Digital PCR is a third-generation technology based on the subdivision of the analytical sample into numerous partitions that are amplified individually. This review presents the major applications of digital PCR (dPCR) technology developed so far in the field of plant science. In greater detail, dPCR assays have been developed to trace genetically modified plant components, pathogenic and non-pathogenic microorganisms, and plant species. Other applications have concerned the study of the aspects of structural and functional genetics.Digital PCR (dPCR) is a breakthrough technology that able to provide sensitive and absolute nucleic acid quantification. It is a third-generation technology in the field of nucleic acid amplification. A unique feature of the technique is that of dividing the sample into numerous separate compartments, in each of which an independent amplification reaction takes place. Several instrumental platforms have been developed for this purpose, and different statistical approaches are available for reading the digital output data. The dPCR assays developed so far in the plant science sector were identified in the literature, and the major applications, advantages, disadvantages, and applicative perspectives of the technique are presented and discussed in this review. Digital PCR (dPCR) is a breakthrough technology that is able to provide sensitive and absolute nucleic acid quantification . It is aThe first pioneering achievement of this technique was realized in 1992 by Sykes et al. , dated tCurrently, several different platforms are used for PCR reaction compartmentalization, such as active partitioning platforms , passive partitioning platforms (based on fluidic effects to create sub-volumes), self-digitization platforms (which combine both passive filling and partitioning), and droplet-based platforms (in which aqueous droplets act as microreactors). The main differences among platforms are in the number of partitions and in the number of samples that can be processed in a run.In dPCR, the detection of the target is achieved similarly to in qPCR, with two main families of chemistries: DNA intercalating dyes and hydrolysis-based probes. As in qPCR, there is the possibility to organize multiplex reactions to simultaneously follow more than one target.In 2004, Gachon et al.  publisheMore than twenty years after the start of their commercialization, biotech crops currently cover around 200 million hectares worldwide . DiffereSample preparation and DNA extraction.Digital PCR analysis for the amplification of the target transgenic sequence.Digital PCR analysis for amplification of a native, reference sequence.Data evaluation.The starting point of dPCR assays for GMP detection is the availability of primers/probes targeting a specific transgenic sequence and of primers/probes targeting an endogenous gene of the plant. This latter is not the modified gene. It is a single-copy, native gene that is strongly conserved in the species object of the analysis. The right choice of such an endogenous sequence is of great significance because it provides vital information on the stability and reliability of the detection system and it permits the quantification of genetically modified ingredients in mixtures. The analytical pipeline for the quantification through dPCR of GM components is not substantially different from that for qPCR and can be summarized in the following modules:The ratio between transgene copy number and reference gene copy number provides the GM percentage present in a sample. However, EU legislation requires that the amount of GM content is estimated as a mass fraction, and a conversion factor was therefore established for each event to convert a copy number ratio into a mass fraction Digital PCR approaches for transgene detection and quantification have been evaluated in cultivated GM crop and reference materials.Various relevant points can be highlighted from the experiences made on dPCR-based GMP detection.Iwobi et al.  comparedGlycine max lines were developed by Kosir et al. [Several dPCR assays have been validated through proficiency studies in comparison with qPCR, and, in general, a similar performance has been found between the two techniques ,18,19,21r et al.  and valir et al. .In conclusion, dPCR has several positive characteristics, such as its precision, accuracy even at very low target concentrations, suitability for routine analytics, and lower sensitivity to PCR inhibitors . HoweverA number of publications have reported the use of dPCR for the characterization of transgenic plants, here understood as experimentally transformed lines not proposed for cultivation and marketing. In this case, therefore, dPCR has a distinct use compared to what was reported in the previous paragraph. The purpose of dPCR analysis is, in fact, in this case not directed to the identification of transgenic components in food and feed but rather to the description and characterization of experimentally obtained transformed plants. In transformation projects, in fact, it is central to verify the success of transformation and to assess the inserted transgene copy number. Transformed lines carrying a single, full-length copy of the transgene are usually desirable to ensure a Mendelian inheritance of the transgene and to avoid silencing problems. Southern blot and qPCR analyses have been traditionally used to do such characterizations. However, the first technique is expensive and very laborious, and the second lacks the accuracy and precision needed to confidently characterize low copy number events. On the contrary, dPCR is emerging as a technology that is able to accurately identify transgene copy numbers, to discriminate between single and low transgene copies, and to do such characterization even in transgenic plants with large genomes. Moreover, the technique has been evaluated even for the determination of genome editing rates in CRISP/Cas9  mutated plants . Table 1CNV refers to genome structural variations in which a specific DNA segment is repeated, and the number of repeats varies among different genotypes. In polyploids, CNV can also be defined as variations in allele dosage at a locus. The different allele dosages can be correlated with different phenotypes, e.g., barley genotypes with an increased copy number of HvCBF4 and HvCBF2 transcription factors showed greater frost resistance . RecentlArabidopsis thaliana loci and found strong variations in their copy numbers among natural ecotypes, demonstrating the high plasticity of the Arabidopsis genome. These authors compared two different analytical approaches for CNV determination, namely MLPA (multiplex ligation-dependent probe amplification) and ddPCR and concluded that ”dPCR should be the method of choice for discriminating loci with high copy numbers”. Two studies applied dPCR to the high-level polyploid sugarcane, a challenging crop species because of its complex genetics. McCord [Bru1 gene, characterized by a higher level of resistance to sugarcane brown rust. The information gained with such assay is now used in USDA breeding programs. Sun and Joyce [Saccharum species. Jouanin et al. [Triticum aestivum. The authors concluded that dPCR is suitable for the high-throughput screening of gene-edited and mutated wheat plants.The results obtained in the leading field of human genetics have indicated that dPCR could serve as a sensitive and less technically challenging method in respect to those above-reported to detect CNV. It is a particularly promising approach, both in diploid and polyploid plants. Zmienko et al.  used dPC. McCord  positivend Joyce  developen et al.  tested tAmaranthus cruentus [Beta vulgaris [Castanea [Hordeum vulgare [A. thaliana microRNAs were evaluated in different Plutella xylostella tissues with the aim to study the cross-kingdom functions of microRNAs derived from host plants in insect herbivores [Several technologies, such as real-time PCR, sequencing, and hybridization-based assays, are currently used for gene expression studies. The suitability of digital PCR for gene expression analysis has been demonstrated in several plant species, such as cruentus , Beta vuvulgaris , CastaneCastanea , and Hor vulgare . The exprbivores .Triticum aestivum and to differentiate this species from Triticum durum. This analytical tool has a great practical value in the Italian pasta production chain: the use of Triticum durum is, in fact, mandatory for pasta production, and Triticum aestivum is considered a contaminant whose percentage cannot exceed a maximum level of 3%. The assay developed by Morcia et al. [T. aestivum traceability along the whole pasta production chain from grains to pasta. Digital PCR has been used as a tool to evaluate the quality and amplifiability of DNA extracted from a challenging matrix, such as olive oil [Plant species traceability in food and feed chains can play a pivotal role in defending quality and safety. A dPCR assay is available to quantify a et al.  was demolive oil . Assays live oil  and Donglive oil . Starch live oil  developelive oil .dPCR is becoming an important new tool for use in the plant pathogen diagnostics and crop protection. Many examples of diagnostic assays ex novo developed or translated from similar qPCR assays have been recently published, as reported in Interestingly, Dreo et al.  demonstrAspergillus populations in soil with the aim to quantify the ratio between aflatoxigenic and atoxigenic strains. Zulak et al. [Blumeria graminis Cyp51 gene, which is able to confer high levels of resistance to triazoles in the field. The identification of such mutations in the pathogen field population has a key role in crop protection. In fact, the identification of resistant pathogenic strains carried out in an early stage of growth of the crop allows one to better modulate the consequent fungicidal treatments. Digital PCR has even been applied to the identification of non-pathogenic specific microorganism strains or populations present in the soil. A droplet digital PCR assay was developed by Xie et al. [Bacillus subtilis, a typical plant growth-promoting bacterium, in rhizosphere samples. Its efficiency in the analysis of soil samples is one of the strengths of digital PCR that is considered less susceptible than qPCR to the PCR inhibitors present in DNA extracts. The success in DNA amplification is of paramount relevance for the soil samples [In addition to the identification of pathogens at the species level, dPCR can be applied to the identification of specific strains. An example is the study of Hua et al. , in whick et al.  used dPCe et al.  for the  samples  and dPCRIn addition to the macro categories discussed in the previous paragraphs, additional uses of the dPCR have been proposed both in applied and basic agricultural research. For example, dPCR has been applied to the single and/or multiple detection of SNPs linked to useful agronomic traits. An example in this sense is the study of Stevanato et al. , which aIn support of the scientific community that uses the dPCR, a dMIQE2020  was recently published by the dMIQE Group , and it In conclusion, digital PCR is, at least in the plant science sector, still a young but very promising technique.The technology has many advantages and few disadvantages, which are summarized in Speaking of the advantages, some are unique to the dPCR and particularly strong. One is the greater resilience of the reaction to the presence of inhibitors, and this characteristic therefore makes the technique particularly valuable for the study of environmental and soil samples . MoreoveThe main advantage of dPCR, however, remains the fact that the analysis operates an absolute quantification that is free from the need for standard reference curves for the quantification of the target sequence. This characteristic is fundamental in various fields of plant science—in particular for GMP detection, for which the transition from qPCR assays to dPCR ones is often considered advantageous. The EURL GM FF  is maintaining a database of species-, element-, and event-specific qPCR assays  Analysis  for moreIn human genetics, the technique has experienced great development in the last ten years, progressing from an expensive and niche approach to a plethora of applications. In plant science, as previously reported, the main applications are currently observed in the two sectors of GMO traceability  and path


