Limited information exists on the anatomically-specific early stage events leading to clinically detectable mineral aggregates in the renal papilla. In this study, quantitative multiscale correlative maps of structural, elemental and biochemical properties of whole medullo-papillary complexes from human kidneys were developed. Correlative maps of properties specific to the uriniferous and vascular tubules using high-resolution X-ray computed tomography, scanning and transmission electron microscopy, energy dispersive X-ray spectroscopy, and immunolocalization of noncollagenous proteins (NCPs) along with their association with anatomy specific biominerals were obtained. Results illustrated that intratubular spherical aggregates primarily form at the proximal regions distant from the papillary tip while interstitial spherical and fibrillar aggregates are distally located near the papillary tip. Biominerals at the papillary tip were closely localized with 10 to 50 μm diameter vasa recta immunolocalized for CD31 inside the medullo-papillary complex. Abundant NCPs known to regulate bone mineralization were localized within nanoparticles, forming early pathologic mineralized regions of the complex. Based on the physical association between vascular and urothelial tubules, results from light and electron microscopy techniques suggested that these NCPs could be delivered from vasculature to prompt calcification of the interstitial regions or they might be synthesized from local vascular smooth muscle cells after transdifferentiation into osteoblast-like phenotypes. In addition, results provided insights into the plausible temporal events that link the anatomically specific intratubular mineral aggregates with the interstitial biomineralization processes within the functional unit of the kidney. Pathological mineral formations occur in various organ systems within the human body. Within the kidney, these are most commonly identified as a urinary tract stone. Little is known about the early stage biominerals that lead to clinically detectable stones, despite their increasing prevalence  and assoThe medullo-papillary complex is one of the 8–12, paraboloid-shaped functional units within a kidney. At the tip of the complex is the papilla, where the end-product, urine, drains into the urinary collecting system. Fundamentally, the renal medullo-papillary complex consists of both uriniferous and vascular tubules of various lengths and diameters where exchange of ions and water occurs along the length and across the tubules. Therefore, from a bioengineering perspective, the medullo-papillary complexes in a kidney can be thought of as biofilters in which solutes are separated from the solvents.Investigations on the etiology of calcium oxalate-based kidney stones have focused on their attachment to biominerals known as the Randall’s plaque (RP) and are commonly observed  at the tip of the complex . Stone fRP has been characterized at multiple length-scales using various microscopy and spectroscopy techniques . HoweverSeveral hypotheses involving cell-based and/or physical chemistry-based mineralization have been proposed to describe RP formations and the process of calcium-based stone formation and the association of the stone with RP , 8, 12. NCPs are known to sequester inorganic ions on organic matrices –26 and tWe aim to better understand how NCPs  affect the mineralization process in RP and subsequent stone formation. In this study, NCPs will be first localized within the anatomy of human renal medullo-papillary complexes. This is in contrast to prior studies that have focused exclusively at the tip of the renal papilla. In addition, we will examine the location of these NCPs relative to the location of vasculature and uriniferous tubules to provide insights into whether they are synthesized by renal epithelial cells or delivered through the vasa recta. Furthermore, it is equally critical to gain insights into how the location of the NCPs in the intratubular and interstitial regions affects the corresponding cascades of progressive, locally occurring biomineralization processes and subsequently the functional form of the complex.All participants provided both verbal and written consents to participate in this study. Institutional approval for both the study itself and the manner in which consent was obtained from Human Research Protection Program (HRPP) at the University of California, San Francisco, was recorded under institutional review board (IRB) approval number 14–14533. Participants discussed the details of the study at their clinical visit where questions were answered by research team members and their verbal and written consents to participate were obtained. All study consents were then scanned and recorded into our data warehouse and the paper copies were destroyed in a secure fashion in accordance with institutional standards. Papillary specimens (n = 14) were obtained from patients undergoing nephrectomy for the diagnosis of renal mass, presumed to be renal cell carcinoma. Patients who were thought to have transitional cell carcinoma were excluded from this study. During harvest of the papillae, an anatomically normal portion of the kidney, distant from the renal mass, was selected. In accordance with current dogma, renal tissue distant from renal cell carcinoma is generally thought to be normal unless subject to mass effect from the tumor. Typically, if a lesion is small enough and it is possible to reconstruct the kidney after mass excision, partial nephrectomy is preferred according to AUA guidelines . This isIn this study, immunohistochemistry, fluorescence, and electron microscopy techniques were chosen to study the anatomy-specific biominerals in human renal complex. Mapping of biominerals at multiple length scales was performed by using X-ray, light, and electron imaging modalities to enable contextual visualization of vascular and uriniferous tubules at the level of the complex, and localization of globular proteins in the tubules and surrounding matrices through 3, 3'-diaminobenzidine (DAB), and gold-particle tagged probes at the level of a tissue (interstitium). Anatomy-specific and multiscale mapping of mineralized regions and localization of NCPs was performed in three zones (I-III) of the complex .Of the 14, one of the specimens was stained with iodine. The renal complex was carefully isolated from a fresh nephrectomy specimen, fixed overnight in 10% neutral buffered formalin , washed three times in 1× phosphate-buffered saline (PBS), and was scanned using micro-CT before staining with iodine. Following scanning, the same papilla was stained with 1.5% iodine  for 2 hours. After washing with ethanol three times, the stained papilla was scanned using micro-CT . Following scanning, the specimen was embedded in paraffin, and was sectioned and examined using a light microscope (Olympus BX51). Micro-CT was performed at 4X magnification, 1200 slices, providing a resolution of 5 μm/pixel. X-ray scanning of specimens before and after iodine staining and superposition of the respective tomograms provided location and anatomical association of mineralized regions in those that were stained with iodine. It is known that vasculature can be revealed by iodine staining or iodinated contrast agents filling . IntratuMedullo-papillary complexes containing RP (RP+) were dehydrated using graded (50–100%) ethanol solutions. The specimen was scanned using a micro-CT followed by infiltration of LR-white resin . The infiltrated specimen was kept in a gelatin capsule (Electron Microscopy Sciences) and polymerized for 2 days at 60°C. 90 nm thick tissue sections were cut with an ultramicrotome . The ultrasectioned block surface was sputter coated with a thin layer of gold/palladium, and was used for structural analyses using