive profiles early in the syndrome may be the extent to which the distribution and loading of pathology and atrophy/hypometabolism are localized in the TPJ/IPL and neighbouring regions.Given the paucity of evidence on correspondence between clinicopathological and cognitive performance data in lvPPA, a direct answer to this question is currently hard to establish. For example, one study noted that PPA with Alzheimer’s disease pathology  is associated with episodic memory preservation, despite these patients presenting significant TPJ/IPL and medial temporal atrophy.133–135 These deficits often interfere with patients’ ability to understand complex verbal instructions. However, there is compelling evidence that many nonverbal cognitive deficits observed in lvPPA are not simply a reflection/by-product of the patients’ progressive aphasia. Patients with lvPPA can show marked deficits on cognitive functions that circumvent language demands, such as nonverbal episodic memory, nonverbal auditory object processing, spatial orientation, spatial working memory and visuospatial functioning.137 Likewise, lvPPA patients exhibit impaired performance on verbal memory, and visuospatial and executive processing tasks, even after statistically controlling effects of aphasia severity.109 In this regard, detailed lvPPA case reports are particularly insightful. In one report, the earliest recorded complaints involved calculation and sustained attention difficulties occurring a few months prior to the emergence of a florid lvPPA aphasic profile.87 In another study, Pozzebon et al.138 interviewed spouses of five lvPPA patients and found all carers to report incipient apathy and social withdrawal, plus deficits of episodic memory and sustained attention emerging almost 1–3 years before spousal recognition of frank expressive language deficits. These deficits were not core of the patients’ presentation, which was of language impairment, thus meeting the primary criteria for progressive aphasia. These findings indicate that non-linguistic deficits in lvPPA can emerge independent of and in parallel to primary aphasia, even where the patient’s core complaint is that of language difficulty.Primary verbal degradation in lvPPA greatly influences performance on tasks taxing working memory and phonological abilities, such as reading, verbal fluency and verbal episodic memory.139 this hypothesis is not a sufficient explanation the early non-linguistic deficits in many other people with lvPPA. Even at the mildest clinician-indexed disease stages , difficulties in calculation and spatial working memory can be evident in lvPPA.8 Likewise, poor nonverbal episodic memory performance remains prominent in lvPPA, even after statistically controlling for disease severity.94 The confounding issue of disease severity has been addressed by recent data-driven studies parcellating its effects. For example, when overall language performance is employed as a categorical proxy for disease severity, the magnitude of general cognitive impairment scales with aphasia severity.140 A key limitation of this approach is that categorizing lvPPA patients first on aphasia severity and then on non-linguistic cognitive performance overlooks linguistic and non-linguistic difficulties occurring in parallel. Using step-wise classifications, therefore, may bias our determination of the absence/magnitude of non-linguistic complaints.As with other progressive disorders, relatively ‘pure’ cases with circumscribed clinico-anatomical presentation evolve to show clear and increasing multidomain dysfunction as the underlying pathology propagates. Although longitudinal profiles of some lvPPA patients neatly fit this pattern,106 The principal component analysis revealed multiple, concurrent sources of variance across neuropsychological performance in lvPPA. Extraction of orthogonal dimensions of performance variation allowed (i) exploration of confounds associated with disease and aphasia severity that otherwise pervade across measures; and (ii) situation of patients along independent dimensions of verbal and nonverbal cognitive performance thereby capturing their graded, inter-individual variations in cognitive impairment. The results indicated that the cognitive profile of lvPPA is best characterized along two orthogonal dimensions: (i) speech production and verbal memory deficits; and (ii) visuospatial and executive changes  whether additional non-linguistic cognitive dysfunction is representative of the syndromic designation of lvPPA; (ii) whether ‘logopenic progressive aphasia’ might better reflect the cognitive profile of such patients; or (iii) whether such cases may represent a new subtype/subcategory of lvPPA. How do we then best capture these terminological differences and inter-individual differences in cognitive performance?Given the evidence for multidomain cognitive dysfunction, it has been argued whether the term ‘lvPPA’ be reserved for ‘pure’ cases , while ‘Logopenic Progressive Aphasia’ might better capture individuals displaying a dynamic, multifaceted phenotype beyond the linguistic constraints of a pure