Background: Tecar therapy (TT) is an endogenous thermotherapy used to generate warming up of superficial and deep tissues. TT capability to affect the blood flow is commonly considered to be the primary mechanism to promote tissue healing processes. Despite some preliminary evidence about its clinical efficacy, knowledge on the physiologic responses induced by TT is lacking.Objective: The aim of this quantitative randomized pilot study was to determinate if TT, delivered in two modes (resistive and capacitive), affects the perfusion of the skin microcirculation (PSMC) and intramuscular blood flow (IMBF).Design: A randomized controlled pilot feasibility study.Subjects: Ten healthy volunteers  from a university population were recruited and completed the study.Intervention: All subjects received three different TT applications  for a period of 8 min.Outcome measures: PSMC, IMBF, and the skin temperature (ST) were measured pre- and post-TT application using power Doppler sonography, laser speckle contrast imaging (LSCI), and infrared thermography.Results: Compared with placebo application, statistically significant differences in PSMC resulted after both the resistive (p = 0.0001) and the capacitive (p = 0.0001) TT applications, while only the resistive modality compared with the placebo was capable to induce a significant change of IMBF (p = 0.013) and ST (p = 0.0001).Conclusions: The use of power Doppler sonography and LSCI enabled us to evaluate differences in PSMC and IMBF induced by TT application. Most of them reported encouraging results in decreasing pain and improving function in different musculoskeletal clinical conditions as low back pain,4 insertional tendinopathies of the Achilles, the patellar, and the wrist extensor common tendons.5 Its capability to affect the blood flow, as a consequence of its thermotherapeutic effect, is commonly considered one way in which TT supports the healing processes of injured/dysfunctional tissues.6 Nevertheless, a substantial lack of knowledge exists on the question: does TT affect blood flow in superficial tissue layers?The Tecar (capacitive and resistive energetic transfer) is an endogenous thermotherapy that uses electrical currents, induced by a 448 kHz capacitive/resistive monopolar radiofrequency, to generate warming up of deep tissues.2 To the best of knowledge, this pilot project is the first experimental study evaluating the effect of TT (CAP and RES separately) on the perfusion of skin microcirculation (PSMC) and intramuscular blood flow (IMBF) using laser speckle contrast imaging (LSCI) and power Doppler sonography. The aim of this quantitative pilot study is to determine if TT, administered in two modes, affects the IMBF, PSMC, and skin temperature (ST) in healthy subjects. Furthermore, the authors want to estimate variability to determine the sample size for future clinical trials evaluating the physiologic responses of TT.The Tecar device provides two different treatment modes: capacitive (CAP) and resistive (RES). These modes are normally delivered with different probes (electrodes), made of medical stainless steel. According to Tecar's developers, the two treatment modes induce different tissue responses depending on the resistance of the treated tissue. When the active electrode is provided with an insulating ceramic layer, acting as a dielectric medium, (CAP) the energetic transmission generates only heat in superficial tissue layers, with a selective action on low-impedance (water rich) soft tissues, for example, adipose tissue, muscle, cartilage, and lymphatic system. If the active electrode has no insulating layer, (RES) the radiofrequency energy passes directly through the body in the direction of the inactive electrode, generating heat in the deeper more resistant (low water content) tissue layers, for example, bone, muscular facia, capsules, and tendons. A recent study on healthy volunteers concluded that the delivery of the TT in a mixed mode (described as a “capacitive/resistive”) enhances blood flow volume in muscle tissue.n = 4 females, n = 6 males; mean age 35.9 ± 10.7 years, mean height 175.7 ± 9.3 cm, mean weight 72.8 ± 12.6 kg) was recruited from a university population. The first 10 volunteers meeting the inclusion criteria were admitted to the study. The inclusion criteria were nonpainful full active range of motion for the right shoulder, elbow, wrist, hand, and cervicothoracic spine. Exclusion criteria were lack of consent in receiving TT, use of pacemaker, epilepsy, angina pectoris, cardiovascular pathologies, pregnancy or breastfeeding, skin lesions, current or recent neck or upper extremity pain (at least 3 consecutive days in the past 6 months), nervous system disorders, diabetes mellitus, thermal sensitivity dysfunction, upper extremities, breast or cervical spine surgery, drug or alcohol abuse, tumors, radiation therapy or chemotherapy in the past year, metallic implants, and internal infection with encapsulated abscess.A sample of 10 healthy subjects . Ethical approval was granted by the Ethics Committee of Canton Ticino (2018-00271/CE3327), and the procedures were conducted according to the Declaration of Helsinki. All subjects signed written informed consent before the study. The proposed methodology was developed according to the Consolidated Standards of Reporting Trials statement (2010) containing an extension for a pilot and feasibility trial.8–10A Tecar device  was used to administer the treatment. LSCI  and a power Doppler  with a linear probe (LA 533) were used to assess PSMC and IMBF, respectively, according to previous studies investigating similar outcome variables.Heart rate (HR), blood pressure (BP), and ST were measured, respectively, with a digital sphygmomanometer  and an infrared thermography device . A digital hygrometer and thermometer  were used to measure room temperature and room humidity. A wooden frame for the right upper arm was used to standardize the position during all the experimental procedures.Three experimental sessions were planned for each participant to test the three different Tecar modes separately: RES, CAP, and placebo (PLAC) separately. To prevent carryover effects, a wash-out period of 1 week between the treatment sessions was considered to be more than sufficient. The order of treatment modality was randomized by asking each participant to choose between three sealed envelopes at the beginning of session I and II. All Tecar applications were performed by the same Tecar-certified physiotherapist.TT was administered in the RES modality using a round-shaped low-impedance electrode made of medical stainless steel, while the CAP modality TT was delivered using a high-impedance electrode made of medical stainless steel with ceramic coating. In the PLAC modality, TT was delivered by alternating between the high- and low-impedance electrodes with the device switched off. The PLAC application was included to test the potential variation of the blood flow related to the mechanical effect of the probe manipulation and not to the physiologic effect of the TT itself.TT was applied to the same region of the right forearm during each of the three sessions. The area was standardized using a reference system based on anatomical landmarks and defined with four strips of tape , appliedThe Tecar's plate electrode (inactive electrode), coated with conductive cream, was positioned under the right scapular region before the subjects were placed in a supine position. Following the completion of all premeasurements, a sufficient amount of conductive cream was applied to the skin of the volar forearm to facilitate optimal gliding of the Tecar's probe, and an 8-min TT application was administered. The round-shaped probe was handled with a roto-translatory movement. Each movement cycle lasted 1 sec. A metronome was used to ensure a precise handling of the probe.The intensity of each mode was set according to a previous pilot testing to ensure the highest possible tolerable treatment intensity. In detail: RES (70%), CAP (40%), and PLAC (0%). In the event that the heat generated by the TT was no longer tolerable, the intensity was decreased as follows: RES (35%) and CAP (20%). After each application, the conductive cream was gently removed from the skin using a cotton towel.A standard explanation of the experimental procedure was given to the subjects. They were instructed to communicate the heat intensity during the TT application. Subjects were in a supine position with legs straight and both upper arms beside the body. A soft pillow was positioned under the head to avoid discomfort. The right upper arm was positioned at 20° of shoulder abduction, the elbow in full extension, and the forearm was held in full supination by fixing the wrist to the wooden positioning frame with a Velcro strap. Subjects were asked not to move their hand and fingers, to avoid interference with the effect of the Tecar application, respectively, to impair the accuracy of the LSCI and Doppler measurements.10 before the measurements all subjects respected a 20-min acclimatization period before any experimental procedure was started. During this time, the subjects rested for 20 min in a supine position in a quiet and darkened room to guarantee stable HR and BP values and the stability of these parameters for the duration of the measurements.According to similar previous studies,All measurements were performed under standardized laboratory conditions. To compare the effect of TT, pre- and postmeasurements of PSMC, IMBF, HR, BP, and ST were conducted immediately after the TT application (post 1), at time interval 2 min (post 2), and after 10 min (post 3).The power Doppler images were assessed in two different positions on the volar forearm and arm: Position 1: between the distal and the middle 1/3 of the volar forearm and Position 2: proximal third part of the volar forearm . In bothTwo B-mode images, one in each position, were taken before assessing the power Doppler clips. Six power Doppler clips (5 sec long), three in each position, were taken before and immediately after the Tecar application (post 1). The same procedure was repeated at time interval post 2 (2 min) and post 3 (10 min) post-TT application.a priori specified region of interest (ROI) was marked on the volar forearm to obtain standardized values. High-resolution LSCI images (752 × 580 pixels), at a frame rate of 25 Hz , were recorded. The LSCI device uses arbitrary units that reflect the mean flux of an area of interest. The flux is related to the concentration of moving red blood cells in the tissue sample volume, where the level