a field emission scanning electron microscope  at 5 keV. The ultrasections were collected on formvar/carbon-coated Ni grids (Electron Microscopy Sciences) for immunohistochemical localization of NCPs. An energy dispersive X-ray spectroscopy (EDS) detector  was used for elemental analyses of histology sections from iodine stained papilla without RP (RP-) placed on ITO (indium tin oxide) coated glass coverslips. Using SEM and EDS analysis, the region rich in iodine can be correlated to the specific structures from morphological analysis.Following micro-CT, sections of papillary tissue showing RP were imaged using polarized and fluorescent microscopy (Olympus BX51) before and after 1 hour of 4% EDTA treatment. Rabbit polyclonal antibody against human OC (sc-30044), mouse monoclonal antibodies for BSP (sc-73630) and OPN (sc-21742) were purchased from Santa Cruz Biotechnology, Inc. . Mouse monoclonal antibody against human CD31 (ab9498) was acquired from Abcam, . Rabbit polyclonal antibody against human BSP (LF-84) was received as a gift from Larry W. Fisher, . Goat polyclonal antibody against human OPN (AF-1344) was obtained from R&D System, Inc. .The medullo-papillary complexes with (RP+) and without visible RP (RP-) (5 specimens) were processed using 15% and 30% sucrose solutions and then embedded in optimal cutting temperature (OCT) resin. 6–7 μm thick sections were cryo-sectioned and collected on glass slides. Immunohistochemistry (IHC) labeling of NCPs and endothelial cell adhesion protein CD31 was performed on sections decalcified overnight in 4% EDTA solution. The sections were incubated in antibody solution . As for IHC staining of CD31, a heat epitope retrieval process in citrate buffer (pH = 6) at 80°C for 20 min was applied. The control test was performed without addition of primary antibody in parallel. Afterwards, the sections were stained using secondary antibody kits  and DAB substrate kit . After counter-staining using hematoxylin, the sections were dried and imaged using a light microscope . The localization of NCPs was similar among all specimens and representative data are shown in this manuscript see .Immunolocalization of NCPs was performed through immunogold labeling of thin sections on nickel grids using polyclonal antibodies . The ultIntratubular mineralized structures were consistently observed in the proximal, peripheral regions distant from the papillary tip in all specimens, irrespective of whether RP was identified at the papillary tip. The superposition of tomograms acquired before and after iodine-staining  suggesteAn additional five unstained RP- papillae were analyzed using the same approach. The combined data indicated that the majority of mineralized tubules within these proximal regions have a diameter between 20–60 μm  and the Tubules preferentially stained with iodine histologically examined  and wereScanning electron microscopy (SEM) images and energy dispersive X-ray spectroscopy analyses of iodine stained histology sections indicated that the erythrocytes in vasa recta showed significantly higher concentrations of iodine than in other cell types or structures . These oMineralized tubules were clearly observed on histology sections owing to their natural opacity to light . After tLight microscopy images of demineralized RP+ sections immunostained with NCPs such as OPN, OC, and BSP suggest their strong localization in the mineralized walls of patent tubules . BSP andMineralized papillary tissues in the kidney clinically identified as RP, are believed to be the nidus from which most calcium-based kidney stones form , 8. In tWhile there have been no known prior studies evaluating the proximal intratubular mineralization within the medullo-papillary complex, the cause for interstitial mineralization has several postulates. These include the oxidative stress theory  and diffHistomorphometry through post analyses of 3D digital reconstructions of whole human medullo-papillary complexes from micro-CT  indicateIt is interesting to note that proximal mineralization is limited to the tubules, while distal mineralization is within the interstitium leaving the tubules patent Figs . Regardlin vivo studies [Previous studies have found that mineralization occurs in the basal membranes of uriniferous tubules and vasa recta at the papillary tip . NCPs su studies . It rema studies . Howeverin vivo studies [in vitro system in which renal cells were cultured in a media rich in calcium. The structure of OPN is rich in serine and glutamic acid with a characteristic polyaspartic motif [Localization of NCPs in mineralized tubules of RP+ tissue suggests a strong correlation between NCPs and the mineralized nanoparticles. NCPs are ubiquitous in organic tissues and normally aid in favorable biomineralization –60. The  studies . To dateic motif . The aciic motif , 66–68. in vitro study suggested that the simultaneous synthesis of BSP and OPN by renal epithelial cells in the inner medullary collecting duct can result in calcified nodules. Several groups have reported the localization of OPN either in RP+ tissues or renal calculi [Based on principles of thermodynamics, a recent study also suggested that the flexible structure of intrinsically disordered NCPs is crucial for the mineralization . An in v calculi , 28. Howin vitro studies of individual NCPs suggest OPN and OC can inhibit or delay apatite crystal formation, while BSP facilitates nucleation [With OC, the γ-carboxyglutamic acid groups are responsible for binding of calcium ions as well as to apatite crystals , 70, 71,cleation . Howeverin vivo models [in vitro studies [in vitro studies suggest that OPN and OC can form a complex and promote mineral formation [The exact roles of NCPs are elusive due to naturally built in functional redundancy in NCPs and their compensatory behavior in the absence of another –76. NCPso models , 77, 78  studies , 73. The studies . Similar studies . However studies . As for ormation , 83. Theormation . The strThe histomorphometric analyses at multiple length scales has allowed us to associate mineralization within the two types of tubules, however, the biomineralization process is yet to be investigated. Results from this study indicated that vasa recta may also be affected by vasculature mineralization which is dominant in the interstitial region, while intratubular mineralization in the proximal regions of the medullo-papillary complex of the renal papillae could be vascular and/or urothelial tubules—the functional units of the renal papilla. Future studies will link intratubular with interstitial mineralization within the medullo-papillary complex using the tenet, form guides function .S1 File(DOCX)Click here for additional data file.S2 File(MPG)Click here for additional data file.S1 FigThe red squares represent the three zones  of interest and correspond to the most proximal, the mid-region of medulla, and the tip of the medullo-papillary complex.(TIF)Click here for additional data file.S2 Fig(a) Mineralized tubules (green) in unstained specimen, (b) iodine stained tubules (blue), (c) void tubules (grey) in the stained specimen following digital inversion, (d) stained (blue) and void (grey) tubules are shown. The tubules with larger diameters are highlighted. The red blob is an imaginary kidney stone at the papillary tip.(TIF)Click here for additional data file.S3 FigI: The void area in a 3D volume is marked as a tubule. II: Segmentation of tubules. III: The 3D volume is rotated to a direction which is most perpendicular to all the tubules. IV: Slices are generated one by one along this direction. V: The tubule diameter  is determined by the minor axis length of the smallest eclipse which can cover all the pixels of the tubular cross section as show by the blue lines.(TIF)Click here for additional data file.