PPA.141–143 we propose that cognitive changes in individual lvPPA patients are best conceptualized as graded, individual-level differences within a multidimensional space  may represent a subsample of individuals within this larger space. What is important to recognize is that ‘pure’ lvPPA as a clinical entity does not occupy the entire multidimensional space, but rather locates in an area of a larger space spanning variable clinical presentations of the syndrome. Variations in atrophy/functional disruptions within the TPJ/IPL cortices, and connected regions, will generate graded variations in the type and severity of the individual verbal and nonverbal profile, determining locations of individuals within this space. In short, phenotypic deviations from prototypical lvPPA may be graded rather than absolute, and reflect the variations in local pathology .94 While the discriminative use of other non-linguistic tests remains to be explored, co-reliance on linguistic and non-linguistic measures uniquely stressing functions supported by TPJ/IPL may significantly improve accurate diagnosis of lvPPA. For practising clinicians, detailed testing of verbal and nonverbal cognition in the syndrome can improve understanding of the structure of this multidimensional space and help identify measures that are highly representative of each axis of space. In turn, this information can be used to cull lengthy test batteries and retain those efficient and effective measures that capture the most important cognitive-behavioural variation in lvPPA. The success of this method has recently been demonstrated in post-stroke aphasia148; how informative it is in PPA, where aetiology, cognitive performance and progression patterns are complex and highly variable, remains to be better understood. At the point of diagnosis, specific quadrants/locations within this space may also inform selection, titration and timing of management approaches, therapies and treatments, as well as inclusion and stratification of specific individuals into trials.First is the need to assess both language and non-linguistic features in PPA diagnosis. Naming, spontaneous speech and paraphasic/lexical error patterns, as individual measures, are increasingly recognized as providing poor specification of PPA type.150 As for the IPL, emerging evidence in PPA indicates that a combination of IPL neurostimulation and speech and language training can induce (i) improved naming and verbal fluency performance; (ii) transfer of gains to unlearnt items and select cognitive domains ; and (iii) benefits sustaining for up to 2 weeks.152 These findings indicate the need for more studies exploring interventions targeting the parietal cortex for linguistic, non-linguistic cognitive and functional improvement in lvPPA. Moreover, TPJ/IPL and general parietal dysfunction are largely considered to form the common neuroanatomical denominator associated with phenotypic diversification across several posterior cortical neurodegenerative syndromes, many of which share Alzheimer’s disease pathology.154 Shifting the focus to reveal the importance of parietal integrity in Alzheimer’s disease will move the field a step closer to achieving a full understanding of the interplay between neurodegeneration and cognitive dysfunction in multiple clinical variants of Alzheimer’s disease.Second, given their importance in the multifaceted clinical profile of lvPPA, TPJ/IPL and parietal cortex seem likely to become neuroanatomical targets of symptomatic intervention in this syndrome. In this regard, recent work has revealed links between excitatory neurostimulation of prefrontal regions and facilitation of oral and written language improvement in lvPPA.As we make progress on the aforementioned steps, it seems likely that we will uncover deeper layers to the lvPPA cognitive and behavioural profile. Therefore, a final, important step forward is to build integrative frameworks of cognitive and brain mechanisms that underpin the broader symptom complex of lvPPA, with relevance to other neurodegenerative dementia syndromes affecting the posterior neocortex. This larger programme requires a two-pronged approach, with each step informing the other. What we first need is a better understanding of how psychological and pathophysiological changes in lvPPA relate to changes in local neural populations and their neurochemistry, genetics and neuropathology, and neuroimaging in the syndrome. This will allow integration of new knowledge regarding brain, behaviour and biological changes in lvPPA with current neurocognitive models. Next, testing the stability and interpretability of these renewed models requires international, multi-centre studies to establish large lvPPA cohorts with deep neuropsychological and neuroimaging phenotyping, and identify candidate neurophysiological biomarkers in the diagnostic process. We acknowledge that this future direction is ambitious and requires navigating recruitment challenges and nosological disagreements. However, we hope that our framework can aid this process by accommodating terminological discrepancies and diverse clinical-cognitive presentations within a multidimensional space, and potentially offer a common research framework to springboard future efforts towards a complete understanding of the lvPPA phenotype.