of flux is scaled from blue (low perfusion) to red (high perfusion). LSCI is a relatively new valid method to assess blood flow in the microcirculation of the skin. Compared with laser Doppler flowmetry and laser Doppler imaging, LSCI has some potential advantages. The macroscopic noncontact measurements, with a high spatial and temporal resolution, allow large full-field imaging of the skin microcirculation in real time due to a faster signaling processing.12 Especially in experimental settings where surface contact is undesirable and microcirculatory perfusion measurements of large skin areas are required, the reproducibility of cutaneous blood flow seems to be superior when measured with LSCI.14The LDCI measurements were performed with a wavelength of 750 nm in a supine position. One day before the measurements, the LDCI system was successfully calibrated. To minimize the risk of confounding factors, daylight and other sources of light were diminished as well as movements of the system during the measurements. The subjects were instructed to breathe normally and not to talk or move during the measurements. The laser aiming function of the device was used to obtain the optimal distance between the measured skin area and the LSCI-system. An The Shapiro–Wilks test revealed a non-normal distribution, and thus, data were described using median and interquartile range. The Friedman test was used to analyze the data for statistically significant median differences between pre- and postmeasurements within the three Tecar modes. Bonferroni correction for multiple comparisons was applied. Averages, medians, and standard deviations of all parameters were calculated to describe pre- and postmeasurements as well as their difference.p < 0.05.All statistical analyses were executed using the SPSS statistical package software . The significance level was set to Clips from the power Doppler were analyzed to extract three parameters: (1) area of blood vessels, (2) number of identified vessels, and (3) median power intensity of blood flow. The procedure for the extraction of these three parameters is described in the following paragraphs. The clips from power Doppler were recorded at 11 fps and were 10 sec long, to include at least three systolic and three diastolic movements. Each frame consisted of an image with two superimposed layers: b-mode and blue shade power Doppler image. The pixels with blue shades referred to an arbitrary scale ranging from 0 to 255 according to the ratio between blue and red color intensity in the RGB color code. An arbitrary threshold was set to 100 to avoid measuring background noise that was always present in the images.The number of pixels with a blue color intensity above 100 was computed for each frame. Since the pixel area curve was increasing during systole and decreasing during diastole, the authors used that curve to identify the systolic and diastolic cycles. The frame indexes corresponding to three systolic and three diastolic movements were identified from the curve of the blood vessels' area and those frames were used to extract the variables of interest. The area of blood vessels was evaluated as described above. To estimate the number of vessels from the images, the individual areas with at least 10 pixels not connected to each other were counted. A threshold of 10 pixels was set to avoid the measurement of background noise.An example of this procedure of noise filtering and calculating the area is presented in Due to fluctuations in the values between the two BP phases, it was decided to analyze only those images taken during the diastole. Using the statistical function from the moorFLPI Review V5.0 analysis software, the mean perfusion values of the ROI from the five recorded frames for every measurement interval (pre- and post-TT) were calculated .For the following variables, PSMC, IMBF , HR, and mean arterial pressure (MAP), the median difference between pre- and postintervention measurements was calculated and reported as a percentage. The MAP was calculated from systolic BP (SBP) and diastolic BP (DBP), using the formula as follows: MAP = (SBP +2 × DBP)/3.p < 0.05) of the median difference between each pre- and postintervention measurement were found for PSMC , IMBF distal , and ST (PLAC—RES). For both the control variables, HR and MAP, no statistically significant differences were found  measured immediately before and after the Tecar application were stable enough to assume that any potential difference between pre- and postmeasurements was not influenced by them. Statistically significant differences  for a period of 8 min, with an interval of 1 week between applications. PSMC, IMBF, and the ST were measured pre- and postintervention. To minimize the risk of bias, standardization of the procedures  was planned carefully. Compared with the PLAC application, both the RES and CAP modes were found to be effective in inducing a change in the PSMC. Unexpectedly, the CAP mode induced a slight decrease, while the RES mode, as expected, induced a moderate increase in the PSMC. Regarding the change in IMBF, a significant increase was found for the RES mode at the proximal third of the forearm. In comparison with placebo, no significant changes in IMBF were found for either the CAP or for the RES mode in the measurement at the distal middle third of the forearm.Although TT has been widely used in physical therapy practise as a physical therapy agent for almost 20 years, there are only a few studies that have investigated its clinical efficacy.2 stating that TT has the capability to induce a response in the deep tissue without an excessive increase of ST, a remarkable feature for any intervention with a thermotherapeutic effect.2 A limitation that might have influenced the decrease in ST between the RES mode and PLAC mode is the use of a “cold” (room temperature) steel electrode and conductive paste during the application in the PLAC mode. This might have influenced the ST and could have caused a vasoconstrictive effect on the PSMC. The use of thermotherapy is still quite common among physiotherapists, and also of interest to researchers to evaluate the forms of thermotherapy in the management of a variety of musculoskeletal disorders.15–18It should be noted that the significant difference in IMBF, for the RES mode, was only found proximally , and 10 min after the TT application. A possible explanation for this could be the larger cross-sectional area and consequently vascularization of the forearm  in comparison with the position 1 region . The onset of change in IMBF needs further investigation. Changes in PSMC and IMBF seem not to be related to any systemic cardiovascular responses, since both HR and MAP did not vary between pre- and postmeasurements, suggesting that TT affects blood flow only at a local level. Interestingly, the difference between pre- and postvalues of ST was only significant when comparing the RES mode with the PLAC mode, with an increase and a decrease of ∼2°C, respectively. These results are in line with Kumaran and Watson15–22 Although tendons are poorly vascularized and tendon nutrition is more reliant on synovial fluid diffusion than vascular perfusion, the role of changed vascularization during healing of tendon healing is still not clear.23 It remains questionable if TT can affect blood flow in other anatomical structures such as tendons and joint capsules. A previous published study of Kumaran and Watson2 suggested that TT application can affect the volume and velocity of IMBF. Compared with this research, some methodological differences can be pointed out. The main difference was in the way in which TT was applied. Kumaran and Watson2 delivered TT combining 10 min of the RES mode immediately followed by 5 min in the CAP mode, for a total treatment period of 15 min,2 although this option is more similar to commonly used clinical treatment sessions. Combining the two modes does not allow researchers to discriminate if the changes in blood flow differ or depend on the mode delivered. Furthermore, the intensity of the treatment was not standardized for all subjects but was set depending on the perceived local thermal sensation that can be potentially different for every participant. Defining the treatment intensity this way implies a risk of increasing the heterogeneity of the blood flow variations, as it is likely that increasing the treatment intensity might also affect the response size.The ability to induce a thermotherapeutic effect in the deep target tissue  without generating an excessive increase of the superficial (skin) temperature makes TT highly tolerable for the patients and suitable for the treatment of a variety of musculoskeletal disorders. Therefore, TT applications could be a treatment option especially in conditions where a dysfunction in blood flow plays an important role in generation and persistence of pain and dysfunction .2 Conversely, in the present study, the probe was placed perpendicularly to the forearm to detect the transverse section of regional vessels. This method allowed us to detect blood flow changes occurring not only in one large vessel but also in the smaller ones. This could be considered to a more accurate and suitable method to detect changes in blood flow within the muscle tissue.24Concerning the use of pulsed wave Doppler for blood flow measurement, some differences between the two studies exist, particularly concerning the probe placement. In each participant, Kumaran and Watson used the most prominently identifiable pulsatile  flow, then following this, the probe was placed parallel to the longitudinal axis of that arteria, to detect its longitudinal section.2 reported similar results concerning the potency of the TT to affect IMBF. Although the operator's efforts to utilize the same amount of pressure when placing the Tecar's probe on the treated area, no precise measurement of the pressure applied on the skin was carried out during the TT application.Despite all the methodological differences, it is encouraging that both the present pilot study and the study of Kumaran and WatsonThis limitation might have partially affected the variations in blood flow during the three application modes. Other limitations could be the chosen measurement intervals after the TT intervention and the fact that the authors could only measure PSMC before and directly after TT application. The present study found a significant increase of IMBF for the RES mode, which provides support for mechanism of action. It should be stated that this does not necessarily mean that this difference is clinically important or meaningful to patients. Future studies investigating the effect of TT on IMBF and PSMC postintervention measurement should be evaluated in 1-min intervals to get more precise information on the onset of microcirculation blood flow changes.In conclusion, these results indicate that TT applications in RES and CAP mode significantly effect PSMC in comparison with a TT PLAC application. A significant change in IMBF and ST was found only for the TT RES mode in comparison with the PLAC application. As the changes in PSMC and IMBF were not related to any systemic cardiovascular responses, TT seems to affect blood flow only at a local level. The applied method using LSCI and the power Doppler technique enabled us to estimate the change in PSMC and IMBF. To improve the clinical efficacy of TT, future studies should focus on physiologic differences between the different TT treatment modes.