We sought to assess cortical function in amyotrophic lateral sclerosis (ALS) using noninvasive neural signal recording.Resting-state magnetoencephalography was used to measure power fluctuations in neuronal oscillations from distributed cortical parcels in 24 patients with ALS and 24 healthy controls. A further 9 patients with primary lateral sclerosis and a group of 15 asymptomatic carriers of genetic mutations associated with ALS were also studied.Increased functional connectivity, particularly from the posterior cingulate cortex, was demonstrated in both patient groups compared to healthy controls. Directionally similar patterns were also evident in the asymptomatic genetic mutation carrier group.Increased cortical functional connectivity elevation is a quantitative marker that reflects ALS pathology across its clinical spectrum, and may develop during the presymptomatic period. The amelioration of pathologic magnetoencephalography signals might be a marker sensitive enough to provide proof-of-principle in the development of future neuroprotective therapeutics. Neuroprotective strategies, particularly considering presymptomatic ALS, will require proof-of-principle biomarkers.2; RSN disturbances are associated with diverse neurodegenerative diseases,4 altering functional connectivity (FC) . The correspondence between RSN topography in health, and spatial patterns of both pathologic atrophy and protein deposition, suggests RSNs reflect the neuroanatomical substrate of diseases.6Resting-state networks (RSNs) reflect the functional organization of the brain7 While this may be compensatory, cortical hyperexcitability is a characteristic feature of ALS,8 perhaps reflecting loss of inhibitory interneuronal influence,9 in turn associated with increased FC.10 A specific spatial pattern of increased FC was demonstrated in asymptomatic gene carriers at high risk of ALS,11 suggesting increased FC as an early pathologic feature amenable to neuroprotective strategies.MRI resting-state activity (rs-fMRI) exposes significant increases in FC among patients with ALS, correlating with progression.12 alongside global reorganization of inferred cortical network topology.13 Magnetoencephalography (MEG) extends the localization capabilities of noninvasive neurophysiology,14 negating dispersive effects of skull and scalp. The temporal resolution of MEG offers advantages, relevant in the assessment of dynamic cognitive processes, such as movement preparation, clinically affected by neurodegeneration.15 MEG simultaneously can explore degenerating cortical networks16; MEG-derived RSNs appear consistent with rs-fMRI.17 We therefore applied resting-state MEG to investigate increased FC as a consistent feature of ALS pathogenesis, to consider the clinical implications of FC topography in ALS and to explore FC structure before symptoms manifest.Recent EEG studies have also described FC increases,C9orf72 and SOD1) were recruited through contact with the Oxford Centre or through prior enrollment in the Pre-fALS Study based at the University of Miami.18 Healthy controls were distributed into age-matched groups.All patients were recruited from the Oxford Motor Neurone Disease Care and Research Centre, diagnosed by experienced neurologists according to standard criteria . A range of patients with ALS, plus a small group with the consistently slowly progressive upper motor neuron–only variant, primary lateral sclerosis (PLS), were included after the necessary exclusion of any credible mimic disorder. Asymptomatic carriers of ALS-linked genetic mutations (AGCs)  at the Oxford Centre for Human Brain Activity. The participant was seated comfortably in the scanner and instructed to remain still and awake, but without performing any specific task. Immediately prior to acquisition, a Polhemus 3D tracking system  recorded 4 fixed coil positions relative to nasion and preauricular fiducial landmarks, alongside distributed points (∼100) covering the scalp and in later cases the nose (∼50 points) surface. Coils were intermittently energized to obtain a continuous record of head position with the MEG helmet. The recording was made in the “eyes open” state with participants' visual fixation directed to a black cross printed on white paper 90 cm away. Blinks and saccades were monitored continuously using a combination of surface electrooculography and infrared eye tracker. ECG was monitored at the wrists. Participants underwent a structural MRI for coregistration purposes, typically on the same day as MEG (or within 1 month), using a Siemens Trio 3T  .19 Source-space covariance maps were created in NIfTII format and inspected for each individual using the FSL tool FSLview. At this stage, a limited number of participants were identified as having suboptimal source-space MEG data, either because of remaining contamination by artifact or failure of the beamformer algorithm to perform adequately. Raw data were reinspected for these individuals but no correctable source of noise was typically identified; it was noted that several individuals without structural MRI available for coregistration fell into this category. Participants unable to complete MRI examinations  were therefore excluded. In total, the number of additional participants excluded from further analysis because of qualitatively suboptimal source-space datasets were 1 ALS, 1 PLS, 2 AGCs, and 1 healthy control . Post MaxFilter, the filtered data were converted to SPM8, down-sampled to 250 Hz, and again visually inspected. Bad segments were manually marked in the continuous data stream. Data were then cleaned using a semiautomated SPM8 script as follows. Data were initially high-pass filtered at 0.3 Hz. The independent component analysis (ICA) components corresponding to physiologic artifact generated by blink and cardiac cycle were identified and removed from the data. Coregistration was then performed using either SPM8 or a bespoke version of the SPM8 coregistration tool adapted to include the registration of headshape points on the nose . Coregistration fit was checked for each individual, and errant headshape points were manually removed if necessary before repetition. Data were then source-transformed using a linearly constrained minimum variance beamformer, which combines information over both magnetometers and gradiometers and uses principal component analysis to regularize the data covariance matrix estimation and account for the reduction in dimensionality caused by MaxFilter, with a single-shell forward model into MNI  space at a 3-dimensional gridstep of 6 mm.21 For the purposes of the connectivity analyses, functional activity across the cortex was summarized using 39 fMRI-derived cortical ROIs. We used a data-driven, functional parcellation so that the boundaries of each region could be determined by recorded functional activity, rendering them directly relevant to our analysis. The parcellation was created from a 100-dimensional group-ICA decomposition of rs-fMRI data from the first 200 participants in the Human Connectome Project.22 These parcels therefore represent contiguous functionally specific local modules, which enables the analysis of a comprehensible functional network. Most ROIs have a counterpart in the opposite hemisphere, apart from those on the midline; therefore, the 39 regions provide complete coverage of cortex within a relatively low-dimensional structure. A single time series was decomposed from each region to represent the functional activity of the parcel. The first principal component of all voxels in the ROI was taken after weighting by the strength of the fMRI spatial map. The data were then orthogonalized regarding all other parcels using a symmetric multivariate approach that eliminates spurious zero-lag connectivity between neighboring parcels while maintaining a close approximation to the original time series. This novel robust approach was recently applied to successfully quantify the heritability of cortical functional architecture among related healthy participants.23 The time series was converted into a band-limited power time course by taking the Hilbert envelope of the “broadband” signal  and down-sampling with a 2-second sliding window filter.25 This power envelope time course was then correlated between parcels