In vivo human neuroimaging, particularly at ultra-high field (UHF), offers unprecedented opportunity to examine multiscale dysconnectivity in psychosis. In this review, we provide an overview of the literature to date on UHF in psychosis, focusing on microscale findings from magnetic resonance spectroscopy (MRS), mesoscale studies on structural and functional magnetic resonance imaging (fMRI), and multiscale studies assessing multiple neuroimaging modalities and relating UHF findings to behavior. We highlight key insights and considerations from multiscale and longitudinal studies and provide recommendations for future research on UHF neuroimaging in psychosis.Schizophrenia and related psychoses are complex neuropsychiatric diseases representing dysconnectivity across multiple scales, through the micro (cellular), meso (brain network), manifest , and social  levels.  Schizophrenia, coined term for “split mind,” is a complex neuropsychiatric illness characterized by a range of positive, negative, and cognitive symptoms . PositivPsychotic disorders can be understood as disorders of connectivity across multiple scales, ranging from micro (cellular level) to meso (brain network level) to manifest  to social  scales . At the In vivo human neuroimaging can provide insight into multiscale dysconnectivity at the microscale  and mesoscale , while simultaneously allowing for acquisition of data at the manifest  and social  scales.Beyond identifying dysconnectivity at multiple scales, a complete picture of psychosis is only possible through understanding the way in which these scales interact and influence one another. For example, micro-level dysfunction , may present itself in meso-level dysfunction , which manifests as abnormal speech and behavioral patterns that affect the interpersonal relationships of individuals living with schizophrenia . MultiscUltra-high field (UHF) neuroimaging  refers to magnetic resonance imaging using a magnetic field strength (b0) of greater than or equal to 7.0 Tesla. UHF offers many benefits relative to lower fields strengths  that can facilitate multiscale dysconnectivity research in psychiatric populations , 12. QuaA specific advantage offered by UHF-MRS systems is the ability to resolve the neurometabolic spectrum along with high SNR. The SNR gain from 3T to 7T is especially pronounced for glutamate, glutamine, and gamma aminobutyric acid (GABA). Glutamine and glutamate are molecules in a dynamic equilibrium, reflecting the integrity of distinct cellular compartments (neurons and astrocytes) in the brain, but separating their spectra is challenging at lower field strengths. The reduced error rates in quantifying these molecules at UHF translates to a reduced need for large sample sizes to observe a given effect. This is especially helpful in the study of schizophrenia where neurobiological heterogeneity is a prime suspect for poor therapeutic progress.1HMRS) has overcome several methodological limitations evident at lower field strengths, such as separating highly coupled spins, improving SNR of metabolites with low in vivo concentrations, increased spectral dispersion, lower uncertainty in spectral quantification such as smaller Cramer lower bounds (CRLB) and coefficients of variance (CV). For example, glutathione is typically isolated as a secondary metabolite to GABA at lower field strengths using a single shot with spectral editing which can lead to inconsistent results  stage, experiencing first episode psychosis (FEP), or with established schizophrenia using UHF-1HMRS  of interest could help eliminate and/or better estimate the effects of macromolecule contaminations. At lower field strengths, especially with the use of spectral editing, studies report GABA as GABA+ to indicate the macromolecule contamination. Using higher field strengths, high order shimming and local power optimization is crucial to minimize field-dependent inhomogeneities and to ensure accurate RF pulse frequency profiles. Inaccurate RF pulse profiles are common among pulses with larger flip angles such as 180° and to achieve the exact 180° flip can be difficult resulting in inaccurate isolation of metabolites. Furthermore, with increasing field strengths, transverse (T2) relaxation times and J-modulation effects decrease at a faster rate relative to lower field strengths. Hence, the quality of results is dependent on MRS pulse sequence use and effective echo times to ensure better detection. In the case of using a long echo time to ensure detection of GABA, 7T poses higher specific absorption rate, also known as tissue heating, when using a continuous pulse along with signal decay with shorter T2 relaxations. This poses a problem when detecting internal references such as creatine and water. Sequences have become robust to target signal loss via shorter echo times, however, the quality of data using CRLB or CV have reported higher than the accepted 