KIT and/or NRAS mutation, known as frequently mutated genes in GCTs, in 3/12 (25%) patients. We also found significant differences in the abundance of 15 metabolites between control and GCT, with unsupervised hierarchical clustering analysis. Metabolites related to the TCA cycle were increased in GCTs. Urea, ornithine, and short-chain acylcarnitines were decreased in GCTs. Moreover, we also detected several metabolites  that displayed significant differences in abundance in patients with germinomas and NGGCTs. Our results suggest that ctDNA and metabolites in CSF can serve as novel biomarkers for CNS-GCTs and can be useful to differentiate germinomas from NGGCTs.Serum and cerebrospinal fluid (CSF) levels of α-fetoprotein and β-subunit of human chorionic gonadotropin are used as biomarkers for the management of central nervous system (CNS) germ cell tumors (GCTs). However, additional discriminating biomarkers are required. Especially, biomarkers to differentiate non-germinomatous germ cell tumors (NGGCTs) from germinomas are critical, as these have a distinct prognosis. We investigated CSF samples from 12 patients with CNS-GCT patients (8 germinomas and 4 NGGCTs). We analyzed circulating tumor DNA (ctDNA) in CSF to detect mutated genes. We also used liquid chromatography-mass spectrometry to characterize metabolites in CSF. We detected  The incidence is different among races, being higher in Asians  than in Whites 2. GCTs occur in higher proportion in male patients, with a male: female ratio greater than 3:12. GCTs in the CNS predominantly occur in children and adolescents and usually occur in midline structures, typically in the pineal region, third ventricular region, and neurohypophysis. Germinoma is the most common type of GCTs and it is highly radiosensitive. The 5-year survival rate of germinoma exceeds 99%, according to a recent study3. In contrast, non-germinomatous GCTs (NGGCTs) require intense chemotherapy, craniospinal irradiation, and sometimes salvage surgical resection5, and have an unfavorable prognosis with a 5-year survival rate of 71.4–87.3%3. Thus, distinguishing between these tumor types is essential to determine the appropriate treatment strategy.Germ cell tumors (GCTs) in the central nervous system (CNS) are rare comprising less than 3% of primary brain tumors6 and in some cases, initial treatment is based on the levels of AFP, β-HCG, and PLAP without tissue confirmation. The pineal region is one of the most frequent sites in which GCTs occur and they often cause non-communicating hydrocephalus.Serum and cerebrospinal fluid (CSF) levels of α-fetoprotein (AFP) and β-subunit of human chorionic gonadotropin (β-HCG), and placental alkaline phosphatase (PLAP) levels in CSF are used as biomarkers for current clinical management of CNS GCTs8. Histologically, GCTs are classified as pure germinoma or non-germinomatous germ cell tumors (NGGCT). However, NGGCT can contain germinoma components, which makes accurate histological diagnosis on small biopsies challenging, and in some cases, inaccurate. Moreover, some patients undergo treatment based on the levels of AFP and β-HCG, without biopsy or surgical resection. Therefore, additional diagnostic biomarkers for GCTs are of critical importance in neuro-oncology. Even though germinomas and NGGCTs are managed with chemotherapy and radiotherapy (not with surgery), therapeutic regimens such as radiation dose, are different and germinomas respond better to treatment5. Therefore, the distinction between germinoma and NGGCTs is crucial in providing patients with the optimal treatment.Intraventricular endoscopy is a feasible and minimally invasive technique, allowing both, access to the lesion for histological analysis as well as CSF diversion via endoscopic third ventriculostomy (ETV). However, biopsied samples for histological analysis, particularly from neuro-endoscopy are often small, representing only a partial region of the tumor, which can lead to inaccuracies in histologic diagnosis due to sampling bias13. We have previously reported on the analysis of circulating tumor DNA (ctDNA) and metabolites in the CSF of patients with lymphoma, gliomas, and metastatic CNS tumors16. In this study, we analyzed both ctDNA and metabolites in CSF from CNS GCT patients, with the aim of identifying biomarkers that could potentially be utilized in clinical practice.In recent years, CSF has been shown to be a relevant source of informative biomarkers for CNS tumorsThe biomarker levels of patients are summarized in Fig. KIT, NRAS, and MAP2K1  and 5.13 ng (range 2.25–20.0), respectively. Only two samples reached the manufacturer’s recommended amount of 20 ng  in the CSF samples. We found differences in the abundance of 15 metabolites between control patients and intracranial GCT patients FDR < 0.25) . We also identified a MAP2K1 mutation with a low MAF (case #20). This mutation was not observed in the CSF sample acquired at diagnosis in the same patient (case #8). Therefore, this mutation could have been acquired during tumor progression and/or treatment and might represent tumor evolution. Our validation experiments using CSF samples from patients without CNS tumors indicate that we can confidently identify mutations at a MAF greater than 0.25% (data not shown). Therefore, we favor that the MAP2K1 change is an acquired mutation that represents tumor evolution.Our study shows that genetic alterations frequently present in GCTs are detectable by analysis of CSF-ctDNA. We detected 19. A recent study from Japan with 124 CNS GCTs identified mutations in MAPK and/or PI3K pathways such as KIT (27.4%), MTOR (6.5%), KRAS (5.6%), and NRAS (3.2%)17. The rate of mutation detection in CSF-ctDNA in our study (25% of cases) was lower when compared to the literature showing that ~ 54% of GCTs had mutations in at least one of the genes involved in MAPK or PI3K pathways18 which are mostly covered by the Oncomine NGS assay. However, it is unclear what mutations were present in the tumors from patients in this study because mutation analysis of tumor tissues was not available.The mutations detected in CSF-ctDNA from patients with GCTs included in our study are consistent with reported mutations in GCTs tissue. Several studies have reported that mutations in MAPK and/or PI3K pathways are the dominant genetic drivers of GCTsAn average of 1.71 ml of CSF from tumor patients was utilized to extract ctDNA, and the median amount of extracted cell-free DNA (cfDNA) for library preparation was 5.13 ng , and PEP levels were also demonstrated in the CSF of IDH-mutant glioma patients, in our previous study24. A study showed that plasma levels of acetylcarnitine negatively correlated with tumor grade in patients with hepatocellular carcinoma24. Studies on plasma metabolites indicate that changes in the MAPK or PI3K pathways are associated with alterations in metabolites including amino acids, acylcarnitines, and phosphatidylcholines26. Therefore, we assumed that GCTs, often associated with mutations in either MAPK or PI3K pathways, exhibited decreased levels of acetylcarnitine and butyrlcarnitine. However, samples were not available for us to explore this relationship further with tissue-based experiments.Acylcarnitines are essential for the oxidative catabolism of fatty acids. They transport fatty acids into the mitochondria, and fatty acids are oxidized (β-oxidation) and converted to energy through the TCA cycleN-acetyltransferase (Asp-NAT)27. Therefore, NAA is used as a neuron-specific marker, for instance, lower NAA concentration in CSF have been reported to correlate with worse clinical functioning and lower brain volume in multiple sclerosis patients28. We detected increased NAA in the CSF of most GCT patients. However, the interpretation of NAA levels should be done with caution, since NAA levels in CSF can be influenced by age and intracranial pressure29. The majority of GCT patients in this study were children or adolescents with non-communicating hydrocephalus with slightly or moderately increased intracranial pressure. In contrast, many of the control CSF samples in our study were elderly patients with NPH. Interestingly, recent studies have shown high expression of NAA metabolism enzymes in brown adipocytes suggesting that that NAA may be involved in lipid synthesis and histone acetylation. Higher expression of NAA synthesizing enzyme and increased amount of NAA, have been associated with worse survival in various cancer types30. Elevated NAA was also seen in the CSF from patients with both IDH-mutant and IDH-wildtype gliomas in our previous study14.NAA is the second most abundant metabolite in the brain, and it is synthesized from aspartate and acetyl-coenzyme A in neurons, by the enzyme aspartate 5. Therefore, reliable diagnosis differentiating germinoma from NGGCT is essential to determine optimal patient treatment. Identification of the teratoma/immature teratoma component is also important because these components can cause large cystic masses (growing teratoma syndrome), during treatment31. We detected significantly elevated levels of betaine in NGGCT , our findings indicate that several metabolites have the potential to discriminate germinomas from NGGCT. Therefore, the analysis of CSF metabolites could be instructive in identifying the presence of GCTs with malignant components that should be treated more aggressively.The levels of several metabolites were significantly different between germinomas and NGGCT. Differentiation between germinomas and NGGCTs is of crucial clinical importance. Increased levels of AFP in serum/CSF indicate that the GCT contains a component of a yolk sac tumor or immature teratoma. Except for germinoma with syncytiotrophoblastic cells, elevated β-HCG suggests an association with choriocarcinoma, embryonal tumor, or immature teratoma. GCTs with a highly malignant component require intensive chemotherapy and corticospinal irradiationCT Figs. B and 3. In conclusion, our results for the first time suggest that ctDNA and metabolites in CSF can serve as novel biomarkers for CNS GCTs, and can be useful in differentiating germinomas from NGGCTs Fig. . Such biWe included intracranial GCT patients treated at Hiroshima University Hospital, Hiroshima, Japan, from 2015 to 2018, or at Memorial Hermann Hospital/University of Texas Health Science Center at Houston, Houston, Texas, from 2018 to 2019. Samples from patients (n = 12) and controls (n = 9) were utilized. The cohort consisted of 8 germinomas, 1 mature teratoma, 1 immature teratoma, and 1 yolk-sac tumor. The control group consisted of 6 normal pressure hydrocephalus (NPH) patients, 1 cytomegalovirus infection, 1 toxoplasmosis, and 1 granulomatous inflammation. CSF was collected during the endoscopic intraventricular intervention (biopsy/ETV) or lumbar puncture (LP) as part of routine clinical practice. Demographic information is summarized in Fig. This study was approved by the institutional review board (IRB), at Hiroshima University Hospital and University of Texas Health Science Center at Houston; respectively, in accordance with the declaration of Helsinki and its later amendments. Informed consent was obtained from all patients.g for 10 min at 4 °C. The cell pellet was discarded, and the supernatant was immediately stored at -80 °C until the time of the analysis.CSF samples were collected from the endoscopic intraventricular approach intraoperatively or lumbar puncture as part of routine clinical practice. Samples were processed within 3 h from the collection and centrifuged at 1,000 AKT1, ALK, AR, ARAF, BRAF, CHEK2, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, RAF1, RET, ROS1, SF3B1, SMAD4, and SMO. Although the recommended cfDNA input amount is 20 ng, the majority of our tumor samples did not reach 20 ng, the cfDNA input ranged from 2.25–20 ng. We chose 2 control samples with the highest cfDNA concentration. The final ctDNA libraries were purified using AMPure XP magnetic beads  and quantified using 4,200 TapeStation . Templating and sequencing were performed using the Ion 540 Chip on the Ion Chef and S5 XL systems with tag sequence technology . NGS data was analyzed with