Little information is available on current practice in beta-lactam dosing during continuous renal replacement therapy (CRRT). Optimized dosing is essential for improving outcomes, and there is no consensus on the appropriate dose regimens. The objective of the present study was to describe current practice for beta-lactam dosing during CRRT in intensive care units (ICUs).We conducted a nationwide survey by e-mailing an online questionnaire to physicians working in ICUs in France. The questionnaire included three sections: demographic characteristics, CRRT practices, and beta-lactam dosing regimens during CRRT.157 intensivists completed the questionnaire. Continuous venovenous hemofiltration was the most frequently used CRRT technique, and citrate was the most regularly used anticoagulant. The median prescribed dose at baseline was 30 mL/kg/h. The majority of prescribers (57%) did not reduce beta-lactam dosing during CRRT. The tools were used to adapt dosing regimens during CRRT included guidelines, therapeutic drug monitoring (TDM), and data from the literature. When TDM was used, 100% T > 4 time the MIC was the most common mentioned pharmacokinetic/pharmacodynamic target (53%). Pharmacokinetic software tools were rarely used. Prolonged or continuous infusions were widely used during CRRT (88%). Institutional guidelines on beta-lactam dosing during CRRT were rare. 41% of physicians sometimes consulted another specialist before adapting the dose of antibiotic during CRRT.Our present results highlight the wide range of beta-lactam dosing practices adopted during CRRT. Personalized TDM and the implementation of Bayesian software appear to be essential for optimizing beta-lactam dosing regimens and improving patient outcomes.The online version contains supplementary material available at 10.1186/s12882-022-02678-x. Beta-lactams are the most widely prescribed antibiotics in critically ill patients. Optimized dosing of beta-lactams is required to deal with pharmacokinetic changes and frequent underdosing – especially during the early phase of sepsis –3. RenalGiven the absence of data on the beta-lactam dosing prescribed during CRRT in France, we designed a survey to assess current practices. It was developed by a pharmacy resident with help from a critically ill clinical pharmacist and an intensivist, after a review of the literature. We performed an online, nationwide, cross-sectional survey between July and September 2019 by emailing a questionnaire to 1423 senior physicians working in intensive care units (ICUs) across France. The survey included 22 questions on the respondents’ characteristics, CRRT practices, and beta-lactam dosing regimens during CRRT. The English version of the questionnaire and the results are given in Table The results were presented as the frequency (percentage) for qualitative variables and the median [interquartile range (IQR)] for quantitative variables. For statistical comparisons of different groups, we applied Pearson’s chi-square test with Yates’ correction. All tests were two-sided, and the threshold for statistical significance was set to p<0.05. Statistical tests were performed using SAS® software .Of the 1423 physicians contacted, 157 (11%) replied. Physicians working in university hospitals accounted for 50% of the respondents, whereas 45% of the respondents worked in public-sector general hospitals and 5% worked in private for-profit or non-profit hospitals. They had a median of 10 years  of experConcerning renal replacement therapy (RRT), CRRT was preferred to intermittent hemodialysis (70%). The CRRT techniques used by intensivists were variously venovenous hemofiltration (73%), venovenous hemodiafiltration (57%), venovenous hemodialysis (55%), and Sustained Low-Efficiency Dialysis (8%). Half of the physicians (52%) reported prescribing continuous venovenous hemofiltration preferentially, followed by continuous venovenous hemodialysis (35%). 64% of the respondents used citrate as the anticoagulant. 77% of the physicians prescribing hemofiltration reported using a combined predilution and postdilution modality. The median prescribed dose at initiation was 30 mL/kg/h, and 75% of the prescribers considered the total body weight (with 39% for body weight on admission and 36% for body weight on the day of the CRRT prescription). 12% of the respondents reported prescribing a flow effluent irrespective of body weight. Polyacrylonitrile was the most commonly used membrane material , followed by polysulfone (39%).Guide Prescription et Rein) (74%), TDM (62%), and data from the literature (35%) to adjust the beta-lactam dosing regimens. Only 4 physicians reported using pharmacokinetic software tools. When TDM was used, 100% T > 4 MIC was the most common pharmacokinetic/pharmacodynamic (PK/PD) target. 41% of the respondents sometimes consulted another specialist when deciding whether or not to adjust the beta-lactam dose during CRRT: this was variously an infectious disease specialist (29%), a microbiologist (10%), a pharmacist/pharmacologist (8%), a nephrologist or toxicologist (5%). The replies to the clinical vignettes highlighted a broad range of dose adaptation practices - particularly for meropenem  and a call to other specialists for advice on antibiotic dosing regimen adjustment during CRRT (p<0.0001)  did not adjust the doses. 17% and 9% of them prescribed full doses for 24 and 48 h, respectively, before reducing the dosing regimens. 13% of respondents reported prescribing a single loading dose before dose adjustment and 4% reported that their use of a reduced dose or a full dose depended on the antimicrobial agent in question. Only three physicians reported using reduced doses all the time. A dose adjustment could be either empirical or adapted according to the TDM results. In 85% of cases, the respondents did not adapt the beta-lactam dosing as a function of the CRRT dose or the effluent flow (85%). The physicians used mainly the French renal prescription handbook  were included in the survey. The majority were intensivists or anesthesiologists, which reflects the fact that anesthesiologists are qualified for critical care medicine in France.In order to review beta-lactam dosing in CRRT, we analyzed compliance with the French guidelines and Kidney Disease: Improving Global Outcomes (KDIGO) guidelines , 22. OurPhysicians may not be sufficiently aware of the need to maintain beta-lactam full doses during the initial phase of treatment. Administration must be optimized in order to maintain effective antibiotic concentrations for a sufficiently long time and thus maximize the chances of therapeutic success. This is especially true in the initial phase of sepsis when cardiac output and capillary permeability increase and protein binding is altered; this leads to increased clearance and a larger volume of distribution, inducing low serum and tissue beta-lactams concentrations , 30. UndOur survey highlighted the broad implementation of extended and continuous infusions and so showed that physicians were well aware of the time-dependent nature of the beta-lactams’ activity. Even though the guidelines recommending the use of extended and continuous infusions for all compounds (to increase the probability of target attainment), these modalities are still mainly used for a few beta-lactams only and adherence to guidelines is suboptimal , 20. ThiThe prescription tools used by the majority of physicians are not designed to recommend personalized dosages, and the use of pharmacokinetic software for finer dosage adjustment is rare. However, beta-lactam TDM is increasingly being used in CRRT, in line with the guidelines , 20. TheThe diversity of PK/PD targets emphasizes the uncertainties in the literature data, even though most physicians are well aware that a beta-lactam concentration over several times the MIC is required throughout the dosing interval . HoweveThe duration of the beta-lactam infusion was not defined in the questionnaire’s clinical vignettes; this limited the interpretation of our respondent’s practices but highlighted the diversity of dosing regimens used. Although meropenem is the best-studied beta-lactam in CRRT, its dosing regimens differed most significantly among our respondents  was explained by recruitment bias, since the survey was conducted during the summer vacation. Thirdly, we did not have an exhaustive list of intensivists in France. However, all types of hospitals  were represented. Despite these limitations, we undertook the largest yet study of this type in French ICUs. The diversity of replies to our questionnaire highlights the uncertainties regarding dosage adjustments required in CRRT and the lack of harmonization of PK/PD targets and emphasized the need for further research on a topic that is crucial in critically ill patients.The diversity of beta-lactam dosing regimens and the tools used to adjust it may be responsible for significant morbidity and mortality. Optimal antimicrobial dosing during CRRT remains challenging. Personalized TDM and the use of Bayesian softwares appear to be fundamental for optimizing beta-lactam dosing regimens and improving patients’ outcomes. However, low availability and a lack of clinical validation limit the implementation of these tools.Additional file 1.