for each individual, resulting in a Fisher z-normalized score for each connection. These communication links between cortical regions (nodes) correspond to “edges” in a graph theory network model, and this specific approach for connectivity estimation shows greater repeatability than a wide range of other choices.20 Group contrasts  were appraised against 5,000 permutations of the group-membership labels and corrected for multiple comparisons on a per-contrast basis (thus accounting for the number of edges contributing to each group contrast). The p values (p < 0.05) corrected using family-wise error were deemed significant. Groups were then further contrasted regarding node weight, representing the sum of all edge strengths connected to each cortical parcel in turn. The resulting (per participant) vectors of 38 node weight values were compared between groups via nonparametric permutation inference as implemented in FSL's randomise, with family-wise correction over the maximum t-stat distribution to account for the number of parcels.FC between a predefined set of regions of interest (ROIs) was computed using correlations between band-limited power time courses in each ROI, after applying a correction for spatial leakage confounds.All participants provided written informed consent. The study was approved by the National Research Ethics Service South Central Oxford Research Ethics Committee B (08/H0605/85) and South Central Berkshire Committee (14/SC/0083).Relative to healthy controls, FC was increased throughout the cortical networks in patients with ALS and, to a lesser extent, in patients with PLS and AGCs. Connectivity matrices representing the study average and group differences in FC are presented in The FC differences were most marked in the group of patients with ALS. A degree of spatial selectivity was noted; FC increases particularly involved edges from the posterior cingulate cortex (including the connection to the right motor cortex) and edges within the right visual and occipital regions. The patients with PLS showed more subtle increases in FC, particularly between posterior and parietal nodes bilaterally. No difference in increases in FC was noted in the AGCs, affecting an overlapping spatial distribution. No edges showed decreased FC relative to age-matched healthy controls.The group differences were further confirmed in consideration of node weight. Global connectivity to the posterior cingulate cortex and other posterior cortical parcels was significantly increased in the ALS group relative to healthy controls; similar but less extensive findings were revealed in the PLS group but not significantly so among the AGCs. Clinical measures  did not correlate significantly with the connectivity strengths as described by a global mean of the interparcel edges.Both ALS and PLS phenotypes showed widespread increases in FC, as measured by the degree of correlation between band-limited power envelopes across spatially distributed parcellated regions of cortex. Limited evidence was obtained in support of alterations to FC prior to the development of overt symptoms. Such abnormalities may have considerable mechanistic importance given the key role of neural oscillations in the long-range synchronization of information across the brain and might contribute to the development of accessible multimodal biomarkers, which remain a key unmet need of therapeutic trials in ALS.26 further discordance in FC degree and distribution is not unexpected with a change in neuroimaging modality, particularly considering the diverse neural activity, including modulation of oscillatory power, that may contribute to the BOLD (blood oxygen level–dependent) response. Early reports suggested a mismatch between ALS-related deficits in structural connectivity and apparently preserved functional organization,27 but a more complex pattern emerged with changes to FC extending beyond the primary motor cortices.29 Fewer studies have considered patients with PLS. A seed-based approach detected increased FC, notably cerebrocerebellar,30 while an ICA-based approach detected increased FC in the sensorimotor network  and increased FC to the right superior frontal gyrus .31 The present findings therefore reaffirm shared pathophysiologic mechanisms between ALS and PLS.Given the range of FC alterations reported in ALS via rs-fMRI,32 supported in vivo by neurophysiology findings of accentuated cortical beta-desynchronization during movement preparation15 and diminished postmovement beta-rebound.33 Studies in healthy volunteers combining magnetic resonance spectroscopy with rs-fMRI have found evidence of the balance of glutamatergic and GABAergic neurotransmitter concentrations influencing the strength of FC.34 These findings may, however, also reflect direct modulation by GABA of the hemodynamic response function, rather than solely alterations to the underlying neural activity.35Increased FC in ALS has been hypothesized to result from loss of intracortical inhibitory influence,36 Increased FC of the precuneus and parietal cortex was previously described,28 and the occipital poles are reported to acquire a stronger network prominence in ALS as measured by the voxel-wise graph theory FC metric, degree centrality.37 FC increases related to the sensorimotor cortex may only be prominent earlier in the symptomatic course of ALS, being notably present in a study of patients without corticospinal tract diffusion tensor imaging changes.38 Rather than being directly pathogenic, FC increases might therefore alternatively reflect a physiologic response to functionally compensate for early pathology, which becomes overwhelmed with further disease progression. This would be in keeping with the more marked FC increases between cortical regions less vulnerable to structural damage in motor neuron disease, i.e., posterior and parietal nodes, that was demonstrated in both patients with ALS and PLS in the present study. However, no corresponding increase in sensorimotor FC is noted within the AGC group, though the sensitivity to detect such changes in a small and genetically heterogeneous group is likely to be low.FC changes extending beyond the primary motor networks are in keeping with ALS as a multisystem cerebral disorder with considerable cortical burden beyond the core motor phenotype.17 Appraisal of multiple, specific frequency bands would necessitate further multiple comparisons correction and (particularly in higher gamma-frequency ranges) potentially diminish signal-to-noise ratio. The FC analysis was repeated after data decomposition into 3 frequency bands familiar to clinicians: theta (4–8 Hz), alpha (8–12 Hz), and beta (15–30 Hz). FC strength differences between patients with ALS and healthy controls were not restricted to any one frequency band, reaffirming the utility of the preceding broadband reduction . Furthermore, the selection of nodes for the parcellation analysis could alternatively be guided by anatomical landmarks, but this may have negative consequences since multiple oscillatory sources might be combined within one anatomical parcel or split across a functionally irrelevant anatomical divide. Selecting neural sources on the basis of decomposed functional data is therefore closer to a physiologic parcellation, but uncertainty remains over the total number of components to be raised—this remains a balance between interpretability, uniqueness, and potentially arbitrary subdivision of sources . Inevitably, therefore, the selection of an RSN “of interest” is nontrivial, and the interpretation of the functional significance of alterations in FC within and beyond RSNs remains speculative.A broad (4–30 Hz) frequency band was selected for the power envelope, since most resting-state structure and connectivity is present within theta, alpha, and beta bands.The acquisition took place as participants rested with eyes open, as fixation is desirable to minimize saccade artifacts in the MEG signal, but many rs-fMRI datasets, including our group's most recent analysis, have been conducted with eyes closed. Since Berger's (1873–1941) description, posterior alpha power is notably increased on closing the eyes; the effect on band-limited envelope coherence is not certain but likely to be positive for occipital regions at