Ion Reporter Software (version 5.10).A range of 0.38–2.88 mL of thawed CSF supernatant was used for the extraction of ctDNA. We used QIAamp Circulating Nucleic Acid Kit , following manufacturer instructions. The cell-free DNA (cfDNA) concentration was quantified using the Qubit dsDNA High Sensitivity Assay kit . For library preparation, we used Oncomine Pan-Cancer Cell-Free Assay  to detect mutations in CSF-ctDNA as per manufacturer’s instructions. This next-generation sequencing (NGS) panel covers alterations in 52-genes, including hotspot (SNVs) and short indels in 34. Targeted metabolic profiling was carried out by LC–MS/MS, 6,495 QQQ Mass spectrometer  equipped with electro spray ionization (ESI) source. Single Reaction Monitoring (SRM) mode of ions with mass to charge ratio for metabolites were utilized for relative quantitative analysis of metabolites. We measured the metabolites using three different chromatographic methods (see supplementary methods), in each method metabolites were normalized with the spiked internal standards and data were log2-transformed on a per-sample, per-method basis. For clustering analysis, we performed the moderated t-test in the LIMMA package, to compare the differential metabolite levels between two groups35. Differentially expressed metabolites were identified after adjusting p-values for multiple hypothesis testing using the Benjamini–Hochberg method36 and a false discovery rate (FDR) of < 0.25. Unsupervised clustering was used in this study and the analysis was conducted in R (version 3.5.2). AUC (area under the receiver operating characteristic curve) and its 95% confidence interval were evaluated by the “DeLong” method with EZR (version 1.40)37. For the box plot, differences in metabolites between two groups of patients were analyzed using the Mann–Whitney U test or two-sample t-test, depending on whether the Kolmogorov–Smirnov test showed a significant difference from the normal distribution in each data.For metabolomic analysis, 0.12 mL of CSF supernatant from each patient was used for the extraction of metabolites as described previouslySupplementary information


TNFRSF1B gene encoding TNFR2 and levels of its soluble form (sTNFR2) affect Alzheimer disease (AD) biomarkers and clinical outcomes. Data analyzed included 188 patients in the Alzheimer's Disease Neuroimaging Initiative (ADNI) who had mild cognitive impairment (MCI) and AD dementia. Further, a replication study was performed in 48 patients with MCI with positive AD biomarkers who were treated at a memory clinic. Cerebrospinal fluid (CSF) sTNFR2 levels along with two related TNFRSF1B gene single nucleotide polymorphisms (SNPs) rs976881 and rs1061622 were assessed. General linear models were used to evaluate the effect of CSF sTNFR2 levels and each SNP in relationship to CSF t-tau and p-tau, cognitive domains, MRI brain measures, and longitudinal cognitive changes after adjustments were made for covariates such as APOE ε4 status. In the ADNI cohort, a significant interaction between rs976881 and CSF sTNFR2 modulates CSF t-tau and p-tau levels; hippocampal and whole brain volumes; and Digit Span Forwards subtest scores. In the replication cohort, a significant interaction between rs976881 and CSF sTNFR2 modulates CSF p-tau. A significant interaction between rs976881 and CSF sTNFR2 also impacts Clinical Dementia Rating Sum of Boxes scores over 12 months in the ADNI cohort. The interaction between TNFRSF1B variant rs976881 and CSF sTNFR2 levels was noted to modulate multiple AD-associated severity markers and cognitive domains. This interaction impacts resilience-related clinical outcomes in AD and lends support to sTNFR2 as a promising candidate for therapeutic targeting to improve clinical outcomes of interest.Tumor necrosis factor receptor 2 (TNFR2) promotes neuronal survival downstream. This longitudinal study evaluated whether the  APOE ε4 and other genes that may be related to the various MRI and clinical endophenotypes of AD  is paradigmatically characterized by the presence of amyloid β plaques and tau neurofibrillary tangles in the brain. However, the cognitive and behavioral phenotypes of AD and their related CSF and MRI biomarker signatures can vary among patients, and molecular factors beyond amyloid β and tau likely play a role in this heterogeneity  and its variants have previously been linked with inflammatory responses, inflammatory disease outcomes, and rate of cancer progression , we evaluated the reliability of the findings by assessing the same factors in a group of patients with MCI-AD who were treated at a memory clinic.We therefore sought to determine whether sTNFR2 and related For the ADNI study, all patient consent was obtained according to the Declaration of Helsinki, and the study was approved by local Institutional Review Boards. For the replication memory clinic cohort, all patients provided written informed consent, and CSF, plasma, and DNA samples were collected for the Lou Ruvo Center for Brain Health Aging and Neurodegeneration Biobank following approval by the Cleveland Clinic Institutional Review Board.TNFRSF1B variants, CSF sTNFR2, and CSF AD biomarkers and cognitive variables were available. The CSF data used in this analysis were cleaned and quality controlled according to methodology described previouslyA multistage study design was used to collect and replicate findings in two independent cohorts (the ADNI cohort and the replication memory clinic cohort) for which data regarding http://adni.loni.usc.edu/methods/documents/). Briefly, eligible participants were aged 55 to 90 years, spoke either English or Spanish, and had an informant who was able to provide an independent evaluation of functioning. Eligible participants had also completed at least 6 years of education .ADNI is a longitudinal multicenter study designed to develop clinical, imaging, genetic, and biochemical biomarkers for the early detection and tracking of AD. The eligibility criteria for ADNI-1 (the first phase of the project) are described in the ADNI-1 protocol  v.1.0 panel , which uses a Luminex platform (Myriad Genetics). Data obtained from “Biomarkers Consortium CSF Proteomics Project RBM Multiplex Data and Primer.”TNFRSF1B SNPs: rs976881 and rs1061622, which were found to be in linkage equilibrium in all populations  according to LDlink analysis  and a missense coding variant in exon 6 (rs1061622). These SNPs were chosen based on their association with TNFRSF1B-related clinical outcomes and peripheral sTNFR2 levels , and intensity data were processed with GenomeStudio v2009.1 (Illumina). For this study, we analyzed two In ADNI-1 patients with MCI or dementia, results from 1.5T MRI scans were performed at baseline, and these results (taken from the ADNIMERGE primary table) were used for this analysis   were recruited from a specialized memory clinic at Cleveland Clinic . Recruitment details have been described previously  dispensed into 96-well plates for TaqMan  allelic discrimination assays for the detection of SNPs associated with APOE alleles . PCR was performed using a 9700 Gene Amp PCR system (Applied Biosystems) and an end-point read in a 7500 Real-Time PCR system (Applied Biosystems)  were characterized. TNFRSF1B variant allelic discrimination was performed using the 7500 Real Time PCR System and TaqMan SNP Genotyping Assays. The genotypes were determined based on sample clustering using the autocaller function of the Genotyping Application within Thermo Fisher Connect software.Genomic DNA was extracted from peripheral whole blood using standard protocols. Two MMSE and Montreal Cognitive Assessment (MoCA) scores  transformation allowed Pearson correlations to be fit; the sTNFR2 and AD biomarker levels described are therefore dimensionless. Along with estimates of correlation, 95% CIs and TNFRSF1B variant of interest on CSF t-tau and p-tau, MRI volumetric measures, and cognitive variables. Third, we tested whether the interaction effects between sTNFR2 and each TNFRSF1B variant and between sTNFR2 and t-tau were present for CSF t-tau and p-tau, MRI volumetric measures, and cognitive variables. Levene's test of equality of error variances, lack-of-fit F-test, and residual plots were used to assess model linearity and fit.First, we tested the effect of sTNFR2 levels on CSF t-tau and p-tau, MRI volumetric measures, and cognitive variables. Second, we tested the effect of each Model 1, evaluated the dependent variables CSF t-tau and p-tau. The main effects were CSF sTNFR2, TNFRSF1B SNP, and interaction effect between sTNFR2 and TNFRSF1B SNP. Covariates for this model included age, sex, CSF Aβ42, and APOEε4 status.Model 2, evaluated the dependent variables hippocampal volume, ventricular volume, and whole brain volume. The main effects were CSF sTNFR2, TNFRSF1B SNP, CSF t-tau, and interaction effects between sTNFR2 and TNFRSF1B SNP and between sTNFR2 and t-tau. Covariates for this model included age, sex, years of education, CSF Aβ42/p-tau ratio, APOE ε4 status, and baseline intracranial volume.Model 3, evaluated the dependent variables Logical Memory delayed recall score, Digit Span subtest score, Trail Making Test Part B score, category fluency test , and Boston Naming Test score. The main effects were CSF sTNFR2, TNFRSF1B SNP, CSF t-tau, and interaction effects between sTNFR2 and TNFRSF1B SNP and between sTNFR2 and t-tau. The covariates for this model included age, sex, years of education, CSF Aβ42/p-tau ratio, APOE ε4 status, and baseline MMSE score.Model 4, CDR-SB scores were log transformed because Levene's test demonstrated that the variances were significantly different for CDR-SB scores for the TNFRSF1B SNPs. A univariate general linear model was used to evaluate the interaction effect for each TNFRSF1B SNP and CSF sTNFR2 on change in CDR-SB score over 1 year in the ADNI cohort. CDR-SB score at 1 year was the dependent variable; the main effects were disease state (MCI or AD dementia), CSF t-tau, and interaction effect between sTNFR2 and TNFRSF1B SNP. The covariates included age, sex, years of education, APOE ε4 status, baseline CDR-SB score, and CSF Aβ42/p-tau.TNFRSF1B SNP, and interaction effect between sTNFR2 and TNFRSF1B SNP. Covariates for this model included age, sex, CSF Aβ42, and APOE ε4 status.Paralleling Model 1, here the dependent variables were again CSF t-tau and p-tau. The main effects were CSF sTNFR2, p < 0.05. For model 3 (with more than three dependent variables in the outcome), the Benjamini-Hochberg false discovery rate was used to assess significance. IBM SPSS Statistics for Windows, Version 22.0  and RStudio Team  .There weAPOE ε4 carriers and a slightly lower baseline MMSE.A total of 48 participants with MCI-AD were included in the replication memory clinic cohort . There wR2 = 0.1) than C/C (R2 = 0.38), suggesting that sTNFR2 levels are not as closely related to t-tau levels in T/T as in C/C. The direct main effects of sTNFR2 on CSF t-tau and p-tau are shown in A significant interaction effect between rs976881 and CSF sTNFR2 was associated with CSF t-tau levels, but this association with CSF t-tau levels was not seen for the interaction effect between rs1061622 and CSF sTNFR2 . The effA significant interaction effect between rs976881 and CSF sTNFR2 was associated with hippocampal volume and whole brain volume but not with ventricular volume . 8.8% ofThe MRI measures were not affected by rs1061622, nor by the interaction effect between sTNFR2 and CSF t-tau. CSF t-tau did have a significant direct main effect on ventricular volume and whole brain volume but not on hippocampal volume .R2 = 0.33), suggesting that lower sTNFR2 levels (but not CSF t-tau levels) relate to poorer Digit Span Forward scores for this group, unlike for the C/C group (R2 = 0.004). The interaction effect between rs1061622 and CSF sTNFR2 had no effect on these cognitive measures. The Digit Span Forward score was significant even after false discovery rate correction  score . 