Management of patients with lung cancer continues to be challenging during the COVID-19 pandemic, due to the increased risk of complications in this subset of patients. During the COVID-19 surge in New York City, New York University Langone Health adopted triage strategies to help with care for lung cancer patients, with good surgical outcomes and no transmission of COVID-19 to patients or healthcare workers. Here, we will review current recommendations regarding screening and management of lung cancer patients during both a non-surge phase and surge phase of COVID-19. The novel Coronavirus disease 2019 (COVID-19) is a respiratory viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Since first identification of SARS-CoV-2 in December 2019, COVID-19 emerged as a major global pandemic with over 98 million people diagnosed with COVID-19 and >2 million deaths worldwide as of January 2021 . The UniEarly data, primarily from China, demonstrated that 15–20% of infected patients develop symptoms requiring hospitalization with a mortality rate of ~1–4% . PatientCOVID-19 cases continue to increase in number. While the vaccines promises to help slow the spread of COVID-19, new strains pose as another source for continued growth –23. It iIn this review article, we will discuss lung cancer management during COVID-19 pandemic and present our current institutional practice and outcomes. The work-up and management of patients depends on rate of new infections and hospitalizations for COVID-19. At NYU Langone Health (NYULH), we follow a tiered approach based on transmission rate. Transmission of COVID-19 outside of New York City (NYC), with little or no cases in NYC, and no cases at NYULH is deemed level 1. Level 2 is when there are no local transmission, but multiple cases in NYC and few NYULH cases. Transmission in NYULH is level 3, while level 4 is when NYULH surge units are full .If the city and hospital are not in a surge phase of COVID-19 outbreak and have available resources, standard criteria for lung cancer work-up and management should be followed with several added precautions. Patients with lung cancer require frequent clinic visits with multiple specialists, and necessitate diagnostic work up with laboratory blood draws, imaging studies, pulmonary function tests (PFTs), bronchoscopies and transthoracic needle biopsies, radiation treatments, anti-neoplastic infusion treatments and hospital admissions for procedures. These frequent visits increased both the patient risk of exposure to COVID-19, as well as increased risk of exposure to healthcare providers taking care of lung cancer patients .All patients presenting for in-person appointments should be contacted 24–48 h prior to the appointment and screened for flu-like symptoms: cough, dyspnea, fevers, chills, and loss of smell or taste. If patients have any symptoms during the telephone screening, the appointments are canceled. If patients required further evaluation of symptoms, they should be triaged for COVID-19 testing and referred to emergency room when appropriate. If patients test positive for COVID-19, in-person clinic and infusion appointments were deferred until patients were asymptomatic for a minimum of 14 days. Those requiring more immediate attention for their cancer management should be admitted for inpatient management.All healthcare providers must wear N95 masks, eye shield, and gloves for patient contact. Face masks or coverings should be worn by all visitors and patients, regardless of their symptoms. Patient entry into the cancer center should restricted to one point of entry and access was controlled by a team of medical assistants and a nurse. COVID-19 clinical screening questions and COVID-19 test results should documented prior to registration and in-person clinic visit. If a patient screens positive with suspicion for COVID-19, the patient should be shifted to an isolation area. The patient's team would perform a clinical assessment in the isolation area and determine the appropriate action. If patients needed to be tested for COVID-19, the clinic visit and treatments were delayed until the results of the test were available.Previously, there was concerns about bronchoscopy as a super-spreading event , but ourPatients with suspected diagnosis or newly diagnosed lung cancer require a variety of imaging modalities, diagnostic biopsies, and staging procedures. Following announcement of COVID-19 as a national emergency in March 2020, guidelines were issued by the American Association of Bronchology and Interventional Pulmonology and the American College of Chest Physicians , which iAll cases considered for bronchoscopy should be discussed at our multi-disciplinary thoracic oncology conference (MDTOC) to determine the optimal course for diagnosis and staging from the perspective of both the patient and the lung cancer team. Staging endobronchial ultrasounds were discouraged in patients without radiographic lymphadenopathy. In these patients, mediastinal and hilar staging via EBUS guided transbronchial needle aspiration (TBNA) was deferred, given the relatively low likelihood of macro- or microscopic metastases in regional lymph nodes in this setting. This was particularly valid if the standardized uptake value (SUV) of the primary lesion in the lung parenchyma was relatively low  on positron emission tomography (PET). Conversely, patients with compelling evidence of malignant adenopathy were managed without tissue confirmation .Diagnostic biopsies of peripheral lung nodules or masses in the absence of significant lymphadenopathy were performed by transthoracic needle aspiration by interventional radiology. This was considered a lower risk procedure for aerosolization of the SARS CoV-2 virus than navigational or robotic bronchoscopy for biopsy of these lesions.For patients with confirmed peripheral lung cancer on prior biopsy who were being considered for definitive stereotactic body radiotherapy (SBRT) by radiation oncology or percutaneous cryoablation by Thoracic Radiology, we deferred staging EBUS TBNA of hilar and mediastinal nodes in those without significant adenopathy on cross sectional imaging or evidence of mediastinal or hilar FDG avidity on PET CT scan.All patients with suspected new or recurrent diagnoses of lung cancer recommended at MDTOC to undergo EBUS TBNA should undergo SARS-CoV-2 nasopharyngeal swab RT-PCR testing prior to the procedure. Patients with negative SARS-CoV-2 testing could proceed to bronchoscopy with the interventional pulmonology team, with strict precautions within the operating room. These included full use of personal protective equipment by all OR staff , as well as ensuring that only essential staff were present in the operating room for anesthesia induction and securing the airway. Patients scheduled to undergo diagnostic and staging bronchoscopy who had positive SARS-CoV-2 RT PCR testing prior to the procedure were quarantined for 2 weeks and re-tested prior to proceeding with the endoscopic procedure.For those lung cancer patients presenting with symptomatic central airway obstruction , therapeutic bronchoscopy  should be performed as clinically indicated regardless of SARS-CoV-2 status. SARS-CoV-2 testing should still be performed within 3 days of the planned procedure for risk stratification and preparation for safest possible conditions for the interventional pulmonologists and the OR staff .We recommend continuing diagnostic and therapeutic pleural procedures in patients who are candidates for systemic anti-neoplastic therapy. Proceduralists should adhere to strict contact precautions with face shields, N95 masks, impermeable gowns, and sterile gloves. For patients symptomatic from known malignant pleural effusions, we recommend placement of indwelling pleural catheter (IPC) insertion. IPC placement is thought to be associated with lower risk of SARS-CoV-2 aerosolization than talc insufflation via video assisted thoracoscopic surgery (VATS) and could be performed in an expedited fashion on an outpatient basis. When patients had IPC insertions, a family member was remotely trained and counseled on safe and proper drainage of the IPC to obviate requirements for home visiting nurse encounters to minimize risk of COVID-19 exposure.max >2.5, or change on short interval CT scan were deemed higher risk for progression of disease with delay in care and prioritized for resection. Patients on neoadjuvant trials should also undergo resection during this surge phase if there are no other contraindications. The guidelines used by our multidisciplinary group at NYULH echoed the recommendations of the American College of Surgeons and the Thoracic Surgery Outcomes Research Network  of 21 patients was asymptomatic and found to be COVID-19 positive prior to surgery . SurgeryStandard post-operative care regarding chest tube management and discharge criteria should be used, including specific precautions regarding superspreading procedures such as extubation, and peri-operative respiratory therapy such as nebulized bronchodilators, incentive spirometry, high-flow nasal cannula oxygen and non-invasive ventilation. All in-hospital care was provided on specific COVID free operating rooms, ICUs and floors. No patients could have visitors during their hospital stay and follow up after resection was done using telehealth visits. Patients with T1/T2 N0 NSCLC were reviewed in the multi-disciplinary and if stereotactic radiation was an option, it should be recommended and discussed with patients. In patients for whom surgery was delayed, consideration was given for neoadjuvant systemic chemotherapy.Several changes in the clinical workflow should be made to meet adequate social distancing guidelines for lung cancer patients needing radiation. All follow-ups that do not require a physical exam should be changed to a virtual telemedicine platform. Most patients with stage I and II lung cancer considered for stereotactic body radiotherapy (SBRT) are elderly with poor lung function and other comorbidities that preclude surgical management; therefore, SBRT should be post-poned as appropriate during surge phase to minimize COVID-19 infection risk . FurtherGiven the lack of level I evidence and controversy surrounding post-operative radiotherapy for patents with fully resected N2 lung cancer , we recoAll patients should be evaluated for adequate pulmonary function, though this may be challenging as pulmonary function labs may be closed due to infection transmission concerns related to COVID-19. If recent pulmonary function tests are unavailable, we recommend clinical estimation of the pulmonary function using prior pulmonary function