20% threshold.To date, MRS has been the most studied modality with UHF neuroimaging in psychosis, and many of these studies have investigated multiscale associations at two or more levels, from the microscale to the social scale. Though important differences are detectable at smaller sample sizes, future studies with larger samples can ensure representativeness and allow for investigation of potential mediating/moderating effects of sociodemographic  and clinical  variables. Conducting longitudinal research across multiple scales will be essential to understand the therapeutic utility of MRS markers in the long term.in vivo, which is not possible at lower field strengths. In addition, UHF-sMRI can also more clearly visualize smaller structures and subregions. A prime example relevant to psychiatric applications is the hippocampus, its subfields, and adjacent white matter output regions, which are strongly implicated in schizophrenia  is the potential to non-invasively image the laminar structure of the brain ophrenia , 41. QuaTo date, 11 studies have assessed brain structure using UHF-sMRI in schizophrenia-spectrum disorders, including schizophrenia, schizoaffective disorder, first-episode psychosis, and childhood-onset schizophrenia . IwabuchOther UHF-sMRI findings highlight multiscale associations between psychotic symptoms and structural alterations in schizophrenia, particularly within the cingulum bundle and cingulate cortex. Palaniyappan et al.  reportedin vivo, as exemplified by imaging of the hippocampus dentate granule cell layer  enjoys the same advantages as UHF-sMRI in terms of increased spatial resolution, leading to smaller voxel sizes and, thus, reduced partial volume errors. This improves blood-oxygen-level-dependent signal estimates from smaller brain regions, particularly in subcortical structures, and allows for more precise estimates of brain connectivity. Moreover, UHF confers increased temporal resolution as well as shortened acquisition times in some cases, which allows for greater sensitivity and benefits participants with less time in the scanner, which is particularly relevant for those with psychiatric disorders, such as schizophrenia. Novel techniques, including direct imaging of neuronal activity (DIANA-fMRI) recently demonstrated in rodents , offer un = 9) employed first-episode psychosis or first-episode schizophrenia samples. Resting-state findings included both increased and decreased connectivity patterns in psychosis relative to healthy controls (We identified 11 UHF-fMRI studies, including 8 resting-state and 3 task-based studies using either a Stroop or passive listening task . The majcontrols , 54, concontrols  reportedcontrols , 57, thecontrols , 58, 59,controls , 36, 59.controls  identificontrols  demonstrUHF-fMRI in psychosis has primarily leveraged quantitative improvements over lower field strengths, including greater signal to noise ratio and reduced partial volume effects relative to lower field strengths. This leads to greater sensitivity  in modesThough UHF-fMRI brings with it several advantages in terms of spatiotemporal resolution that benefit studies involving small brain regions and connectivity, these have not yet been widely implemented in schizophrenia. Dedicated UHF-fMRI acquisition protocols may help bring out both the quantitative and qualitative advantages of UHF in fMRI research. At the same time, as with UHF-sMRI, head motion is a major consideration for UHF-fMRI studies, as its effects on signal integrity are exacerbated at higher magnetic field strengths, which can be particularly problematic in populations with more excessive motion concerns (e.g., psychosis; ]. SeveraPsychiatric symptoms fluctuate over time as do their underlying neurobiological markers, which underscores the importance of longitudinal multiscale investigations. Though the relative novelty and limited accessibility of UHF scanners have hindered acquisition of longitudinal data, three longitudinal studies have been published to date. These studies primarily focused on metabolite variations between first-episode psychosis patients and healthy controls using UHF-MRS.in vivo imaging of astrocytes, hence the use of myo-inositol as a biomarker to assess astroglial activity. At baseline, FEP patients showed reduced myo-inositol concentration in the ACC relative to controls that normalized after treatment, indicating dynamic astroglial processing in FEP and supporting myo-inositol as a biomarker of psychosis.Jeon et al.  examinedContinuing redox imbalances, glutathione and glutamate have been observed to improve early intervention outcomes among patients that are treatment resistant to dopamine-blocking antipsychotics. Dempster et al.  assessedIncreasing availability of UHF scanners along with pulse sequence and coil developments will facilitate longitudinal studies assessing structural, functional, and chemical metrics in the pathophysiology of schizophrenia across various illness stages. Collaborative efforts to harmonize and acquire 7T data across the emerging global centers will be critical to account for relatively small sample