9.9% ofF = 2.83; adjusted R2 = 0.36). A total of 4.6% of all variance in CDR-SB change over 1 year was attributable to the interaction effect between rs976881 and CSF sTNFR2 (2 = 0.52 vs 0.70). There was no significant effect of rs1061622 on CDR-SB change over 1 year .A significant interaction effect between rs976881 and sTNFR2 was associated with change in CDR-SB scores over 1 year  than the homozygous alternate allele (C/C) (R2 = 0.42) (A significant interaction effect between rs976881 and CSF sTNFR2 was associated with CSF p-tau levels . Eleven  = 0.42) , suggestA significant interaction effect between rs1061622 and CSF sTNFR2 was associated with CSF t-tau and p-tau levels. Fourteen percentage of all variance in CSF t-tau and 14% of all variance in CSF p-tau were attributable to the interaction effect between rs1061622 and CSF sTNFR2 . PairwisTNFRSF1B gene variants contributed to significant differences in AD-associated severity markers such as CSF t-tau and p-tau, MRI measures of interest, and cognitive scores in the ADNI cohort. In a replication memory clinic cohort, the interaction between CSF sTNFR2 and rs976881 was also associated with differences in CSF p-tau biomarkers, with a significantly larger effect size than was seen in the ADNI cohort. In the ADNI cohort, rs976881 T/T allele (homozygous reference) carriers demonstrated slower rates of CDR-SB score changes in relation to corresponding sTNFR2 levels. Additionally, statistical interaction effects demonstrated that the strength of a linear relationship between sTNFR2 and (1) CSF t-tau and p-tau, (2) MRI whole brain and hippocampal volumes, and (3) Digit Forward test score varied for the rs976881 T/T allele compared to the C/C and T/C alleles. Studies in animal models have demonstrated that TNFR2 plays a protective role against neurodegeneration in the CNS  related to higher sTNFR2 levels than the alternate allele (C/C)  scores. These measures assess overlapping but distinct cognitive skills, including working memory, complex attention, and speed of information processing, and can be categorized as contributing to domains of attention/executive functioning and processing speed. In this cohort, rs976881 status was also related to the change in CDR-SB score over 1 year. This is consistent with our previous finding that better initial performance on the Digit Span subtest and related working memory task is associated with a slower rate of functional decline on the CDR-SB test over time in patients with AD  and longitudinal cognitive outcomes in the ADNI population. In the ADNI cohort for this study, 4.6% of the variance in CSF t-tau levels was explained by rs976881 and sTNFR2, whereas 11% of the variance in CSF t-tau was attributed to the same variables in the replication cohort. There were some differences between the cohorts, however. First, the positive correlation between the sTNFR2 and neurodegeneration biomarkers are consistent within each cohort but the replication cohort had 2.2 times higher mean levels of p-tau than ADNI . These other SNPs were not included because of the smaller number of participants within ADNI who had these genetic variants and who also had measured levels of sTNFR2 available. Using multiple SNPs may allow researchers to define a stronger TNFRSF1B haplotype with regard to sTNFR2 levels, and some variables may become more or less salient considering additional SNP interactions. Independent replication using the same biomarkers used in ADNI would allow for further clarification. Type II errors have to be considered for this study, given the small number of patients within some of the allele groups; smaller effect sizes could therefore have been missed.The study is an evaluation of the association between CSF sTNFR level and TNFRSF1B gene variant rs976881 and CSF sTNFR2 affects CSF and MRI biomarkers of neurodegeneration, cognitive profiles, and rate of functional decline over 1 year. These results provide important information regarding the molecular characterization of AD phenotypes and suggest that this genotypic variant could be used as a marker of resilience in AD. Independent confirmation of these results in other cohorts with mutidomain AD biomarkers is warranted.The rs976881 T/T reference genotype has been related to higher levels of sTNFR2 previously and activation of TNFR2 signaling has been posited as a promising strategy for AD therapy  can be found at: The studies involving human participants were reviewed and approved by Cleveland Clinic Institutional Review Board ADNI Individual Site Institutional Review Board. The ethics committee waived the requirement of written informed consent for participation.JP: obtained funding for research project, organization of research project, execution of research project, design the statistical analysis, execution of statistical analysis, and writing the first draft of manuscript preparation. GB: execution of research project, review and critique of statistical analysis, and review and critique of manuscript preparation. MK: execution of research project and review and critique of manuscript preparation. JB: design the statistical analysis and review and critique of manuscript preparation. CB, WB, and JL: review and critique of statistical analysis and review and critique of manuscript preparation. LB: organization of research project, review and critique of statistical analysis, and review and critique of manuscript preparation. All authors contributed to the article and approved the submitted version.JP receives research funding from the National Institutes of Health, Alzheimer's Association, and Keep Memory Alive Foundation. CB receives research funding from the National Institutes of Health. WB receives research funding from the National Institutes of Health. JL has received consulting fees from Acadia, Aptnyx, Biogen, Eisai, GE Healthcare, Sanofi, and Takeda and grant support from the Alzheimer's Association, Alzheimer's Drug Discovery Foundation, Biogen, Department of Defense, GE Healthcare, Genzyme/Sanofi, Lewy Body Dementia Association, Michael J Fox Foundation, and National Institute of Health . The remaining 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.


Delivery of therapeutic agents to the central nervous system is challenged by the barriers in place to regulate brain homeostasis. This is especially true for protein therapeutics. Targeting the barrier formed by the choroid plexuses at the interfaces of the systemic circulation and ventricular system may be a surrogate brain delivery strategy to circumvent the blood-brain barrier. Heterogenous cell populations located at the choroid plexuses provide diverse functions in regulating the exchange of material within the ventricular space. Receptor-mediated transcytosis may be a promising mechanism to deliver protein therapeutics across the tight junctions formed by choroid plexus epithelial cells. However, cerebrospinal fluid flow and other barriers formed by ependymal cells and perivascular spaces should also be considered for evaluation of protein therapeutic disposition. Various preclinical methods have been applied to delineate protein transport across the choroid plexuses, including imaging strategies, ventriculocisternal perfusions, and primary choroid plexus epithelial cell models. When used in combination with simultaneous measures of cerebrospinal fluid dynamics, they can yield important insight into pharmacokinetic properties within the brain. This review aims to provide an overview of the choroid plexuses and ventricular system to address their function as a barrier to pharmaceutical interventions and relevance for central nervous system drug delivery of protein therapeutics. Protein therapeutics targeting the ventricular system may provide new approaches in treating central nervous system diseases. The central nervous system (CNS) is protected by various barriers that are pivotal for maintaining a homeostatic environment for brain functions. One chief example is the blood-brain barrier (BBB) that sheaths the majority of cerebral blood vessels. Another is the barrier formed by the choroid plexuses (CPs) at the interfaces of the systemic circulation and ventricular system, comprising one of the blood-cerebrospinal fluid (CSF) barriers (BCSFBs). Though crucial for the protection of physiological functions in the brain, restricted CNS entry represents an obstacle for the delivery of therapeutic agents to treat CNS disorders. In this review, we will provide an overview of the CPs and ventricular system, including their function as a barrier to pharmaceutical interventions and relevance for CNS drug delivery of protein therapeutics.The cerebral ventricular system is an interconnected succession of spaces that resides within the center of the brain. It is composed of four fluid-filled cavities: the third, fourth, and lateral ventricles . Each laThe ependyma is a single layered epithelium that coats the surface of the ventricular system. Its extent spans the lateral ventricles to the filum terminale of the spinal cord ,4,5. TheSpecialized ependymal cells are present throughout the ventricular system. Tanycytes are one example, which reside within the circumventricular organs (CVOs) bordering the third and fourth ventricles. CVOs are important homeostatic and communication hubs located in the CNS. There are several CVOs: the pineal gland, subcommissural organ, median eminence, vascular organ of the lamina terminalis, and the subfornical organ found within the third ventricle walls in addition to the area postrema located adjacent to the fourth ventricle. All but the subcommissural organ are vascularized by fenestrated capillaries that lack a BBB. They are typically classified by their function as either secretory or sensory CVOs ,22,23,24Another example of specialized ependymal cells is choroid plexus epithelial cells (CPECs). The CPs are tentacular tufts with a leaf-like organization suspended within every ventricle  1,2]. E,2. E1,2]The CPs perform various essential and complex functions that have been discussed by many prominent reviews ,48,49,50CSF content is actively regulated by the CPs, which contrasts with early misconceptions that CSF is a plasma ultrafiltrate ,48,54. CHowever, not all CSF proteins are produced by the CPs. Serum albumin is a prime example of a plasma-derived protein that can cross the CPs and enter the ventricular space ,61,62. TPeptide and protein movement from the blood to the CPECs is driven by anatomical arrangements and morphological characteristics. Upon reaching the choroidal vessels, serum proteins can rapidly traverse the CP vascular endothelium either by movement through endothelial fenestrae or, potentially, via vesicular shuttling. The endothelium is highly permeable to macromolecules such that even ferritin (≈474 kDa) can pass through the choroidal capillaries ,36,50. FThe second potential route for proteins to enter the CSF is to undergo intact transcytosis across CPECs. This can occur following pinocytic uptake at the basolateral and intercellular membranes as shown for horseradish peroxidase , or The transport of small peptides such as di- and tripeptides can be mediated through members of the proton-coupled oligopeptide transporter (POT) family  that belong to solute carrier proteins (SLC15A) ,73. Two Amyloid-beta (Aβ) is a heterogeneous mixture of 37–43 amino acids and its accumulation in the brain is considered a major underlying mechanism for the development and progression of Alzheimer’s disease . In addiThe transferrin receptor (TfR) is a transmembrane glycoprotein that mediates the transport of iron-containing transferrin (Tf) . The TfRTfR in the CNS has been shown to regulate the transport of iron across brain barriers and maintain iron homeostasis in CPECs, brain capillary endothelial cells, and neurons ,86. SincInsulin receptors are ubiquitously distributed in peripheral tissues where they play an essential role in regulating glucose homeostasis. In the brain, the insulin receptor differs in size, glycosylation, and insulin-binding affinity in comparison with the peripheral insulin receptor . The in-Besides the insulin receptor, insulin-like growth factor receptors (IGF1R and IGF2R) are involved in mediating the intracellular effects of insulin, insulin-like growth factor-I (IGF-I), and IGF-II . InsulinThe low-density lipoprotein (LDL) receptor family comprises a group of plasma membrane receptors that play important roles in lipid metabolism and other physiological activities . They arSeveral core members of the LDL receptor family have been identified at the CPs, including the LDL receptor (LDLR), LDL receptor-related protein 1 (LRP1/LRP/α2-macroglobulin receptor), LRP2/megalin/glycoprotein 330, and LRP8/ApoE receptor 2 . LDLR isCompared to LDLR, information on the role of LRP1 at the CP has been provided by additional studies. Previous work has examined LRP1 mRNA and protein expression in humans and rats ,111,112.LRP2, also known as megalin or glycoprotein 330, has been found at the apical and subapical site of CPECs in rats ,116. HowLRP8 is also known as ApoE receptor 2 due to its function in the uptake of ApoE phospholipid discoidal particles or ApoE-enriched high-density lipoprotein . It has The neonatal Fc receptor (FcRn) is a crucial facilitator of Immunoglobulin G (IgG) transport from mother to offspring, providing immunity to the newborn . In addiMonodelphis and mice at very early age, which may be correlated with the higher protein concentrations in fetal CSF compared with adult CSF [The SPARC (secreted protein acidic and rich in cysteine) is a soluble and cell surface albumin-binding glycoprotein that has been implicated in albumin transport from blood to CSF . Higher dult CSF ,130. Thedult CSF . HoweverA variety of methods have been utilized to study protein transport across the CPs. A straightforward, yet indirect example is to administer a labeled or exogenous protein of interest systemically  and measure CSF appearance over time. However, without supporting information such as time-dependent localization studies, this strategy does not directly implicate the CPs as the source of entry into the CSF. Here, we highlight several techniques to examine CP protein handling.Though not fully qualitative, imaging studies can provide crucial insight into the role of CPECs in the bidirectional transport of proteins. Early ultrastructural-level investigations examined the passage of HRP across the CPEC barrier from blood to CSF, and vice versa. These detailed investigations were instrumental in establishing the barrier properties of the CPs . Other eVentriculocisternal perfusions have been employed for decades and were instrumental in delineating the physiology and dynamics of the ventricular system. These experiments typically encompass cannulations of one or both lateral ventricles and the cisterna magna for inflow and outflow, respectively ,136,137.Another model for the study of CP transport is the ovine in situ CP perfusion. This method was originally developed to examine the CP independently of the BBB in a large animal ,138. A mIsolation and culture of primary CPECs remains the most straightforward model to evaluate bidirectional peptide/protein transport across a CPEC monolayer. Primary CPECs have been isolated and cultured from several preclinical species in addition to human tissue with varying success ,143,144.Limitations of primary CPECs are that a good extraction method is required to obtain acceptable viability and purity , they are time-consuming to isolate, and they require fresh tissue which can present a challenge for CPECs from both preclinical species and humans . SeveralBy combining complimentary techniques, investigators can delineate the transport of therapeutic proteins across the CPs into the CSF, the direction that is oftentimes the focus of drug delivery. A good example of such a study was the examination of apolipoprotein A-I entry into CSF . SeveralCNS disorders represent a massive economic and healthcare burden on the global population. Numerous CNS diseases and injuries leave irreparable damage and remain difficult to impossible to treat. As well, in too many cases, current treatment options are only palliative and do not address the underlying pathologic drivers, if they are even known  via the foramina of Magendie and Luschka in a manner that CSF reflux from the SAS back into the fourth ventricle is typically associated with pathophysiology ,164,165.From the subpial and subarachnoid spaces, CSF can also pass into perivascular spaces (PVSs) surrounding arterioles, capillaries, and likely venules within the brain. The PVS is a physical space that facilitates the distribution of fluid and solutes via bulk flow adjacent to brain vessels. It represents a site of CSF and brain interstitial fluid (ISF) exchange. PVS fluid movement is proposed to be highly driven by arterial pulsations ,173,174.PVS fluid can enter the brain parenchyma, but the extent and movement remain hotly disputed and are an active field of neuroscience research. One hypothesis (the glymphatic system) proposes CSF inflows into the arterial PVS in the same direction as blood flow, driven by arterial pulsations as discussed directly above. CSF then enters the parenchyma through astrocytic end feet, which is heavily dependent on aquaporin-4 (AQP4) water channels. This influx of fluid facilitates mixing between CSF and brain ISF. Parenchymal exit occurs via the perivenous PVS and tracts located alongside cranial nerves  ,120,121 CSF flow within the ventricular system is not constant and unidirectional. Rather, it is pulsatile and complex with macroscale and near-wall variations. Ependymal ciliary beating is a major driver of ventricular CSF bulk movement and directionality, with other major influences including cardiac pulsations and inspiration ,206,207.Despite the lack of tight junctions, the ependymal cells should be considered a barrier to protein therapeutics administered intraventricularly or delivered across the CPs ,198. EpeThese statements should not be taken as an ultimatum that proteins cannot enter the brain parenchyma from the ventricular space. The rate and extent of CNS distribution for any compound are dependent on numerous factors. Here, we are emphasizing that the ependyma should be taken into consideration when determining/predicting the biodistribution of protein therapeutics. Similar to protein transport by CPECs, detailed studies are truly needed to better understand ependymal cell biology and their role in regulating the CNS disposition of therapeutic proteins.As discussed above, the PVS is considered a linkage of CSF with SAS and ISF at the brain parenchyma ,177. IncBe that as it may, barriers lie in this compartment to further restrict the exchange of compounds between CSF within the PVS and brain parenchyma. The pial surface is regarded as an important interface between the SAS and brain parenchyma . During Other factors influencing the PVS distribution, such as physiochemical properties, state of consciousness, circadian rhythm, and diseases or surgical intervention, can significantly alter PVS physiology and thereby CNS distribution of protein therapeutics ,167,210.As the systemic or intraparenchymal delivery of protein therapeutics may be greatly hindered by BBB and brain tissue structure, targeting the CSF space, adjacent cerebral structure, or CPs may offer another approach in the treatment of CNS diseases.First, the therapeutic targets may locate at subarachnoid, perivascular, and periventricular spaces and their adjacent tissues. In several neuroinflammatory disorders, such as experimental autoimmune encephalomyelitis , multiplSecond, CSF circulation and PVS pathways may bring administered protein therapeutics to wider areas and deeper brain distributions. Intrathecal administration of protein therapeutics is regarded as a promising delivery method to bypass the BBB to reach brain parenchyma. Examples include the treatment of lysosomal storage disorder mucopolysaccharidosis by intrathecal delivery of recombinant human alpha-L-iduronidase or iduronate-2-sulfatase, which resulted in enzyme accessing the superficial and deep brain tissues in dog and mice ,221. TheAs a medium to carry brain and blood-derived signals to distant targets within the brain, CSF should be taken into consideration for protein therapeutic brain delivery. However, the complex physiology of the subarachnoid, perivascular, and ventricular spaces in addition to the confounding factors influencing CSF flow in health and disease are still not entirely understood. Careful deliberation should thus be included in decision making when determining if CP-mediated delivery is the best option as the CNS delivery strategy.max of cerliponase alfa in CSF was 1260, 1630, and 1390 µg/mL at day 1, week 5, and week 13 after intraventricular infusion, which resulted in CSF/plasma ratio of 1200, 809, and 1320, respectively [0–t in CSF was 393, 340, and 1330 times higher than that in plasma at day 1, week 5, and week 13 after administration. The results of this study indicated a high CNS distribution with limited systemic circulation of cerliponase alfa after intraventricular administration, which may contribute to the reduced decline in motor and language function compared with historical controls. However, serious adverse effects associated with the intraventricular device, including device-related infections and leakage, have been found during the clinical study, leading to interruption of the treatment [Although peptide/protein-based therapies have been introduced in various indications, their application in CNS diseases remains challenging. Based on the summary of approved peptide/protein therapies for CNS diseases , it was ectively . The AUCreatment . 188Re-labeled nimotuzumab and 211At-labeled 81C6 mAb, which enable efficient delivery of radiation doses to the tumor area and limit potential harm to the surrounding normal brain region and distant organs [131I-Omburtamab is a radioimmunotherapy agent that targets the glycoprotein B7-H3 expressed in neuroblastoma and is being assessed for treatment of metastatic leptomeningeal tumors [131I-omburtamab was intraventricularly administered via the Ommaya shunt, which resulted in around 10-fold CSF-to-blood ratios based on PET analysis and generally low radiation doses to other organs besides brain parenchyma and liver [Other potential peptide/protein therapies are under investigation for a wider range of neurological diseases, including Alzheimer’s disease, glioblastoma, and other brain tumors . For thet organs ,228. 131l tumors . In the nd liver . Thus, ind liver .131I-omburtamab are examples for clinical use of peptide/protein therapy targeting CPs, which maximize brain distribution while minimizing systemic side effects. The drawbacks of this delivery method include device-related complications and variability in ventricular distribution.Overall, cerliponase alfa and It is clear from the failures to develop CNS drugs that a better understanding of CNS physiology and fundamental cellular biology is desperately needed. Various CNS barriers are anatomically positioned to obstruct the penetration of therapeutic compounds and proteins at efficacious concentrations. Although targeting the CPs and ventricular system may be an option to deliver drugs to brain parenchyma without impairment by the BBB, other factors deserve attention for this delivery pathway, including CSF fluid dynamics, CPECs, ependymal cells, and PVSs. During the experimental examination of CP delivery, physiological conditions should always be closely monitored as change in CSF dynamics can alter the therapeutic disposition. Due to the developmental changes in CPs and the ventricular system, the experimental model should correspond to the age of the targeted patient population to provide better correlations. Studies on carrier/receptor-mediated transport at CPECs have paved the way for new pathways of peptide and protein therapy delivery. However, further elucidation of the receptor–ligand co-localization and trafficking studies are warranted to provide direct evidence in CPEC transport systems. Further development of inducible knockout models targeting CPEC transporters are needed to better understand CP protein handling. With a better understanding of the physiological processes and comprehensive experimental techniques, the CPs have the potential to be targeted as a promising approach to treat specific CNS disorders.