tests if available, baseline oxygen saturation, virtual walk desaturation testing and stair challenge. The standard radiation treatment time slots were increased from 15 to 20 min to minimize waiting room time as much as possible. All patients should masked, screened for COVID-19 symptoms, checked for elevated temperature, and maintained social distancing protocols. COVID-19 testing should be performed on all patients prior to CT simulation. Patients with active COVID-19 infection were treated with radiation therapy if the initiation or continuation of such therapy was deemed essential following MDTOC review. Such patients were typically scheduled for last patient of the day treatments followed by terminal room and facility cleans. With these protocols in place, we were able to continue offering radiotherapy without significant staff shortages necessitating forced patient delays throughout the pandemic.Management is different based on whether the hospital system is in surge phase and the capacity of the hospital. We have proven that lung cancer surgery can be safely performed in a hospital with census saturated with COVID-19 patients. Furthermore, we have had providers care for both COVID-19 patients and non-COVID-19 lung cancer patients, with a 0% transmission rate. It is not feasible to have dedicated non-COVID centers for oncologic surgery as it decreases available resources in a pandemic; therefore, the recommendations summarized in this review can guide cancer centers to continue to provide lung cancer care during COVID-19 pandemic with minimal added risk to patients and healthcare providers.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intratumoral hemorrhage/apoplexy in clival chordomas is extremely uncommon, with only 16 reported cases. The average age of patients was 46.2 years and slightly more than half were men. In cases published before 1990, all patients died from their disease without any intervention. Since then, 11 patients have undergone resection by a variety of approaches and there have been no deaths. The diagnosis of skull base chordomas relies on a combination of clinical presentation and radiographic features related to the location and invasion of the tumor. Chordomas presenting with sudden-onset symptoms should alarm the surgeon of a possible hemorrhage. As an illustration of this presentation, we describe a 58-year-old woman who presented with acute-onset headache and cranial nerve deficits. Computed tomography and magnetic resonance imaging demonstrated a hemorrhagic clival lesion with cavernous sinus extension. The patient underwent transsphenoidal resection of the lesion that resulted in the resolution of her symptoms. Histopathological evaluation of the lesion was consistent with chordoma with acute hemorrhagic components. Although intratumoral hemorrhage is rarely detected in chordomas, it should be considered a differential diagnosis of such lesions because prompt recognition and treatment are critical for patient survival. Skull base chordomas are aggressive recurrent intracranial lesions thought to originate from primitive notochord remnants and constitute <1% of all intracranial neoplasms . Clival A 58-year-old hypertensive woman with a remote history of breast cancer treated with mastectomy and chemotherapy presented to the emergency department with progressive left-sided acute-onset headache, nausea, vomiting, double vision, and left-sided partial ophthalmoplegia and ptosis of five-day duration. The neurological examination also demonstrated slight anisocoria consistent with partial left oculomotor nerve palsy. The left pupil was in the "down-and-out" position. Other neurologic findings were unremarkable. All pituitary hormones were within normal limits, except for a slightly elevated cortisol level  and a decreased adrenocorticotropic hormone value .Computed tomography (CT) of the head demonstrated a high-density lesion arising from the clivus, with extensive osseous erosion into the left greater wing of the sphenoid and left petrous apex and scattered hyperdensities within the tumor bed Figures -1C. MagnThe patient underwent a microscopic transsphenoidal approach with endoscopic assistance for excisional biopsy to establish a pathologic diagnosis and decompress the cavernous sinus. The left sphenoid sinus and clival recess were exposed. The tumor was removed from the left clival recess and followed into the left cavernous sinus, medial and lateral to the cavernous ICA. The cavernous component of the tumor was visualized and resected with 30° and 45° angled endoscopes. Once adequate decompression was achieved, hemostasis was obtained. Nasal packings were used for closure, and no cerebrospinal fluid leak was encountered.Histological examination revealed a moderately cellular tumor comprising plump cells with well-defined cytoplasmic borders Figure , foamy cThe patient tolerated the procedure well with no complications. Postoperatively, her ophthalmoplegia and ptosis improved dramatically, with no new neurologic deficits. She was discharged home on postoperative day 3 and referred for proton beam radiotherapy. On telephone follow-up seven months after her surgery, she reported complete resolution of her symptoms and no new neurological issues.Skull base chordomas are slow-growing, aggressive, recurrent tumors marked by extensive local invasion and destruction of surrounding structures . AlthougAcute hemorrhage is most commonly intratumoral 75%) % 5-15]  15] but   15] butThe diagnosis of skull base chordomas relies on a combination of clinical presentation, histopathologic, and radiographic features related to the location and invasion of the tumor. Chordomas presenting with sudden-onset symptoms should alarm the surgeon of a possible hemorrhage. Sudden headache 82.3%) ,8,15,17 2.3% ,8,1Skull base chordomas generally present as well-circumscribed, osteolytic, destructive lesions with associated cortical and lytic bone destruction and soft-tissue extension on non-contrast CT scans ,21, whicMacroscopically, chordomas are generally lobulated with a gelatinous matrix or chondroid solid features. Microscopically, chordomas take one of three forms. Chordoma not otherwise specified or classical chordoma is the most common form and is composed of lobules of epithelioid cells separated by septa of fibrous tissue in a myxoid matrix. The cells have vacuolated cytoplasm and are referred to as physaliphorous cells. Chondroid chordoma is the second histological form and comprises an extracellular matrix resembling hyaline cartilage. The last histological form is dedifferentiated chordoma, in which the tumor shows features of both classical chordoma type and undifferentiated spindle cell tumors . This foAlthough the cause of hemorrhage remains largely unknown, various mechanisms have been proposed. Some authors have reported that rapid tumor growth without adequate matched blood supply may cause these small friable vessels to rupture . VasculaDespite the importance of maximal surgical resection, adjuvant therapy remains an essential part of the armamentarium in the management of skull base chordoma, especially in cases where complete resection is not feasible . PostopeHemorrhagic clival chordoma arising from the cavernous sinus is a rare clinical and radiographic presentation. The presence of skull base lesions with mixed T2 hyperintensity and focal areas of hemorrhage and bone erosion should raise a high index of suspicion for hemorrhagic clival chordoma, which presents acutely with apoplexy and resulting neurological deficits, requiring prompt recognition and treatment by skull base neurosurgeons for patient survival.


A universal response to replication stress is replication fork reversal, where the nascent complementary DNA strands are annealed to form a protective four-way junction allowing forks to avert DNA damage while replication stress is resolved. However, reversed forks are in turn susceptible to nucleolytic digestion of the regressed nascent DNA arms and rely on dedicated mechanisms to protect their integrity. The most well studied fork protection mechanism involves the BRCA pathway and its ability to catalyze RAD51 nucleofilament formation on the reversed arms of stalled replication forks. Importantly, the inability to prevent the degradation of reversed forks has emerged as a hallmark of BRCA deficiency and underlies genome instability and chemosensitivity in BRCA-deficient cells. In the past decade, multiple factors underlying fork stability have been discovered. These factors either cooperate with the BRCA pathway, operate independently from it to augment fork stability in its absence, or act as enablers of fork degradation. In this review, we examine these novel determinants of fork stability, explore the emergent conceptual underpinnings underlying fork protection, as well as the impact of fork protection on cellular viability and cancer therapy. These mechanisms rely on stabilizing RAD51 nucleofilaments on the regressed nascent DNA of reversed replication forks. However, recent work has revealed a multitude of additional factors and pathways that directly affect fork protection by either influencing RAD51 nucleofilament formation or through completely distinct mechanisms. In this review, we examine the recent advances in the understanding of the classical fork protection pathways, as well as the emerging determinants of fork protection.The genome is at its most vulnerable during DNA replication since this process seldom progresses completely unobstructed. Several factors of endogenous and exogenous origin can pose obstacles to the progression of replication forks resulting in replication stress. A global response upon encountering replication stress involves the remodeling of replication forks to enable the annealing of nascent DNA strands, a process known as replication fork reversal. Fork reversal, intrinsically coupled to an initial fork slowing response, is thought to safeguard fork integrity by granting tolerance to transient replication stress. Mechanistically, the protective effect of fork reversal is thought to manifest in distinct ways: (i) Fork regression, which accompanies fork reversal, allows for the template lesion to be brought back into a double-stranded DNA configuration permissive to excision repair; (ii) The regressed complementary arms of nascent DNA can potentially allow for the bypass of template DNA obstructions by employing the nascent strand of the sister chromatid as a template; (iii) Fork reversal allows forks under stress to assume a dormant configuration until the replication stress can be resolved, thereby protecting them from