sizes reported in the current literature.This review highlighted key findings, insights, challenges, and future directions of studies to-date of psychosis using UHF neuroimaging. Due to its increased SNR, resolution, and sensitivity, UHF neuroimaging shows promise in uncovering novel neurobiological mechanisms underlying psychiatric disorders and disentangling findings derived from lower field strengths. With the rapid increase in popularity and interest in applying UHF in psychiatric and neurocognitive disorders, there is an expectation for the sophisticated modality to provide new or confirm existing findings at lower field strengths with concrete biomarkers for psychosis. Replication of robust findings at 3T, such as hippocampal volume reductions in schizophrenia and associations with symptoms, demonstrate that potential artifacts at UHF can be addressed. UHF studies that judiciously combine multiple scales of measurements from live subjects are providing greater insight into the cascading effects of dysconnectivity from the synapse-to-the-streets, clarifying multiple manifestations of psychosis. Expanding UHF multiscale studies to combine structural and functional imaging with neuro-metabolite studies will allow researchers to target as-of-yet undetectable metabolites and gather evidence of metabolite imbalances at the brain network level. Incorporating these studies with behavioral and experiential measures at the manifest and social scales will facilitate translational research and broader clinical applications, including precision diagnostics and treatment. UHF research in psychiatry is still in its infancy and most studies are affected by small sample sizes and limited acquisitions, yet its sensitivity in demonstrating neurochemical, structural, and functional changes in psychosis despite these limitations highlights its clinical potential.KL led preparing the review and drafted the initial version along with KK who wrote a part of the first draft. LP conceived, directed the review, and revised the manuscript. All authors approved the final draft and agreed to be accountable for the content of the work.


Liquid biopsies allow for the detection of cancer biomarkers such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Elevated levels of these biomarkers during cancer treatment could potentially serve as indicators of cancer progression and shed light on the mechanisms of metastasis and therapy resistance. Thus, liquid biopsies serve as tools for cancer detection and monitoring through a simple, non-invasive blood draw, allowing multiple longitudinal sampling. These circulating markers have significant prospects for use in assessing patients’ prognosis, monitoring response to therapy, and developing precision medicine. In addition, single-cell omics of these liquid biopsy markers can be potential tools for identifying tumor heterogeneity and plasticity as well as novel therapeutic targets. In this review, we focus on our current understanding of circulating tumor biomarkers, especially in breast cancer, and the scope of novel sequencing technologies and diagnostic methods for better prognostication and patient stratification to improve patient outcomes. Breast cancer is the most prevalent malignancy among women in the United States, with an estimated 287,850 newly diagnosed cases in 2022 . It is pIn the luminal A subtype, which accounts for 50–60% of breast cancer cases, the tumors are positive for estrogen receptor (ER) and/or progesterone receptor (PR) . BecauseThe most ominous subtype is triple-negative breast cancer (TNBC), characterized by the lack of ER and PR expression and HER2 amplification . TNBCs aBreast cancer is thus a heterogeneous and complex disease with a wide range of histologic characteristics, treatment responses, metastatic activity, and patient outcomes. Apart from age and genetic, hormonal, and reproductive components of breast cancer risk, there are modifiable factors such as excess body weight, physical inactivity, alcohol use, and receipt of hormone replacement therapy. Breast cancer incidence in women in the United States increased by an average of 0.3% per year between 2004 and 2018 . In addiLiquid biopsies are emerging as a minimally invasive method for early detection and risk management of breast cancer. The purpose of this review is to examine the utility of liquid biopsy indicators of breast cancer, circulating tumor cells (CTCs), and circulating tumor DNA (ctDNA), as well as current improvements and technological advancements in the field.Tumor biopsies have been the standard method for tumor tissue detection thus far. Tissue biopsy specimens are collected from primary or metastatic tumor sites to generate a histopathological and genetic profile, determine the patient’s prognosis, and guide therapy selection. However, traditional tissue biopsies are limited by their invasive nature, potential risk linked with tumor site, expense of the procedure, difficulties in accessing certain sites, and processing time. In addition, they are insufficient to portray the complete genomic picture and heterogeneity of the