The CellSearch® system has been used to identify circulating tumor cells (CTCs) in cerebrospinal fluid (CSF) to diagnose leptomeningeal metastasis (LM) in patients with epithelial cancers. Using this system, we prospectively explored sequential CSF CTC enumeration in patients with LM from HER2+ cancers receiving intrathecal (IT) trastuzumab to capture dynamic changes in CSF CTC enumeration.CSF from patients enrolled in an IRB-approved phase I/II dose escalation trial of IT trastuzumab for LM in HER2+ cancer (NCT01325207) was obtained on day 1 of each cycle and was evaluated by the CellSearch® platform for CTC enumeration. The results were correlated with CSF cytology from the same sample, along with clinical and radiographic response.neu expression analysis of CTCs was performed in 8 patients; 75% had confirmed expression of HER2/neu positivity in CSF and HER2/neu expression was absent in 25%. Four of 10 patients received 7 or more cycles of IT trastuzumab; in 3 of these patients, increase in CSF CTCs enumeration from baseline was detected 2–3 months prior to changes seen on MRI, and while CSF cytology remained negative.Fifteen out of 34 patients with HER2+ LM were enrolled in CSF CTC analysis; 14 were women. Radiographic LM was documented in 14 (93%) patients; CSF cytology was positive in 6 (40%) and CSF CTCs were identified in 13 (87%). Median CSF CTC was 22 CTCs (range 0–200 +) per 3 ml. HER2/Our study demonstrates that enumeration of CSF CTCs may provide dynamic, quantitative assessment of tumor burden in the central nervous system compartment during treatment for LM and prior to changes on MRI or CSF cytology.Trial Registration: Clinicaltrials.gov: NCT01325207; registered March 29th, 2011.The online version of this article (10.1007/s11060-020-03555-z) contains supplementary material, which is available to authorized users. Diagnosis, prognosis, and treatment of leptomeningeal metastasis (LM) carries a significant implication for oncologists and patients –3. DiagnMajor clinical societies have been working to establish guidelines for a unified approach to diagnose and manage LM. The current European Association of Neuro-Oncology-European Society for Medical Oncology (EANO-ESMO) guidelines provide recommendations regarding the diagnosis and treatment of LM from solid and liquid cancers , which iThe EANO-ESMO guidelines reference several promising new platforms to improve sensitivity for tumor cell detection in the CSF. The LANO group proposes to include CSF CTCs, as well as other platforms that allow for molecular profiling such as CSF circulating tumor DNA. Importantly, it also states prospective studies must include evaluation of liquid biopsies .Further work is needed to establish the clinical utility of these analyses as biomarkers of both disease diagnosis and response to treatment, as well as to incorporate the use of CSF CTCs into clinical practice. The goal of this study was to evaluate the value of CSF CTCs as biomarkers prior to and during treatment in patients with LM from HER2+ cancers who were receiving treatment with intrathecal (IT) trastuzumab on a clinical trial . We usedThis multi-institutional phase I/II dose escalation trial of IT trastuzumab for LM  was condPatients had an intraventricular reservoir (Ommaya) placed prior to initiation of IT therapy. IT trastuzumab was administered via an Ommaya reservoir twice weekly for cycle 1 (1 cycle = 28 days), then once a week for cycle 2 and every 2 weeks thereafter for subsequent cycles. CSF for cytology, as well as for CTC enumeration, was obtained immediately prior to drug instillation on day 1 of each cycle . The Her2/neu CTC assay is not FDA approved, but rigorous performance testing was performed in accordance with Clinical Laboratory Improvement Amendments (CLIA) regulatory requirements and adapted for use on the CellSearch® platform. We were able to identify Her2/neu Ab+, CK-PE+, DAPI+ and CD45- cells using the CellSearch® method. Results were reported as number of CTCs per 3 ml of CSF (CSF CTCs/3 ml). The enumeration of CSF CTCs was limited by standard approach to 200+ CSF CTCs/3 ml though even higher numbers of CTCs were seen. This study was approved after review by the Institutional Review Boards and was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice. All patients provided written informed consent before enrolment.The CellSearch® (Menarini Silicon Biosystems) platform was used to enumerate CTCs in CSF specimens. The CellSearch® Assay is an immunomagnetic isolation technique using antibodies to EpCAM conjugated to magnetic beads for the immunomagnetic capture of EpCAM-expressing epithelial cells. A CTC was defined as a cell that expresses cytokeratin (CK), displays a nucleus when stained with 4,6-diamidino-2-phenylindole (DAPI), and has no expression of CD45. CSF CTCs were also evaluated for HER2/neu CTC assay was performed in 8/15 patients with detectable CTCs, and Her2/neu expression of CTCs was confirmed in 6/8 (75%).Fifteen out of 34 patients were enrolled in the CSF CTC analysis: 14 patients were women with breast cancer and 1 was a man with colon cancer. At trial entry, positive CSF cytology was confirmed in 6 patients and suspicious in 4  a decrease in CSF CTCs count at the time of disease progression was observed, while no CSF CTCs were detected in another patient (patient 1).Four out of eight patients with low CSF CTC enumeration at baseline (range 0–22 CSF CTCs/3 ml) received ≥ 7 cycles of IT trastuzumab Fig. . In 50% This is the largest study incorporating serial longitudinal enumeration of CSF CTCs in patients with HER2+ cancers and LM enrolled on a clinical trial. In this multicenter trial, we obtained CSF for CTCs analysis from multiple participating institutions prior to the initiation of LM-directed treatment, during treatment, and at progression. We compared these samples to CSF cytology samples collected at the same time, as well as MRI scans performed within a few days of CSF collection. Our study demonstrates CSF CTC analysis can easily be incorporated into multi-center prospective studies for patients with LM, given that the CellSave preservation tubes allow the specimen to be stored for up to 72 h and that CSF samples can be shipped unfrozen.CSF CTC enumeration by the CellSearch® platform most likely reflects the overall disease burden in the CNS compartment, which can be easily calculated per 1 ml of CSF. We observed that patients with a higher CSF CTC enumeration were more likely to progress rapidly, suggesting a high burden of disease in the CSF compartment, which is not necessarily seen on neuroimaging. High disease burden in the CNS compartment at the time of diagnosis could correspond with the dismal overall survival of patients with LM, which is typically 3–4 months , 20–22. CSF CTC analysis was optional in this study and not all patients elected to participate, which is a limitation of this and many other studies. In addition, our cohort included patients with both recurrent and newly diagnosed LM with median time between LM diagnosis and the date of enrollment of 250 days. As a result, each group is relatively small and thus not amenable for correlations with overall survival and progression free survival. Another limitation of this study is that some of patients had LM in the brain and spine, however all CSF samples were collected only from an Ommaya reservoir and not via a lumbar puncture which could increase the change of identifying higher CTC count than from an Ommaya reservoir . Regardlneu expression using a specific Her2/neu antibody phenotyping marker reagent is an important advantage to confirm that all cells in the CNS compartment expressed HER2/neu prior to initiation of treatment. This enables real-time confirmation of HER2/neu expression or loss of HER2/neu over time, which has been reported previously at other sites of metastases [neu expression of CSF CTC was identified in 6 of 8 (75%) of patients at trial entry when CSF CTCs were detected. HER2/neu expression of CSF CTC was not identified in 2 patients: 1 with only a single CSF CTC, and another who had 81 CSF CTCs/3 ml. While the reason for lack of HER2/neu positivity in the last patient is not clear, we hypothesize that this could be due either to HER2/neu loss in the CSF during molecular evolution of cancer, or it represents a false positive HER2/neu FISH analysis of the archival tissue. Incorporation of molecular analysis of CSF performed by next generation sequencing analysis could answer this question.The ability to assess CSF CTCs for molecular markers  such as tastases . In our Many oncologists struggle with the decision to place an Ommaya reservoir and recommend IT chemotherapy because some patients decline so rapidly. Our data suggests that CSF CTC enumeration may aid in the detection of LM at an earlier time and may quantify disease burden. The information gained from quantitating CSF CTCs at baseline may help guide these decisions once future, larger prospective trials have further established the role of CSF CTC enumeration to determine disease burden in the CSF compartment.In this study, we demonstrate the value of CSF CTC enumeration in patients with LM enrolled on a phase I/II dose escalation trial of intrathecal trastuzumab. Beyond standard EpCAM-based CTC analysis, we have employed HER2/neu expression analysis of CTCs using a specific Her2/neu antibody phenotyping marker reagent to confirm that pre-treatment CSF CTCs express HER2+. Our findings demonstrate that enumeration of CSF CTCs is a more accurate measurement of tumor burden in the CNS than standard CSF cytology and can be used as a biomarker of treatment response. CSF CTC analysis might be incorporated in clinical trials and in the clinical care of patients with LM from epithelial cancers.Supplementary file1 (DOCX 337 kb)—Supplementary Fig. 1 Changes in CSF CTCs over time in patients who remains on study for ashort period of timeSupplementary file2 (DOCX 15 kb)Below is the link to the electronic supplementary material.