least. Whether this would differentially influence clinical groups remains to be shown. ALS, in common with most neurodegenerative diseases, causes a subtle slowing of dominant alpha, contributing to increased low-frequency power. These differences were appraised in the current resting-state MEG dataset but not found to be significant after the same family-wise error correction for number of parcels included.39 partially mitigated by reliance on spouses volunteering for inclusion as healthy controls. Finally, CNS active medications have the potential to accentuate the group difference in FC. Patients continued any regular medications for the study since withdrawal of agents providing symptomatic relief was considered unethical. Most importantly, approximately half of the patients with ALS were taking riluzole on a long-term daily basis. However, splitting the ALS data according to riluzole use did not reveal any marked differences in FC, in keeping with the short-term effect on cortical hyperexcitability as measured by transcranial magnetic stimulation.40A further potential confound could arise from insufficient matching of participants according to intellectual achievement,15 The directionally similar findings in carriers of high-risk genetic mutations suggests these changes may also predate the onset of symptoms. Larger cohort numbers may prove more successful in distinguishing the clinical effect of FC increases, and longitudinal repetition is a prerequisite to full realization of any prognostic capability. MEG is a well-tolerated, noninvasive, and easily repeatable investigation with potential to add sensitivity to biomarker-driven neuroprotective studies, strengthening the envisaged multimodal characterization of presymptomatic ALS. MEG therefore provides a plausible candidate metric for ALS, in the form of cortical FC, that may permit pharmacodynamic appraisal of future therapeutic interventions aimed at restoring seemingly harmful cortical hyperexcitability.This study reinforces previous rs-fMRI findings of increased FC in ALS and builds on recent MEG data implicating cortical hyperexcitability.


Many unhealthy behaviors often begin during adolescence and represent major public health challenges. Substance abuse has a major impact on individuals, families, and communities, as its effects are cumulative, contributing to costly social, physical, and mental health problems. We conducted an overview of systematic reviews to evaluate the effectiveness of interventions to prevent substance abuse among adolescents. We report findings from a total of 46 systematic reviews focusing on interventions for smoking/tobacco use, alcohol use, drug use, and combined substance abuse. Our overview findings suggest that among smoking/tobacco interventions, school-based prevention programs and family-based intensive interventions typically addressing family functioning are effective in reducing smoking. Mass media campaigns are also effective given that these were of reasonable intensity over extensive periods of time. Among interventions for alcohol use, school-based alcohol prevention interventions have been associated with reduced frequency of drinking, while family-based interventions have a small but persistent effect on alcohol misuse among adolescents. For drug abuse, school-based interventions based on a combination of social competence and social influence approaches have shown protective effects against drugs and cannabis use. Among the interventions targeting combined substance abuse, school-based primary prevention programs are effective. Evidence from Internet-based interventions, policy initiatives, and incentives appears to be mixed and needs further research. Future research should focus on evaluating the effectiveness of specific interventions components with standardized intervention and outcome measures. Various delivery platforms, including digital platforms and policy initiative, have the potential to improve substance abuse outcomes among adolescents; however, these require further research. Adolescence is recognized as the period for onset of behaviors and conditions that not only affect health limited to that time but also lead to adulthood disorders. Unhealthy behaviors such as smoking, drinking, and illicit drug use often begin during adolescence; they are closely related to increased morbidity and mortality and represent major public health challenges. Unemployment, poor health, accidents, suicide, mental illness, and decreased life expectancy all have drug misuse as a major common contributing factor Globally, tobacco use is the leading preventable cause of premature death and most adult smokers initiate smoking in adolescence Efforts should be concerted on early identification, awareness and prevention programs, and routine monitoring of adolescent health data. Given the prevailing burden and impact of substance abuse in children and adolescents, it is essential that effective interventions and delivery platforms on enhancing social skills, problem-solving skills, and self-confidence are identified and implemented This article is part of a series of reviews conducted to evaluate the effectiveness of potential interventions to improve adolescent health and well-being. We developed a conceptual framework based on existing conceptual frameworks In this article, we conducted a comprehensive overview of systematic reviews for the effectiveness of substance abuse interventions for adolescents and various delivery platforms.We systematically reviewed literature published up to December 2015 to identify systematic reviews on interventions for substance abuse in adolescent population. For the purpose of this overview, the adolescent population was defined as aged 11–19 years; however, since many reviews targeted youth (aged 15–24 years) along with adolescents, exceptions were made to include reviews targeting adolescents and youth. We did not apply any limitations on the start search date or geographical settings. We considered all available published systematic reviews on interventions for adolescent substance abuse. A broad search strategy was used that included a combination of appropriate keywords, medical subject heading, and free text terms. Search was conducted in the Cochrane Library and PubMed. The abstracts (and the full sources where abstracts are not available) were screened by two abstractors to identify systematic reviews adhering to our objectives. Any disagreements on selection of reviews between these two primary abstractors were resolved by the third reviewer. After retrieval of the full texts of all the reviews that met the inclusion/exclusion criteria, data from each review were extracted independently into a standardized form. Information was extracted on (1) the characteristics of included studies; (2) description of methods, participants, interventions, and outcomes; (3) measurement of treatment effects; (4) methodological issues; and (5) risk of bias tool. We extracted pooled effect size for the outcomes reported by the review authors with 95% confidence intervals (CIs). We assessed and reported the quality of included reviews using the 11-point assessment of the methodological quality of systematic reviews (AMSTAR) criteria 1.Intervention for smoking/tobacco use (n = 20)2.Interventions for alcohol use (n = 8)3.Interventions for drug use (n = 2)4.Interventions targeting combined substance abuse (n = 16)Our search identified 614 potentially relevant review titles, of which 110 full texts were reviewed. Finally, 46 reviews were deemed eligible and meeting the inclusion criteria . We clasWe report findings from a total of 20 systematic reviews focusing on various interventions for smoking/tobacco use among adolescents. Of these 20 reviews, three reviews focused on school-based interventions; three reviews focused on family-/community-based interventions; four reviews focused on digital platforms; four reviews focused on policy interventions; one review focused on the effect of providing incentives; while five reviews focused on multicomponent interventions for smoking/tobacco use among adolescent age group. The AMSTAR rating for the reviews ranged between 5 and 10 with a median score of 8. Meta-analysis was conducted in nine of the included reviews.We