adverse outcomes of unrestrained progression such as fork breakage. However, in certain genetic backgrounds or under conditions of prolonged fork arrest, reversal can render forks susceptible to nucleolytic resection. Excessive nucleolytic resection  of reversed forks is often associated with the accumulation of chromosomal abnormalities. Fork degradation is thus a prominent mechanism of genome instability. In addition, fork degradation is considered a major predictor of chemosensitivity and cellular fitness. The best described mechanism ensuring protection against fork degradation involves the BRCA proteins  by MUS81, triggering a POLD3-mediated fork restart—highly reminiscent of the break induced replication (BIR) pathway of fork rescue . Indeed,Recent studies have revealed the existence of several previously unknown factors involved in the protection of stalled forks from degradation. Interestingly, these factors employ fork protection mechanisms often distinct from the FA/BRCA pathway. However, several of these mechanisms still rely on the fork protection activity of RAD51. A relatively well-studied enabler of RAD51-mediated fork protection is BOD1L, which operates in the FA pathway of genome stability . HoweverA unique player in the RAD51-mediated fork protection pathway is RAD52. Despite its role in promoting MRE11-mediated degradation in BRCA2-deficient cells , recent The occurrence of fork degradation independently of defects in a RAD51-mediated fork protection mechanism was documented in HR-proficient cells subjected to prolonged HU-mediated fork stalling . As mentInterestingly, recent work has revealed novel players which operate specifically in protecting replication forks from undergoing degradation through the DNA2-WRN pathway. One such fork protection factor is ABRO1, a paralog of ABRAXAS, a BRCA1-interacting protein . ABRO1-mWe recently uncovered an unexpected role for PCNA ubiquitination in protecting stalled replication forks from degradation through the DNA2-WRN pathway . In lineRecent work has revealed roles for AND1 and TIM1, members of the fork protection complex  in protecting stalled forks from MRE11-mediated degradation. A role for TIM1 in fork protection was identified through the characterization of the PCNA-interacting genome surveillance protein SDE2 and its function in preventing fork degradation ,27. SpecA unique fork integrity pathway depends on the Y-family polymerase POLK. Recent studies showed that inactivation of POLK results in MRE11-dependent degradation of stalled forks . NotablyComponents of the non-homologous end joining (NHEJ) pathway of DSB repair have also been implicated in protecting the stability of stalled replication forks. Recent works have revealed a role for the NHEJ effector RIF1 in protection from the DNA2-WRN pathway of degradation ,35. NotaDespite the well-documented role of the loss of 53BP1, a master regulator of NHEJ, in restoring HR in BRCA1-deficient cells, 53BP1 loss has not been previously reported to exhibit functions in fork protection ,37. InteSchizosaccharomyces pombe (S. cerevisiae) and CtIP (human) functioning to trigger the endonuclease activity of MRE11 at protein-blocked 5′ DNA ends  bearing a homozygous genetic knockout of BRCA2 (BRCA2-KO) . ImportaIn line with the role of fork protection in restoring viability, the further enhancement of fork degradation was shown to contribute to synthetic lethality in BRCA-deficient cells. BRCA-deficient cells were found to be hyperdependent on FANCD2 for fork protection and survival ,17. HoweRestoration of fork stability often correlates with resistance to PARPi Figure . Restorain vivo.A specific context in which PARPi sensitivity correlates with fork protection defects is that of the activation of ssDNA-induced innate immune response. Nucleolytic processing of stalled replication forks was shown to induce activation of the cGAS-STING pathway triggered by the presence of cytosolic ssDNA . FurtherA significant body of work identified restoration of DSB repair as a major mechanism of acquired PARPi resistance. Our recent work showed that loss of E2F7 promotes PARPi resistance in BRCA2-deficient cells, and this correlates with both fork protection and HR restoration . BRCA2 DAnother mechanism implicated in PARPi-mediated cytotoxicity is the trapping of PARP1 and PARP2 proteins on the chromatin. Trapped PARP1/2 could directly pose obstacles to DNA replication, and thereby potentially underlie fork degradation and DSB formation Figure ,B. The cX. laevis egg extracts established a role for RAD51 and BRCA2 in suppressing gaps both at the junctions of replication forks, as well as MRE11-dependent gaps behind them . We further examined the downstream effects of the loss of fork protection upon the inactivation of these pathways on genome stability, as well as the impact of their combined inactivation with the BRCA pathway on cell viability. We also explored the emerging role of nucleosome remodeling and chromatin dynamics as determinants of stalled fork resection. Importantly, histone modifications and nucleosome remodeling may have a direct impact on the accessibility of nucleases to stalled fork substrates, thereby influencing fork resection.The inability to protect replication forks is associated with a vulnerability to treatment with chemotherapeutics in BRCA-deficient cells. Conversely, the artificial restoration of fork stability in BRCA-deficient cells has been shown to restore chemoresistance to these cells. This highlights the challenges in the treatment of BRCA-deficient cancers. In the last section, we examined mechanisms underlying the restoration of fork stability to BRCA-deficient cells. These include restoration of the RAD51 fork protective function, inactivation of replication fork reversal, increasing the reliance of BRCA-deficient cells on proteins which bolster fork stability . Finally, we evaluated fork degradation as a mechanism contributing to PARPi-mediated synthetic lethality. Recent findings showed that fork degradation, though often associated with PARPi sensitivity, may not be universally causative of PARPi-mediated cellular lethality. Instead, a more significant correlation may be found between PARPi sensitivity and the inability to mitigate replication-associated ssDNA gaps. These gaps may arise as a result of the effect of PARPi in suppressing key functions of PARP1 in mediating replication fork slowing upon stress encounter, as well as inducing PARP-trapping as a result of the aborted resolution of misincorporated ribonucleotides during DNA replication. We suggest an importance of unligated Okazaki fragments as lesions potentially contributing to PARPi-mediated cellular lethality. Collectively, these observations imply a complex role of fork protection in determining progression as well as therapeutic outcomes in BRCA-mutant cancers. Furthermore, the breadth of the emergent determinants of fork stability discussed here, may indicate a more significant role of fork stability in cancer etiology than previously anticipated.


The transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the culprit pathogen of coronavirus disease 2019 (COVID-19), has resulted in more than 0.43 billion cases and 6 million deaths globally. Compared with other existing variants of concern, the Omicron variant is characterized by significantly greater infectivity (Ro ≈ 6), greater tropism of the upper respiratory tract and lower severity of human infections [Different policies have been adopted worldwide during the COVID-19 era. The community-based dynamic zeroing policy has played a pivotal role in minimizing the number of laboratory-confirmed cases and deaths in mainland China. The rapid roll-out of vaccinations, as well as herd immunity, have formed the fundamentals of the ‘total opening’ of some developed countries. This, however, does not necessarily justify rapid and total opening in mainland China. Omicron confers a considerably higher mortality risk than seasonal influenza, and would likely result in social instability and the emergence of other novel variants with a greater infectivity.The dynamic zeroing policy has been adopted for maintaining effective disease prevention and control. However, China needs to reopen so as to normalize socio-economic development and adapt to global reopening. Prolonged dynamic zeroing cannot be pursued in the long run. There are several recommendations on how China could reopen in an orderly and effective manner.First, enforcing nationwide vaccination is crucial to safeguarding herd immunity. As of 28 February 2022, China has vaccinated 87.64% of the whole population with at least two doses of SARS-CoV-2 vaccines, with 40.32% completing the third-dose boosting vaccination. However, the vaccination rate was still markedly lower than 83% in the Chinese population aged >70 years in mainland China and even lower in Hong Kong  or the aSecond, efforts to reduce the risk of progression to critical illness and death would benefit from the acceleration of research and development of targeted medications and potent neutralizing antibodies. In addition to paxlovid and molnupiravir, the therapeutic effects of medications (e.g. VV116) and neutralizing antibodies (BRII-196 and BRII-198) have provided hope with regard to improving clinical outcomes, although conducting phase three studies within mainland China would be challenging due to limited patient sources. International collaboration should therefore be encouraged.Third, rapid antigen testing should be prioritized in community settings. Antigen testing has been associated with significantly shorter turnaround times and less reliance on medical facilities and personnel compared to nucleic acid testing. This ensures timely identification of the source patient, on which basis population-based screening with viral nucleic acid testing can be reserved for close contacts (particularly the imported cases).Fourth, we should strengthen longitudinal follow-up investigations of infected cases during the latency and convalescent periods. In-depth analysis of the infectivity of imported cases (including those showing re-positive test results) would inform the policy maker with regard to the minimal duration of quarantine and the management of re-positive cases after hospital discharge.Finally, performing pilot investigations in certain designated cities or regions, as well as adjusting the policy according to the epidemic characteristics of imported cases, will be equally vital for verifying the outcomes of the transition towards safe and orderly social reopening in China.nwac054_Supplemental_FileClick here for additional data file.