tumor. These limitations necessitate the development of alternative blood-based markers for therapeutic use.Liquid biopsies were developed as a minimally invasive and less expensive alternative to tissue biopsies ,13. LiquThe first blood-based circulating tumor marker to be discovered was CTCs in 1869, found in patients with metastatic cancer; circulating nucleic acids were discovered in 1948 ,15. LateCTCs are disseminated cancer cells from the primary or metastatic tumor(s) that are found in the peripheral blood. Their origin from multiple tumor sites, some of which may undergo the phenomenon of epithelial-to-mesenchymal transition (EMT), makes CTCs a diverse population reflecting the spatiotemporal heterogeneity of the disease . CTC idectDNA is fragmented DNA originating from the tumor and released into the blood; it is a subset of cfDNA, which can originate from any cell in the body. ctDNA, like other circulating blood indicators, can be produced by apoptosis, necrosis, or active shedding of tumor cells. However, apoptosis is the most common method, and hence cleaved DNA is usually around 140–180 bp in length ,21,22. TPCR-based ctDNA detection requires knowledge of the mutations or alterations to be measured, mostly by tissue biopsies or frequently targeted hotspot mutations. Whole-genome and whole-exome sequencing are used in untargeted techniques. Some PCR techniques include digital drop polymerase chain reaction (ddPCR), amplification refractory mutation system PCR, peptide nucleic acid/locked nucleic acid-mediated PCR, pyro phosphorolysis-activated polymerization, and beads, emulsion, amplification, and magnetics (BEAMing) ,28,29,30Early stage breast cancer is more commonly diagnosed than more advanced breast cancer, but around 20% of these patients have recurrence . CTCs inMany researchers have looked at the detection and characterization of CTCs in early stage breast cancer peripheral blood. For HER2-positive patients with primary breast cancer, the GeparQuattro clinical study examined neoadjuvant chemotherapy (NACT) that included trastuzumab . The resAnother group evaluated the prognostic significance of cytokeratin (CK-19)-positive CTCs in early stage breast cancer after NACT and identified it to be an independent risk factor . Sandri A large prospective trial of primary breast cancer patients revealed the independent prognostic value of CTCs before and after NACT . The BEVInterestingly, in a secondary analysis performed on a randomized clinical trial of patients with localized breast cancer for five or more years following diagnosis, detection of CTCs was related to a greater probability of recurrence in HR-positive patients . FollowiMany approaches have been used in the past decade to detect and measure ctDNA in patients with early stage breast cancer. For example, Beaver et al. detected ctDNA in the plasma of early stage breast cancer patients using primary breast tumors with matched pre- and post-surgery samples . PIK3CA Researchers have assessed ctDNA amounts in patients with early stage breast cancer for the purpose of reducing mortality by early detection and therapy modification. Riva et al. investigated the presence of ctDNA in a cohort of patients with nonmetastatic TNBC to examine whether ctDNA was associated with response to NACT and measure MRD after surgery . Using dA principal caveat of tissue biopsies is their inability to track changing genomic profiles over time, which liquid biopsies can overcome through serial sampling. Longitudinal fluid biopsy sampling allows precise monitoring of therapeutic efficacy and tracks the development of treatment resistance. Many recent studies have attempted sequencing ctDNA to generate mutation profiles to identify gene alterations in the resistant clones. Such mutation tracking showed ctDNA to be associated with relapse in early stage breast cancer ,52,53. dRothe and colleagues looked at the relationship between ctDNA and response to anti-HER2 therapy and discovered that HER-2-enriched tumors with no ctDNA had the greatest pathologic complete response rates at baseline, indicating that ctDNA can be used as a biomarker for NACT response in HER-2-amplified breast cancer . Along tResearchers also looked into the use of ctDNA analysis for diagnosing early stage breast cancer after mammography results . They anAnother study examined the efficacy of ctDNA to predict relapse in TNBC patients with residual disease after NACT . Using tAlthough significant strides have been made toward improving breast cancer survival rates, resistance to treatment develops in many patients and eventually leads to death from metastatic breast cancer. CTCs are released into the bloodstream of patients with solid tumors, functioning as seeds for subsequent metastasis. Elevated levels of CTCs during cancer treatment are an indicator of cancer progression and therefore can reveal the mechanisms of metastasis. CTCs in metastatic breast cancer provide more information than those in early stage breast cancer because they reflect the dominant clones at metastatic homing sites and aid in quantifying the remaining tumor burden. Thus, CTCs