report findings from three systematic reviews focusing on school-based interventions for smoking/tobacco use among adolescents We included three systematic reviews evaluating the impact of family-/community-based interventions for smoking/tobacco use among adolescents We report findings from four systematic reviews evaluating various digital platforms for smoking/tobacco use among adolescent age group We found four reviews reporting the impact of smoking/tobacco use policy initiatives We found one review evaluating the impact of incentives  to prevent smoking among adolescents We found five reviews addressing multicomponent interventions for smoking/tobacco use among adolescents We report findings from a total of eight systematic reviews focusing on various interventions for alcohol use among adolescents. Four reviews focused on school-/college-based interventions while one review each focused on family-/community-based interventions, digital platforms, policy interventions, and multicomponent interventions. The AMSTAR rating ranged between 7 and 10 with a median score of 8.5. Meta-analysis was conducted in five of the included reviews.We report findings from a total of four reviews focusing on school-/college-based interventions for alcohol use We found one review evaluating the impact of universal family-based prevention programs  in preventing alcohol misuse in school-aged adolescents We found one systematic review reporting the efficacy of computer-delivered interventions (CDIs) to reduce alcohol use among college students We found one review that evaluated restriction or banning of alcohol advertising via any format including advertising in the press, on the television, radio, Internet, billboards, social media, or product placement in films We found one review evaluating the effectiveness of universal multicomponent prevention programs in preventing alcohol misuse in school-aged children We report findings from two systematic reviews focusing on various interventions for drug use among adolescents. Both the reviews focused on school-based interventions. The AMSTAR rating for the reviews ranged between 8 and 10 with a median score of 9. Meta-analysis was conducted in both the included reviews.We found two reviews evaluating school-based interventions for drug use We report findings from a total of 16 systematic reviews focusing on various interventions for combined substance abuse among adolescents. Of these 16 reviews, four reviews focused on school-based interventions, one review focused on family-/community-based interventions, four reviews focused on digital platforms, three reviews focused on individual-targeted interventions (mentoring and psychotherapy), and four reviews focused on multicomponent interventions. The AMSTAR rating for the reviews ranged between 6 and 10 with a median score of 7. Meta-analysis was conducted in five of the included reviews.We found four systematic reviews evaluating the impact of school-based interventions targeting substance abuse among adolescents We found one review evaluating parenting programs to prevent tobacco, alcohol, or drug abuse in children younger than 18 years We report findings from four reviews evaluating digital platforms for substance abuse among adolescents We report findings from three systematic reviews evaluating individual-targeted interventions for substance abuse among adolescents; these included mentoring We report findings from four systematic reviews evaluating multicomponent interventions for substance abuse among adolescents We included 46 systematic reviews focusing on interventions for smoking/tobacco use, alcohol use, drug use, and combined substance abuse. Our overview findings suggest that among smoking/tobacco use interventions, school-based pure prevention programs and SFC are effective in reducing smoking initiation and current smoking. However, there is lack of long-term follow-up for the impact of school-based smoking/tobacco use programs. Family-based intensive interventions typically addressing family functioning are also found to effectively prevent smoking. Coordinated widespread community-based interventions have also shown positive impacts on smoking behaviors. Mass media campaigns involving solid theoretical basis, formative research in designing the campaign messages, and message broadcast have shown positive impacts on uptake of smoking given that these were of reasonable intensity over extensive periods of time. Evidence from Internet-based interventions, policy initiatives, and incentives appears to be mixed and needs further research.Among interventions for alcohol use, school-based alcohol prevention interventions including personalized feedback, moderation strategies, expectancy challenge, identification of risky situations, goal setting, and BAIs have been associated with reduced frequency of drinking. Family-based interventions have a small but persistent effect on alcohol misuse among adolescents while CDIs for alcohol are found to reduce the quantity and frequency of drinking among college students. There is lack of robust evidence for or against recommending the implementation of alcohol advertising restrictions and multiple component interventions. For drug use, school-based interventions based on a combination of social competence and social influence approaches have shown protective effects in preventing drugs and cannabis use. Among the interventions targeting combined substance abuse, school-based primary prevention programs that include antidrug information combined with refusal skills, self-management skills, and social skills training are effective in reducing marijuana and alcohol use among adolescents. There is very limited evidence on the effectiveness of mass media and mentoring for combined substance abuse.We adopted an overview of reviews approach for synthesizing existing evidence on adolescent substance abuse. Although an overview of systematic reviews builds on the conclusions of rigorous reviews of studies in different settings and of varying quality, avoids duplication of work and allows for a much faster review, there are some potential limitations. The interventions on which primary data exist, but which have not been covered by a systematic review, will not have been included. Furthermore, an overview of systematic reviews relies on review authors' characterizations of the findings rather than on individual studies and therefore may be affected by selective reporting biases. It also misses upon studies not taken up by included reviews. However, we have quality rated the existing reviews for transparency.Our review findings highlight that school-based delivery platforms are the most highly evaluated platforms for targeting adolescents for substance abuse. Most of the existing evidence for substance abuse interventions comes from HICs. There is lack of data to determine the differential effects of interventions by gender, socioeconomic status, and population density. Meta-analysis could not be conducted in most of the included reviews since the interventions varied in intensity, follow-up periods, and reported outcomes. Furthermore, in reviews where meta-analysis was conducted, not all the data contributed to the pooled effect estimate. There is lack of rigorous data evaluating the sustainability and long-term effectiveness of substance abuse programs targeting adolescents. Future research should focus on evaluating the effectiveness of specific intervention components with standardized intervention and outcome measures. There is a need to evaluate relative effectiveness and cost-effectiveness of various delivery platforms targeting adolescents for substance abuse interventions. Various delivery platforms, including digital platforms and policy initiative, have the potential to improve substance abuse outcomes among adolescents; however, these require further research. Future trials should focus on reporting separate data for gender and socioeconomic subgroups since the impact of such behavior change interventions might vary among various population subgroups. Lastly, there is a dire need for rigorous, higher quality evidence especially from low- and middle-income countries on effective interventions to prevent and manage substance abuse among adolescents.