Perinatal white matter injury (WMI) is the most common brain injury in premature infants and can lead to life-long neurological deficits such as cerebral palsy. Preterm birth is typically accompanied by inflammation and hypoxic-ischemic events. Such perinatal insults negatively impact maturation of oligodendrocytes (OLs) and cause myelination failure. At present, no treatment options are clinically available to prevent or cure WMI. Given that arrested OL maturation plays a central role in the etiology of perinatal WMI, an increased interest has emerged regarding the functional restoration of these cells as potential therapeutic strategy. Cell transplantation and promoting endogenous oligodendrocyte function are two potential options to address this major unmet need. In this review, we highlight the underlying pathophysiology of WMI with a specific focus on OL biology and their implication for the development of new therapeutic targets. Preterm birth, defined as being born before 37 weeks of gestation (gw), is associated with significant adverse neurological outcomes. White matter injury (WMI) refers to myelin deficit in the developing white matter. It is the most common non-hemorrhagic neuropathology in preterm infants, especially in those born before 28 gw . WMI is + premyelinating oligodendrocytes (pmOLs). pmOLs are particularly vulnerable to hypoxic and inflammatory insults (Hypoxic-ischemic injury (HI) and inflammation are two major risk factors leading to WMI . Preterm insults . Limited insults .Currently, the management of WMI is limited to supportive measure and symptomatic relief. In this review, we summarize the current understanding of oligodendrogenesis and myelination during normal white matter development, as well as the pathophysiology of WMI. Furthermore, we discuss the current state of experimental therapeutic approaches aiming to restore myelination.Myelin, the multilayered glial membrane surrounding axons, is paramount to axonal conductivity and health in the jawed vertebrate nervous system. In addition to enable saltatory and fast conduction of action potentials, myelin supplies axons with energy-rich metabolites such as lactate and pyruvate through the monocarboxylate transporters . Importa+ precursors in the lateral ganglionic eminence (LGE) at ∼E15.5 , myelin is formed by specialized glial cells called OLs, which arise from a lineage-restricted proliferative pool of OL precursor cells (OPCs) . OPCs art ∼E15.5 . The thind birth ; this land birth , althoug+ (PDGFRα+) OPCs emerge in the forebrain at around 10 gw and distribute throughout the developing cerebral cortex during the next few weeks. However, a higher number of OPCs appears only around 15 gw, when they are most numerous in the ganglionic eminences and in the cortical ventricular zone/subventricular zone , acquiring a larger cell body, and extending their processes. pmOLs are O4he axons . The estFollowing establishment of primary glial-axon interaction, pmOLs differentiate into mature OLs that are characterized by the expression of galactocerebroside /O1 and myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin associated glycoprotein (MAG), and transmembrane protein proteolipid protein (PLP).Several transcription factors are involved in the regulation of OL lineage differentiation, among which helix-loop-helix (HLH) family members have been extensively studied. Olig2 acts as a central node to which many pathways converge to drive oligodendrogenesis and maturation . For insEpigenetic mechanisms including DNA methylation, histone modification, and regulatory non-coding RNAs play permissive roles in OL biogenesis . HistoneOnce physical interactions between OL and axon occur, the initial layers of myelin rapidly wrap around the axons. Simultaneously, the myelin sheath extends longitudinally along the axon and the myelin membrane layers compact their cytoplasm to form mature myelin. During the myelin sheath growth, actin filaments turnover is the driving force by regulating repetitive cycles of leading edge protrusion and spreading . An indiTo date, two distinct modes of myelination—axonal activity-dependent vs. independent—have been proposed . In actiWMI was historically named as periventricular leukomalacia (PVL) . HistoloSince 1980’s, the presentation of WMI has changed from cystic PVL to milder diffuse WMI (dWMI), thanks to the advancement of medical technology and improved clinical management of premature babies . Magneti+/O4–) and pmOLs   . pmOLs a4+, O1–) . Anatomi4+, O1–) .+ cells was observed with degenerating O4+ cells in the core and intact O4+ cells in more superficial zones of the lesions   being an exemplar disease for cell-based therapy using various cell sources. PMD is an X-linked disorder caused by mutation in the proteolipid protein-1 (PLP1) gene. It is a progressive congenital disorder of myelin formation, which results in severe neurological disability. There is no effective treatment to date. An open label phase I clinical trial with allogenic human NSCs transplantation was conducted in four individuals with PMD (1391637) , 2019. T1391637) . At the 1391637) . On the 1391637) . The lesIn preclinical animal models of hypomyelination disorders, OPCs, NSCs, glial progenitor cells (GPCs), human amnion epithelial cells (hAECs), human umbilical cord blood cells (UCBC), and mesenchymal stem cells (MSCs) have showed beneficial effects in re-establishing myelination and/or function ; Table 1Despite significant progress, there are major concerns regarding the use of therapeutic cell-based approaches in humans, particularly in non-fatal disorders. The report of tumors developing several years after human fetal brain-derived cell transplantation has heightened anxiety about the potential for neoplasia , in addiThe goal of cell-based therapy for WMI varies. Some sought to directly protect myelinating cells through immunomodulation/trophic supports and others to functionally replace the damaged cells . In realclinicaltrials.gov  transplantation is a less invasive method . HoweverLoss of pmOLs during the acute phase of WMI is followed by a significant increase in these cells, suggesting that OPC deficit may not be the major cause of pathology later in life. Instead, dysregulation of pmOL maturation may be the main mechanism underlying neurologic disability in preterm infants . Thus, tClinicalTrials.gov NCT01096784). Further studies are needed to explore the potential neuroprotective effects of IGF-1 with respect to dWMI .Erythropoietin (EPO), originally recognized for its role in erythropoiesis, has also been extensively studied in neurological conditioned . EPO recHigh-throughput screening (HTS) platform allows for the identification of approved compounds for repurposing therapy as well as drug discovery . Indeed,For instance, HAMI3379, initially developed as a cysteinyl-leukotriene CysLT2 antagonist to treat cardiovascular and inflammatory disorders , has theGPR56/ADGRG1 is an emerging member of the GPCR family with considerable therapeutic potential in neurodevelopmental disorders . While tFingolimod (FTY720), the first oral drug approved for the treatment of relapsing remitting multiple sclerosis (RRMS), is a functional modulator of Sphingosine 1-phosphate receptor 1 (S1P1). Administration of FTY720 in neonatal model of oxygen-toxicity is reported to attenuate hyperoxia-induced hypomyelination through reduction of hyperoxia-induced oxidative stress and inflammation accompanied with direct protection of developing OLs .Several other drugs and biological compounds, such as Pioglitazone (PPAR-γ agonist) , ClemastClinicalTrials.gov NCT03555019)  .In addition to optimal early nutrition, the impacts of behavioral interventions and environmental enrichment (EE) have been increasingly appreciated in neurodevelopmental outcomes . Given tSignificant progress has been made in our understanding of OL development and myelination. However, more work needs to be done in both pathogenesis and treatment of WMI. Our knowledge on the leading pathophysiology of perinatal WMI remains two-decade old, which is pmOL maturation arrest . Single MM and XP wrote and revised the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Among biological macromolecules, proteins hold prominent roles in a vast array of physiological and pathological processes. The protein sequence-structure-function paradigm establishes that the amino acid sequence governs the structure that in turn determines the function . Thus, tApart from globular well folded proteins, during the past years intrinsically disordered proteins (IDPs) have attracted a lot of attention. IDPs present a general tendency to aggregate and may form toxic amyloid fibers and oligomers associated with many human pathologies ,3,4. IntPeptides, which are characterized by a smaller size than proteins, represent key elements of cells as well. Peptides can even find diagnostic and therapeutic applications, for instance working as tumor markers . In the This Special Issue comprehends contributions  on the above-described topics and, in detail, it includes structural studies on globular folded proteins, IDPs and bioactive peptides. These works were conducted by utilizing different experimental  and/or computational approaches (mainly molecular dynamics simulations and bioinformatic tools).A broad range of structural biology topics are covered by the Special Issue as summarized below.α-helix backbone conformation as revealed by the analyses of both peptides and IDPs.Proteins under biological conditions unfold and refold several times in vivo showing a marginal structural stability. A detailed molecular-level knowledge not only of the native but also of the diverse non-native conformational states, which are accessible to a protein in solution ), is necessary to fully comprehend its function. Several investigations employed short peptides as models to obtain the canonical features of the DSE . Short pCanavalia ensiformis) urease that possesses entomotoxic and antimicrobial functions [Glycine max) ubiquitous urease. Compared to Jaburetox, Soyuretox contains a higher secondary structure content but preserves similar entomotoxic and fungitoxic properties. Due to the positive toxicity profile, both peptides find biotechnological applications and have in fact been already used to generate transgenic crops giving rise to plants active against insects and nematodes [The Special Issue further includes interesting reviews describing structural and functional features of different proteins. Leonardo L. Fruttero and collaborators focus on intrinsically disordered polypeptides from plant ureases . Jaburetox represents a recombinant peptide derived from the jack bean  isoforms and describe within their review the proteins structural characteristics along with their function in actin stabilization and the relationship to pathological conditions  . In detaAnother interesting review by Christian Roumestand and collaborators is centered on High-Pressure (HP)-NMR. The authors summarize recent advances of HP-NMR and describe how this technique can be employed to characterize, at a quasi-atomic resolution, the protein folding energy landscape . GlobulaHelicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis) [Beat H. Meier, Anja Böckmann and collaborators describe within their review the lessons learned from studies on two ATPases  . The autThe reported information can surely be relevant to researchers conducting studies on other NTPases (Nucleoside TriPhosphatases) .An interesting research article by Michael O. Glocker and collaborators highlights how mass spectrometry techniques have enlarged their horizons by becoming additional tools to study intermolecular interactions . In detaIn this pandemic era, most research efforts are centered on SARS-CoV-2 related studies. Thus, a contribution to the field is also included in this Special Issue. Vladimir N. Uversky and collaborators, by utilizing different bioinformatic tools, analyze amino acid sequences of ACE2 (Angiotensin-converting enzyme 2) receptors from eighteen non-human species and compare them with the human ACE2 sequence pointing out the degree of variability . ResultsAuthors Alberto Perez and Lijun Lang report on the p53-MDM2 (Mouse Double Minute 2 homolog) interaction, which is highly relevant in cancer research. In fact, p53 triggers programmed cell death when cells misbehave, while MDM2 downregulates p53 anticancer activity; inhibitors of the p53-MDM2 interaction thus possess anticancer potentials . The autE. coli alcohol/aldehyde oxidoreductase that, beginning from a wide range of materials, is able to generate relevant bio-renewable fuels and fine chemicals [Work by Rajni Verma, Jonathan M. Ellis and Katie R. Mitchell-Koch focuses instead on molecular dynamics simulations of the enzyme YqhD. YqhD represents an hemicals . The comhemicals . The stuhemicals . This woIn their research article, Richard P. Cheng and collaborators analyze, largely by using NMR analyses, cross-strand lateral ion-pairing interactions and their importance for antiparallel β-sheet stability . The autDavid A. Snyder and collaborators tells us about protein flexibility and its importance for proper protein functioning by performing a comparative investigation of protein flexibility captured by crystallographic B-factors, molecular dynamics and NMR studies . This woβ2-microglobulin. The authors carried out a structural characterization of Nb23. The study points out peculiar structural features of Nb23 with respect to Nb24, which could be linked to diverse target antigen affinity [Finally, Gennaro Esposito and collaborators in their research article report on the structure characterization of a nanobody by utilizing solution state NMR techniques coupling to molecular modelling investigation . Nanobodaffinity .In conclusion, this ensemble of studies clearly stresses how the knowledge of structural features enables the understanding of the multifaceted roles of protein and highlights the importance of flexible regions and disorder in dictating binding events and directing protein functions under normal physiological and pathological conditions. Coupling of experimental and computational work is always necessary to reach a complete and detailed structural portrait of a protein in its isolate state and when it is bound to a ligand . Researchers are still eager to develop better computational tools to keep into account proteins/peptide flexibility and to predict interactions of proteins with large flexible systems, such as peptides, and in the next few years much efforts will likely be devoted towards reaching this goal and optimizing existing tools. Nevertheless, the reported studies also highlight the continuous search by the scientific community for novel improved experimental techniques to a protein/peptide structure, dynamics and interactions.Given the variety of topics embraced by this Special Issue, a great interest from researchers working in the protein/peptide structural biology field is expected.