are invaluable tools for cancer detection and monitoring through a simple, non-invasive blood draw, allowing multiple longitudinal sampling.In early efforts to evaluate the predictive values of CTCs in metastatic breast cancer, CTCs were indeed revealed to be a strong independent prognostic marker for the disease . Martin p < 0.0001) and OS  compared with <5 CTCs/7.5 mL plasma at baseline. Furthermore, increased CTC counts 3–5 weeks and 6–8 weeks after treatment were associated with shorter PFS and OS. These data shed light on the independent prognostic value of CTCs for PFS and OS of metastatic breast cancer patients.A pooled analysis of individual patient data was acquired from 17 European centers to evaluate the clinical validity of CTC quantification in the metastatic breast cancer prognosis . Using aBecause high CTC levels have been linked to poor prognosis, the SWOGS0500 trial was designed to see if switching chemotherapy in metastatic breast cancer patients with persisting CTCs after the first cycle of first-line chemotherapy would improve OS . After 2Cristofanilli et al. tested the predictive utility of CTCs for stratifying the patients with stage IV metastatic breast cancer . In a reThe DETECT study program aimed to assess treatment interventions in metastatic breast cancer patients using CTC phenotypes . The triIn HR-positive, HER2-negative metastatic breast cancer patients, the STIC study was designed to assess the efficacy of CTC-driven vs. clinician-driven first-line therapeutic choices . In thisAnother clinical trial, CirCe01, looked into the efficacy of CTC-based monitoring of patients with metastatic breast cancer after they had completed their third line of treatment . PatientIn metastatic breast cancer, CTCs can give clues about the genomic landscape of the different tumor populations and the tumor burden. Many studies have recently looked into the prognostic significance of ctDNA. Dawson et al. performed a comparative analysis of conventional serum marker CA15-3, CTCs, and ctDNA in 30 women with metastatic breast cancer receiving systemic therapy. ctDNA was detected in 97% of the patients, and their levels had a greater dynamic range and correlation than CA15-3 and CTCs . BettegoShaw et al. examined whether the mutation profiles of cfDNA would capture the heterogeneity exhibited in numerous single CTC profiles . In 112 Murtaza et al. found that ctDNA can represent the clonal hierarchy of breast cancer, making it a valuable tool for detecting inter- and intra-metastatic heterogeneity . They pen = 800, kappa 0.89–0.93). Their findings show that targeted therapies against uncommon HER2 and AKT1 mutations have clinically relevant activity, indicating that these mutations could be used to treat breast cancer. They concluded that with adequate clinical validity for introduction into standard clinical practice, ctDNA testing could provide accurate, quick genotyping that permits the selection of mutation-directed therapy for patients with breast cancer. The study’s results highlight the importance of ctDNA analysis in the development of mutation-directed medicines. Early ctDNA dynamics were found to be a predictor for PFS in advanced breast cancer in the BEECH trial . The BEELiquid biopsies make longitudinal sampling possible through a patient’s treatment period, provide information on the changing mutation profile of the disease in real time, and serve as predictive markers for precision medicine. NGS-based approaches can be used to build cancer mutation profiles, which can then be used to create patient-specific panels for customized treatment. Coombes and colleagues developed a tailored ctDNA profiling method for detecting breast cancer recurrence . A patieButler et al. conducted whole-exome sequencing on the cfDNA and primary tumor of two metastatic patients . They diThe PI3K/AKT/mTOR pathway is frequently dysregulated in cancer and was shown to play a critical role in tumorigenesis and treatment resistance . The losPARP inhibitors are synthetically lethal to TNBC tumors harboring BRCA1/2 aberrations by impairing the DNA repair mechanisms . DetectiAlthough the field of liquid biopsies is an attractive area of investigation because markers are easily accessible in biological fluids, it is in the process of early development and faces overarching challenges. The main challenge is that most clinical studies of liquid biopsies are retrospective and limited by a small patient cohort. In addition, there is a lack of uniform protocols for sample procurement, storage, and handling as well as for isolation, quantification, and analysis tools. Likewise, standardized methods for biomarker sensitivity and specificity assessment are unavailable. These limitations emphasize the importance of large-scale studies to validate liquid biopsies for clinical application and to standardize protocols and methodologies.Despite substantial evidence from several analyses that high CTC levels can be an independent prognostic factor, data from the SWOG0500 trial demonstrated that switching chemotherapy in patients with persistent CTC levels has no benefit . AnotherOne of the challenges to the implementation of CTC-based liquid biopsies is the heterogeneity of CTCs, which is affected by many factors. For example, CTCs released into the bloodstream from the primary tumor often differ from those released from metastases. Some CTCs may undergo EMT during intravasation and mesenchymal-to-epithelial transition (MET) during the extravasation . During The CellSearch is an immuno-based method for detecting and isolating CTCs from blood and is the only method to have received FDA approval . It invoCTCs also vary in size, typically from 8 to 20 microns in diameter, similar to or smaller than white blood cells but larger than the other blood cells ,92. In aThe rarity of CTCs in billions of blood cells makes isolating high yield, high purity CTCs difficult. CTCs in metastatic breast cancer revealed mainly mesenchymal and epithelial-mesenchymal phenotypes, suggesting that EpCAM- and cytokeratin-based enrichment approaches may not capture cells undergoing EMT . A cocktCompared with CTCs, the prognostic value of ctDNA needs to be further investigated, as few driver mutations have been identified in breast cancer and low amounts of ctDNA have been detected in early stage breast cancer. Additionally, pinpointing the tumor source of circulating DNA fragments remains difficult. While cfDNA levels in a healthy individual can be 3–15 ng/mL of plasma, they can vary from 10 to >1000 ng/mL of plasma in a patient with advanced disease . Even thctDNA sequencing is a non-invasive method for detecting cancer mutations that reduce biopsy-related costs and inconveniences in patients with breast cancer. For sensitive detection of known molecular alterations, methods such as ddPCR and BEAMing are more appropriate. However, ctDNA sequencing using NGS enables a greater detection rate of mutations per patient sample and copy number variations. In addition, NGS of ctDNA is convenient for investigating the presence of genetic aberrations, and this approach is an increasingly favorable method in precision oncology, where tissue biopsies are often inadequate for molecular characterization.In the past decade, immense progress in the field of NGS has expedited the development of liquid biopsy markers for diagnostic assays. Recent studies indicate that CTCs can be used to predict late recurrence and hence guide treatment selection. However, a major challenge in the field has been the conflicting reports involving CTCs and ctDNA analysis in metastatic breast cancer . These dEmerging technologies such as the single-cell sequencing of CTCs can be a potential tool to identify spatiotemporal tumor heterogeneity, plasticity, and novel pharmacological targets for predicting clinical outcomes and treatment response ,108. SinProspective studies of biomarker measurement in large patient cohorts and healthy at-risk individuals will help overcome some of the existing drawbacks of using liquid biopsies for diagnosis and prognosis. Moreover, a combined application of blood-based biomarkers such as cfDNA, ctDNA, and CTCs could benefit their clinical application . The comKeup et al., performed an integrative statistical analysis of multiple liquid biopsy analytes (LBAs) in metastatic breast cancer . To examCirculating markers have a vast potential in prognosis, therapy monitoring, and guiding precision medicine for patients. However, there is an urgent need to develop more sensitive and accurate diagnostic methods in the early stages of breast cancer when minute liquid biopsy markers are detected. Recent breakthroughs in molecular technologies such as NGS and ddPCR have paved the way for developing microfluidic chip-based cell separation techniques with diagnostic potential. Early detection of breast cancer in its early stages would be conceivable with such solid and sensitive approaches, and current research is headed in that direction. A complementary multi-marker analysis for generating personalized therapeutics is needed to improve liquid biopsy markers’ accuracy and diagnostic capabilities. Liquid biopsies have great potential for prognosis, therapeutic monitoring, and integrative data processing methodologies, and developments in sequencing technologies will speed up the era of precision medicine.In early stage disease, CTC and ctDNA monitoring will be a significant addition to currently used tools aimed at detecting and subtyping the disease for alignment with therapies. Liquid biopsy monitoring will help identify patients who are at high risk of relapse. Another important element of an overall strategy to reduce the risk of relapse will be novel therapies that are safe and effective, and therefore can be offered as early as possible after diagnosis. Considering a high degree of intratumoral heterogeneity, which is also reflected in liquid biopsies, such therapies will likely be phenotype-based rather than driver-based. In this regard, we have proposed a strategy for modeling a phenotype in cell culture that is crucial in poor prognosis minimal residual disease, i.e., an opportunistic switching of cancer cells between quiescence and proliferation ,116,117.