Many factors, including histopathologic parameters, seem to influence the prognosis of patients undergoing resection of colorectal cancer liver metastases (CRCLM), although their relative weight is unclear. Histopathologic growth patterns (HGPs) of CRCLM may affect sensitivity to antiangiogenics. We aimed at evaluating differences in histopathologic parameters of response according to the use of bevacizumab or cetuximab as first-line targeted agents, and at exploring the prognostic and predictive role of HGPs.N = 103) vs cetuximab (N = 56) in five first-line no-profit clinical trials.We performed a comprehensive histopathologic characterisation of CRCLM from 159 patients who underwent secondary resection, after receiving triplets FOLFOXIRI  or COI  plus bevacizumab (p = 0.013) and infarct-like necrosis  were significantly higher in the bevacizumab than in the cetuximab group. Achieving major response positively affected relapse-free survival (RFS) (p = 0.012) and overall survival (OS) (p = 0.045), also in multivariable models .Both major histopathologic response , a higher percentage of major response was reported  and an unsignificant advantage from cetuximab vs bevacizumab was evident in RFS (p = 0.116). In the pushing HGP (N = 66), a significant benefit from bevacizumab vs cetuximab (p = 0.017) was observed. No difference was described in the replacement HGP .In the desmoplastic HGP (The histopathologic response is the only independent determinant of survival in patients resected after triplets plus a biologic. When associated with triplet chemotherapy, bevacizumab induces a higher histopathologic response rate than cetuximab. The assessment of HGPs should be further explored as a predictor of benefit from available targeted agents. The management of metastatic colorectal cancer (mCRC) patients with liver-limited disease (LLD) is an intriguing challenge for oncologists, as the optimal integration of systemic and locoregional treatments may maximise survival outcomes and even cure a selected subgroup of patients. In the last years, the availability of active systemic treatments and the development of innovative surgical techniques have increased the percentage of potentially resectable patients, thus widening the horizons of pursuable surgical strategies.3 Therefore, doublets plus an anti-EGFR monoclonal antibody (only in RAS wild-type patients) or the triplet FOLFOXIRI  plus bevacizumab (independently from molecular subgroups) are the standard regimens with highest activity.7 Recent data from phase II studies suggest that the combination of triplet chemotherapy with an anti-EGFR agent is feasible and allows achieving impressive response outcomes in molecularly selected patients.10Recent guidelines recommend the choice of highly active regimens, able to induce early and deeper tumour shrinkage, as the preferable options for patients with initially unresectable or borderline resectable colorectal cancer liver metastases (CRCLM).15 Therefore, the optimal systemic regimen in the setting of liver-limited mCRC should be able to induce not only radiologic, but also histopathologic response. Retrospective studies suggested that the addition of bevacizumab to oxaliplatin-based doublets positively affects the rate of major/complete histopathologic response.17 At the same time, up today no conclusive data about the differential impact of bevacizumab vs anti-EGFRs on TRG were provided, since available series are affected by several bias, including an inappropriate molecular selection of patients treated with anti-EGFRs, and the adoption of heterogeneous chemotherapy backbones.19Although response parameters including early tumour shrinkage and deepness of response highly influence the chance to achieve R0 resections, the balance of several clinical, molecular and pathologic factors may influence patients’ survival outcomes. Among these latter factors, the histopathologic response to the pre-operative treatment, mainly defined in terms of tumour regression grade (TRG), is crucial.20 From a biologic viewpoint, although metastases with desmoplastic and pushing HGPs rely on angiogenesis for their vascular supply, those with a replacement HGP co-opt pre-existing sinusoidal vessels, suggesting an intrinsically resistance to anti-angiogenic drugs.21Recently, three different histopathological growth patterns (HGPs) of liver metastases have been described: desmoplastic , pushing , and replacement .Drawing from these considerations, we performed an extensive histopathologic evaluation of CRCLM resected after triplets and either bevacizumab or cetuximab, aiming at evaluating differences in histopathologic parameters of response according to administered targeted agents (bevacizumab vs cetuximab), assessing the independent prognostic impact of histopathologic parameters, and exploring the potential prognostic or predictive role of HGPs.From July 2008 to September 2016, 677 mCRC patients received first-line FOLFOXIRI or COI  plus bevacizumab or cetuximab in five clinical trials, enrolling patients from 40 Italian Oncology Units. All trials were approved by the local Ethics Committees at all participating centres, and patients provided their written informed consent to receive the treatment and to participate to translational analyses.N = 508), 252 in the FOLFOXIRI plus bevacizumab arm),22 MOMA 23 and MACBETH 10 by Gruppo Oncologico del Nord Ovest (GONO), adopted FOLFOXIRI as chemotherapy backbone; COI-E 9 and COI-B 24 by Fondazione IRCCS Istituto Nazionale dei Tumori (INT), used capecitabine, oxaliplatin, and irinotecan (COI). Bevacizumab was the combined targeted agent in TRIBE, MOMA, and COI-B, whereas cetuximab was used in MACBETH and COI-E. For the purpose of the present analysis, among patients treated with cetuximab, only those centrally defined as RAS and BRAF wild-type were included.TRIBE , whose disease was deemed unresectable by experienced multidisciplinary teams. The adoption of guidelines for defining unresectability Trials by INT included only mCRC patients with borderline resectable liver-limited disease, defined by technical  and/or biologic reasons  predicting high recurrence risk. Four biweekly pre-operative cycles of COI-B or COI-E were planned.In all studies, disease assessment by contrast-enhanced CT scan of chest and abdomen was performed every 8 weeks until disease progression.For the purpose of this analysis, we identified patients with liver-limited disease who underwent secondary resection of their metastatic lesions with curative intent and with available tissue samples of resected metastases.26 then modified for liver metastases.13 This score identifies five TRGs based on the presence of residual tumour cells and the extent of fibrosis. A cut-off of 3 mm of tumour thickness at the tumour-normal interface (TNI) was used to differentiate minor from major/complete pathologic response.27All histopathologic assessments were performed by optical microscope and centralised at Fondazione IRCCS Istituto Nazionale dei Tumori, Milan. Tissue samples were independently evaluated by two pathologists  blinded with respect to clinical information, treatment regimen, and outcome. TRG was scored according to the scheme from Mandard et al.,28We