Recent advances in targeted and immune therapies have enabled tailored treatment strategies for advanced lung cancer. Identifying and understanding the genomic alterations that arise in the course of tumor evolution has become hugely valuable, but tissue biopsies are often insufficient for representing the whole cancer genome due to tumor heterogeneity. A liquid biopsy refers to the isolation and analysis of any tumor-derived material in the blood, and recent studies of this material have mostly focused on cell-free tumor DNA (ctDNA) in plasma. Indeed, liquid biopsy analysis is now expected to expand in utility and scope in clinical practice. In this review, we assess the biology and technical aspects of ctDNA analysis and discuss how it is currently applied in the clinic. Key points: Liquid biopsy is a potentially powerful tool in the era of personalized medicine for guiding targeted therapies in non-small cell lung cancer. EGFR), anaplastic lymphoma tyrosine kinase (ALK), and ROS proto-oncogene 1 receptor tyrosine kinase (ROS1) gene rearrangements provide actionable treatment targets for as well as important prognostic insights [Lung cancers are the most commonly diagnosed tumors and are the leading cause of cancer-related deaths worldwide . A delayinsights ,6,7.EGFR tyrosine kinase inhibitors (TKIs), such as INTEREST  and IPASS (Iressa Pan-Asia Study), have reported that sufficient biopsy samples were available in only 42% and 31%, respectively, of the patient subjects to allow a complete molecular diagnosis, and consequently that multiple tissue biopsies were required for a complete tumor characterization [The diagnosis and genotyping of lung cancer typically rely on tissue biopsy obtained from local sampling. This is a clinically well-validated approach, but is invasive and has some inherent clinical risks. Prior meta-analysis has indicated major complication rates from CT-guided transthoracic lung biopsy of 5.7% and 4.4% for core biopsy and fine needle aspiration, respectively . A rebiorization ,10. HoweImprovements in genomic and molecular technologies have yielded the identification of various new materials in the bloodstream . A liquiWe here review the clinical applications of liquid biopsy in non-small cell lung cancer patients and discuss the challenges and future directions for this methodology.Mandel and Metais first reported the existence of circulating cell-free DNA (cfDNA) in the blood of healthy individuals in 1948 . The oriCirculating cell-free tumor DNA (ctDNA) is a component of the cfDNA detected in the bloodstream of cancer patients . PreviouThe ctDNA levels are influenced by many factors such as disease burden, anatomic locations, vascularity, etc. ,26,27. BThe detection of ctDNA can be challenging because it can represent a very small proportion of the total cfDNA pool, ranging from less than 0.1% to over 10% ,29. StanEGFR, Kirsten rat sarcoma viral oncogene homolog (KRAS), or B-Raf proto-oncogene, serine/threonine kinase (BRAF) genes [EGFR TKI-resistant clones such as T790M can also be detected [EGFR TKIs. These are the Cobas EGFR Mutation test v.2 CE-IVD  and the Therascreen mutation kits . Other highly sensitive targeted assays are also available such as BEAMing  and digital-droplet PCR (ddPCR) [Targeted assays can detect known mutations, such as those in the F) genes ,31,32. Tdetected . Two ctD (ddPCR) ,34. HoweNGS is a high-throughput sequencing method that can simultaneously interrogate variable areas of the genome and detect somatic mutations. NGS-based assays such as CAPP-Seq are designed to detect multiple mutation types, including insertions/deletions or gene rearrangements, and copy number alterations. Hybrid capture NGS is a well-established method ,36 that EGFR TKIs and ALK TKIs, have significantly improved the survival outcomes for patients with advanced NSCLC. ctDNA assays can be used to for initial genotyping of these lung tumors and can monitor the impact of genotype-directed therapies [Recent advances in molecular targeted therapies, such as the use of herapies ,42,43. Hherapies . By assaEGFR mutations are the most commonly tested, and the only variations to be routinely assessed by plasma-based liquid biopsy in clinical practice for NSCLC patients. The specificity of these tests is sufficient to guide the choice of EGFR-TKIs based on the test result however the sensitivity is less than satisfactory. A prior meta-analysis of 27 selected studies which included nearly 4000 patients with NSCLC revealed a pooled sensitivity of 60% and specificity of 94% for the detection of EGFR mutations in the plasma or serum [EGFR T790M mutation, low rates of concordance between the results of a tissue and plasma ctDNA assay were observed in a recent phase III trial of osimertinib [EGFR mutations in the plasma were similar to those in whom EGFR mutations were revealed in a conventional tissue biopsy [EGFR T790M mutation in a plasma ctDNA test were equivalent to patients classified as positive in a tissue-based assay [EGFR T790M mutation based on ctDNA analysis, and that this efficacy was comparable to cases with the EGFR T790M mutation detected in a tissue biopsy [Currently, or serum . In the mertinib . Howevermertinib . In sevee biopsy ,46,48,49ed assay . Remon ae biopsy .EGFR mutational status, resistance to ALK TKIs has been successfully evaluated using a plasma cfDNA test in NSCLC patients. Previous mutational analysis of cfDNA by Bordi and colleagues identified novel ALK point mutations in five out of 20 NSCLC subjects treated with crizotinib who had shown disease progression [ALK and KRAS mutations are associated with acquired resistance to crizotinib in ALK-positive NSCLC, suggesting that an ALK mutation detected in plasma can be a useful marker for response monitoring [ALK-positive lung cancer [ALK-positive NSCLC patients with acquired resistance to ALK TKIs to track the evolution of drug resistance during the treatment course and observed that ALK mutations emerged and disappeared during the sequential administration of ALK TKIs. This correlation between plasma ALK mutations and the response to specific ALK TKIs suggested the potential for using a liquid biopsy to guide ALK TKI selection. According to recently published results from the LBA81_PR-Phase II/III blood first assay screening trial (BFAST), blood-based NGS found 119 (5.4%) ALK-positive disease in 2188 NSCLC patients [In addition to the gression . These anitoring . Anotherg cancer . The autpatients . The oveMET) proto-oncogene emerged as a novel targetable genetic alteration. Drilion et al. studied the efficacy of crizotinib in 69 patients with NSCLC harboring a MET 14 exon alteration and showed plasma-derived ctDNA assays can also detect MET exon 14 alterations in NSCLC patients [MET-exon-14-altered tumors and available plasma samples, 18 (49%) were ctDNA positive in plasma samples. Recondo and colleagues tracked genetic alterations during MET TKI therapy using plasma and tissue NGS in 20 patients with advanced MET exon 14-mutant NSCLC [MET exon 14 skipping mutations with serial tissue biopsy and ctDNA analysis [RET gene fusions. Solomon and colleagues performed the analysis of ctDNA and tissue in patients with RET fusion-positive NSCLC who showed disease progression after an initial response to selpercatinib. Interestingly, RET solvent front and gatekeeper mutations associated with resistance was detected in ctDNA before disease progression [Other oncogenic driver mutations can be detected with ctDNA and used as therapeutic targets. Recently, mesenchymal epithelial transition  and ICIs (N = 34) [The response to immunotherapy can be monitored by changes in the ctDNA level during treatment with immune checkpoint inhibitors (ICIs). Guibert et al. reported that changes in the kinetics of ctDNA from serial plasma samples in advanced NSCLC patients could identify patients who were likely to benefit from anti-PD-1 therapy . Anagnos(N = 34) . In this(N = 34) . BaselinDespite promising data from several clinical trials in immuno-oncology, there are some challenges to overcome before TMB can be used in clinical practice: further evaluation of tissue-plasma concordance should be carried out; different methods measuring TMB, e.g., WES and various kinds of targeted NGS, need to be standardized; and a definitive cut-off point for TMB should be agreed upon .p < 0.001) [Minimal residual disease (MRD) is a concept frequently used with hematologic disorders, which refers to the presence of any tumor-derived material in the blood after curative treatment. The clinical significance of MRD, however, is less well verified for solid tumors. MRD detection in lung cancer using ctDNA has been described and involves measurement of the differences between the presurgical and postsurgical ctDNA levels. A previous prospective study of 41 NSCLC patients measured somatic driver mutations in ctDNA from blood samples that were collected within 10 days before and then after surgical resection . In this< 0.001) .Further studies have shown that ctDNA-based MRD detection can predict the prognosis following a definitive treatment. Newman et al. introduced a highly sensitive ctDNA sequencing method for NSCLC referred to as Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) . In thatSince the cancer stage at the time of diagnosis is an important prognostic indicator in lung cancer, proper screening for this disease is crucial to improving clinical outcomes for affected patients . Lung caThe low sensitivity of ctDNA, particularly for the early-stage detection of lung tumors, is a notable limitation of this methodology as a screening approach. In a previous prospective study of 284 consecutive patients with stage I and II NSCLC, the detection sensitivity cfDNA using ARMS-PCR was only 14.2%, in contrast to a 91.7% specificity . HoweverImprovements are clearly needed therefore in the sensitivities of the currently available assays for cancer detection, particularly for early-stage disease. A wider coverage of these assays which captures all NSCLCs is also needed before liquid biopsy screening methodologies can be implemented for lung cancer in real world settings.EGFR and ALK mutations have already been approved for clinical use. Liquid biopsy testing is also likely to play an important future role in treatment decisions and resistance monitoring for targeted cancer therapies, and advances in this field could bring significant changes to current clinical practice. In addition, liquid biopsy assays can be used to assess blood TMB which is predictive of the responses to ICIs, in addition to detecting MRD which has prognostic implications and guides adjuvant therapy choices. Several clinical trials are currently active, trying to define clinical relevance and utility of liquid biopsy in advanced lung cancer (The clinical utility of liquid biopsy analysis is now expanding to the diagnosis and treatment of NSCLC. The minimally invasive and readily reproducible nature of this methodology makes it highly suitable for the dynamic evaluation of patients and its most widely used application at present is targeted gene sequencing. Indeed, liquid biopsy kits for g cancer .Despite the variety of potential clinical applications of a liquid biopsy, these assays have several limitations at present such as a low sensitivity for early-stage disease due to DNA concentration threshold issues, processing capacity of target mutation numbers, and a high rate of false-positives. However, with the continuing progress in sequencing technology, liquid biopsy testing has already begun to change the treatment algorithms for NSCLC. It is still uncertain how much a liquid biopsy can support or even replace conventional tissue biopsy analysis, and more clinical evidence is needed from large-scale prospective trials to address this.


Brain tumors integrate into the brain and consist of tumor cells with different molecular alterations. During brain tumor pathogenesis, a variety of cell types surround the tumors to either inhibit or promote tumor growth. These cells are collectively referred to as the tumor microenvironment. Three-dimensional and/or longitudinal visualization approaches are needed to understand the growth of these tumors in time and space. In this review, we present three imaging modalities that are suitable or that can be adapted to study the volumetric distribution of malignant or tumor-associated cells in the brain. In addition, we highlight the potential clinical utility of some of the microscopy approaches for brain tumors using exemplars from solid tumors.Although our understanding of the two-dimensional state of brain tumors has greatly expanded, relatively little is known about their spatial structures. The interactions between tumor cells and the tumor microenvironment (TME) occur in a three-dimensional (3D) space. This volumetric distribution is important for elucidating tumor biology and predicting and monitoring response to therapy. While static 2D imaging modalities have been critical to our understanding of these tumors, studies using 3D imaging modalities are needed to understand how malignant cells co-opt the host brain. Here we summarize the preclinical utility of in vivo imaging using two-photon microscopy in brain tumors and present ex vivo approaches  and highlight their current and potential utility in neuro-oncology using data from solid tumors or pathological brain as examples. As spatial molecular profiling technologies become accessible, efforts are focused on determining regional heterogeneity within tumors . In paraBrain tumors exhibit heterogeneity, integrate with the existing functional architecture of the normal brain, and reside within a complex 3D architecture of a tumor microenvironment (TME) . Thus, tTraditionally, two-dimensional 2D) methods relying on thin tissue sections, such as immunohistochemistry (IHC) and immunofluorescence, have been used to study the distribution of cells within tumors. These methods have several shortcomings. First, cross-sectional images do not fully represent tumor heterogeneity. 3D morphological structures such as tumor vasculature or neurons cannot be represented as continuous structures in 2D space. Second, reconstructing thin serial sections can provide high-resolution 3D maps [D methodsIn recent years, there have been remarkable advances in both in vivo and ex vivo 3D imaging techniques. Here, we summarize the current state-of-the-art developments in high-resolution 3D imaging, focusing on in vivo  and ex vivo technologies ) and provide examples of their current or potential applications for brain tumor studies .Two-photon microscopy (2PM) is the most widely used form of multiphoton microscopy and was first demonstrated in 1990 . Unlike The thickness and inhomogeneity of the intact mouse skull present a barrier to high-resolution optical imaging. Therefore, techniques such as a small craniotomy , thinnin2 subregions [2. However, these systems do not provide subcellular resolution in the axial dimension (>12 μm) [IVM studies are typically based on fluorescent proteins or exogenous fluorochromes  ,23,24. O(>12 μm) ,26.Two-photon IVM (2P-IVM) is the most widely used approach for high-resolution 3D imaging in the preclinical brain tumor field, providing valuable insights into growth and invasion patterns ,28,29, v2P-IVM has been useful for characterizing the infiltration of metastatic tumor cells into the brain parenchyma. For example, in preclinical brain tumor metastasis models, circulating tumor cells have been shown to extravasate at vascular branch points, remain proximal to microvessels, and induce perivascular growth through vascular co-option or angiogenesis . TrackinAn intriguing 2P-IVM study showed that intracranial biopsies could stimulate tumor associated microglia/macrophage (TAM) recruitment and trigger glioblastoma (GBM) progression . This fiOne of the barriers to drug delivery to the brain tumor is the BBB. From this perspective, IVM can be a unique and important tool to study drug delivery to the brain. Drug analogs can be fluorescently labeled to allow pharmacokinetic measurements to determine the final destination of specific drugs along with their therapeutic effects on the tumor. Several conjugated drugs are available, such as the PARP-1 inhibitor AZD2281 modified to incorporate the fluorochrome BODIPY  or ibrutGFPCx3cr1 mice [Microglia constitute mostly of immune cells in the brain TME, and it has become clear that TAMs actively influence brain tumor biology . Marker GFP mice . IVM canGFP mice . As the Moreover, 2P-IVM can be correlated with various approaches such as MRI or focus ion beam/scanning electron microscope (FIB/SEM) to obtain multilayered and informative datasets. Iron oxide nanoparticles are preferentially phagocytosed by TAMs can be detected by MRI to monitor immune responses . Using tThe brain was the first organ to be imaged in vivo using 2PM, and methods have since been developed to improve image quality. While in vivo 2PM has shed unprecedented light on pathological processes associated with brain tumors, some limitations of the technique should be considered. Most importantly, deep tissues such as the brain can only be imaged at reduced depth . ImproveIn addition, injury from optical imaging setups  is accompanied by inflammatory responses that may impact studies investigating vascular or immune system involvement in brain tumor pathology . ThinnedTo address some of the challenges described above, methods are currently being developed to improve temporal resolution and reduce photodamage using adaptive excitation , increasTumors or metastases can arise from a few malignant cells, and these single foci are difficult to detect with imaging systems limited by spatial resolution. Simultaneous visualization of multiple cell types or molecular markers will facilitate our understanding of the underlying molecular mechanisms and cellular co-options during tumor progression. In this context, the application of whole-brain imaging techniques to brain tumors may reveal interesting aspects of tumor biology. Robust whole-brain imaging techniques, software tools for volume rendering and 3D data analysis, and anatomical reference atlases for the mouse brain have been developed. These methods have successfully revealed various brain structures and functions in rodents, such as mesoscale projection patterns, single-cell-level neuronal tracing, and neuronal activity patterns . These ax-y-z stage, a z stack of serial optical sections through the volume is obtained , was used for in vivo imaging of transgenic GFP-expressing embryos of medaka  and zebrOther LSFM configurations that allow for laterally unconstrained imaging include dual inverted selective-plane illumination microscopy (diSPIM) and light-sheet theta microscopy (LSTM), which allow imaging of the entire postmortem human brain using thick sections of cleared human brain tissue (~10.5 mm × 14.1 mm × 3 mm) . Althougx-y-z axis. An important consideration for LSFM is optimized tissue clearing methods that render large samples highly transparent to improve light penetration, imaging depth, and contrast.In general, LSFM images chemically cleared transparent tissues with a high resolution and short acquisition time, using only optical sections without mechanical sectioning. Subsequently, the acquired data can be reconstructed for 3D visualization of whole organs or tissues with micrometer resolution in the The concept of organic solvent-based tissue clearing was established in 1914 by Werner Spalteholz . InitialIn the last decade, LSFM combined with optical tissue clearing has significantly impacted various fields such as neuroscience and developmental biology ,83,84,85Modern pathology relies on thin sections (<10 μm-thick) of FFPE  tissue, which are subsequently stained with H&E (hematoxylin and eosin) or other diagnostic markers. However, solid tumors exhibit spatial heterogeneity and consist of various cell types and niches, vasculature, and distinct phenotypic features. Conventional histopathology is only capable of capturing a reduced aspect of the 3D spatial structures of intact tumors. Due to technological advances in LSFM and tissue clearing protocols, several studies have begun to bridge the gap and visualize whole human organs and tumors ,97. For The CLARITY protocol (hydrogel-based) has also been used for 3D imaging of archived FFPE clinical samples from breast cancer tissues , for ima3 processed in less than 48 h [Recently, a new technique called pathoDISCO (hydrophobic reagents) has been developed and applied to breast cancer tissues. This technique uses selective autofluorescence enhancement, rapid chemical tissue clearing, and light-sheet microscopy to obtain virtual sections and 3D reconstructions of resected tumors with tissue size of ~30 × 20 × 5 mmhan 48 h . Selectihan 48 h . This apSeveral challenges must be overcome before 3D imaging protocols can be successfully extended to the clinical setting. Time-efficient protocols for tissue clearing and multi-labeling techniques, as well as more cost-effective light-sheet microscopes and associated analysis software need to be developed. In contrast to the lipid-rich and homogeneous structures of the mouse brain, solid tumors often have an abundance of fibrous tissue and a heterogeneous composition, so visual assessment of tissue clearing between samples can be difficult. In general, the tissue-clearing methods  can affect the morphology of some structures (expansion and shrinkage during the protocol), which can deform connected regions within the tissue. In addition, tissue shrinkage may interfere with accurate pathological assessment in routine clinical diagnostics. Therefore, clearing protocols must be optimized to achieve standardized and reproducible tissue processing methods for research and clinical applications.Mouse xenograft tumor models can serve as preclinical models to optimize antibody penetration and concentration for protein markers of interest. In addition, the time required for tissue clearing and immunostaining needs to be reduced to make this technology useful for drug development and clinical research. There are also limitations in the availability of commercial image analysis software. More sophisticated and applicable software need to be developed to process large datasets along with machine learning algorithms to process feature extraction and meaningful 3D spatial relationships in the TME. Each of these described techniques represents a valuable approach for research purposes. However, they do not yet seem to be suitable in routine clinical histopathology due to long incubation times, the need for large volumes of antibodies along with issues related to antibody specificity, tissue perturbation and shrinkage due to different clearing methods.Nevertheless, the rapid development of protocols for tissue clearing and the commercial availability of light-sheet microscope systems will one day outweigh the challenges in applying whole-tissue 3D imaging in the clinical setting. LSFM is likely to be highly informative for understanding tumor biology in patient samples.Block-face imaging platforms such as STPT generate complete datasets that can be registered to standardized anatomical reference atlases, which are crucial for integrating anatomical data from multiple experiments. In this review, we focus on STPT, which takes advantage of two-photon excitation to optically section the standard paraformaldehyde fixed tissues . Other bSTPT is an automated method developed for high-throughput ex vivo mosaic imaging of fluorescent-labeled brains and can employ three different PMTs . Paraforz-spacing of 50 μm and an x/y resolution of 1 μm/voxel, within ~21 h [z-stack imaging (2.5 μm spacing in z). STPT can provide a dataset ranging from hundreds of gigabytes for spatially down-sampled STPT brains [Automated imaging platforms, such as STPT, generate large amounts of data. The mouse brain can be imaged as a set of 280 serial coronal sections, with a in ~21 h . STPT haT brains ,118 to tT brains . New neuT brains . STPT haT brains ; therefoAlthough STPT is not yet widely used in the cancer field, this microscopy platform offers several advantages for the study of cancers, particularly brain tumors. First, STPT does not require optical clearing for imaging, and tissue morphology is preserved due to the light paraformaldehyde fixation. This aspect may be advantageous for the study of tumor–stroma interactions. In our previous work, we highlighted the usefulness of STPT in visualizing and quantifying the infiltration of implanted GFP-labeled mutant IDH1-expressing astrocytes in the mouse brain relative to reference anatomical structures annotated by the Allen Brain Atlas  Figure .Second, with the fourth generation of the STPT vibratome, tissue sections with a thickness of 20 to 25 μm are captured. This is very useful for the analysis of cell morphology. For instance, the indexed sections can be used for genomics, proteomics or stained for markers using traditional histology approaches after image acquisition. STPT can also be integrated into a tissue processing module, where thin sections from 25 μm upward are captured as they are cut from an agarose-embedded fixed whole organ. This module then transports these sections with a conveyer belt and automatically transfers and mounts them onto glass slides indexed back to their correct position in the overall image. This specific feature of STPT has the potential to reduce handling time of the sections to achieve a high throughput. Any subsequent data obtained from indexed sections can be easily overlaid on top of the 3D reconstructed imaging data to create a rich, multimodal dataset. Indexed sections also allow researchers to relabel and map tissue sections to answer follow-up questions. Since spatial integration of data acquired from multiple modalities is an important initiative in cancer, technologies that allow integration of 3D spatial reconstruction and molecular profiling of indexed tissue sections represent an ideal approach. Therefore, STPT sections can be processed for subsequent applications, providing a promising framework for application to patient samples.Recently, several features of STPT have been improved. For example, to reduce the scan time and thus the acquisition time, a multiphoton, multi-foci scanning approach has been implemented. This approach splits the incoming laser beam into multiple spots scanned simultaneously across the sample and allows the full power of the laser system to be utilized by distributing the power across multiple spots avoiding saturation of the area excited by the laser. It also speeds up the scanning of an individual frame. For example, a 2-foci system can scan a single image twice as fast as a traditional one-foci system. Furthermore, the multi-foci systems do not suffer a reduction in the pixel residence and maintain the same signal-to-noise ratio as a single-foci galvo-based system. Reducing the sample scanning time, the replacement of intermediate optics allows a larger FOV to be scanned while maintaining the same image quality as the original system.In the context of brain tumors, collagen is the major component of the TME and is involved in cancer fibrosis ,124. SecSTPT has some drawbacks; for example, volumetric data acquisition rates for block-face imaging methods are slower compared to light-sheet imaging and multi-color imaging, and antibody labeling have not yet been adapted to STPT. However, tissue clearing in conjunction with antibody labeling protocols will likely be adapted for STPT in the near future. Since the tissue is only lightly fixed, the structure and volume of the sample are preserved, which offers advantages for downstream applications that apply molecular or histological techniques. Furthermore, given the analytical neuroscience pipelines already available, STPT can be readily used to study brain tumors.A comprehensive understanding of brain tumors needs to consider the spatial structures of tumors, their TME, and their localization within the host environment. The rodent brain has been extensively studied to understand its neural circuitry, normal function, and pathology using 3D technologies, and researchers are in the process of studying the 3D structures of extracranial solid tumors from patients using ex vivo 3D imaging approaches. As such, the field of neuro-oncology needs to take advantage of the many advances, as these new methods may have important implications for personalized medicine and can improve our understanding of this often fatal disease. As imaging technologies and analysis pipelines improve, STPT and LSFM are positioned to provide important fundamental insights into the pathology of brain tumors.