distinguished infarct-like necrosis, consisting of large confluent areas of eosinophilic cytoplasmic remnants, located centrally within the lesion and surrounded by fibrosis and foamy macrophages, from tumoural “dirty” necrosis, containing nuclear debris in a patchy distribution.30Lymphocytic intratumoural infiltration and peritumoural inflammatory response were determined using a score ranging from absent (no lymphocytes) to mild (<5 lymphocytes/HPF), moderate (5–10 lymphocytes/HPF), and severe (>10 lymphocytes/HPF).Toxicity-related parameters were evaluated in the non-neoplastic parenchyma and determined based on their presence or absence. Sinusoidal dilatation was graded semiquantitatively as follows: 0, absent; 1, mild (centrolobular involvement limited to one-third of the lobular surface); 2, moderate (centrolobular involvement extending to two-thirds of the lobular surface); 3, severe (complete lobular involvement).20  were recognised by standard H&E stained tissue sections, according to the key histopathologic characteristics of the growth patternsχ2 test, Fisher exact test or Mann–Whitney test as appropriate.Baseline characteristics and histopathologic parameters of response and toxicity reported in patients treated with triplet plus bevacizumab or triplet plus cetuximab were compared by means of p < 0.10) were included in a multivariable Cox proportional hazard model.RFS was calculated from the day of surgical resection to the evidence of disease relapse, or death from any cause. Post-resection OS was calculated from the day of surgical resection until death from any cause. Survival curves were estimated by the Kaplan–Meier method and compared with the log-rank test. The impact of histopathologic response and other prognostic factors on relapse-free survival (RFS) and post-resection overall survival (OS) was firstly assessed in univariate analyses. Significantly prognostic variables  and of radiologic response parameters  and relative 95% confidence intervals (CIs) were calculated. Variables significantly (p < 0.10) affecting the probability of undergoing liver surgery were included in a logistic regression model.We investigated the effects of clinical and molecular characteristics  that was calculated from the day of study entry (registration or randomisation) to the first observation of disease progression according to RECIST, or death from any cause.Liver metastases from 159 patients were analysed. Patients’ baseline characteristics are summarised in Table p = 0.436). In significantly higher percentages of cases in the bevacizumab than in the cetuximab group major histopathologic response  and infarct-like necrosis  were reported. No differences in other parameters of histopathologic response and toxicity were observed (Table RAS and BRAF wild-type cases and those bearing any RAS or BRAF mutation were evident (Supplementary Table RAS-mutated tumours.Table p = 0.015) and of FOLFOXIRI instead of COI , and the deepness of radiologic response  were significantly associated with the probability of achieving a major histopathologic response (Table p < 0.001) and the administered targeted agent  were significantly associated with the probability of achieving a major response.Among investigated baseline characteristics, radiologic response parameters and treatment subgroups, the administration of bevacizumab instead of cetuximab , those achieving major response (N = 41) showed significantly longer RFS  , as well as the nodal status of the primary tumour , retained its prognostic impact in the multivariable model.Histopathologic response according to TRG was the only parameter associated with post-resection outcomes (Supplementary Table 12) Fig. . As showonse N = 1 showed p = 0.045) .Consistently, major response was associated with longer OS  Fig. . In the p = 0.856; RFS log-rank p = 0.783) . Although among patients whose metastases presented a desmoplastic HGPa non-significant advantage for cetuximab was reported  , 66 41%), and 65 (41%) specimens, respectively. In the overall population, no impact of HPGs on survival parameters was observed (OS log-rank 83) Fig. , but a h97) Fig. . Consist1%, and 632 Because of the wide spectrum of potential clinical scenarios, both the design of clinical studies and the applicability of trials’ results in the daily practice are extremely difficult. In fact, in spite of the limited amount of prospective evidence in this field, the management of patients with CRCLM has notably changed in the last decade, with a clinically meaningful absolute survival gain.33 Not only the improvements in innovative surgical and other locoregional techniques, but also the availability of several conversion or neoadjuvant systemic regimens contributed to these advances, that are expected to further improve over time.37 Increasing evidence is collected about the possibility to significantly affect the natural history of the disease through a substantial pharmacological and surgical debulking of the tumour burden, made possible by the multidisciplinary management of affected patients.The landscape of CRCLM is extremely heterogeneous and multiple clinical, molecular, and pathological factors weight on patients’ outcomes.17To this regard, the pathologists’ role in the multidisciplinary team is increasingly important, although further effort is needed to clarify how to exploit histopathologic parameters to drive treatment decisions. Here we confirm the impact of TRG on the post-operative outcome, while no association of necrosis, fibrosis and infarct-like necrosis with survival is reported.It is arguable that TRG mirrors the ability of pre-operative regimens to control the micrometastatic disease, thus reducing the probability to experience disease relapse.Our data strengthen this concept, since the association between TRG and survival parameters is retained in the multivariable model, aiming at catching the heterogeneity of potential clinical presentations and previous responses to systemic treatments. As all patients included in the present analysis received a triplet plus a biologic as pre-operative treatment, it seems that the use of highly active regimens as conversion or neoadjuvant treatments may counterbalance the poor prognostic impact of negative baseline characteristics when achieving a radical resection of metastatic lesions. In other words, clinical and molecular factors weighing on post-resection outcomes are no longer relevant when using these upfront treatments. On the other side, achieving a relevant histopathologic response is a major determinant of clinical outcome and, notably, is significantly associated with deeper radiologic response. The possibility to adopt different post-operative strategies based on histopathologic response results is worth of investigation in properly designed prospective trials.18,19 Notably, all these studies were invariably biased by their retrospective nature, the heterogeneity of chemotherapy backbones and the inappropriate molecular selection of patients treated with the anti-EGFRs.Here we also show that the use of triplets plus bevacizumab had more histopathologic responses than triplets plus cetuximab. Up today, whereas different retrospective analyses with several potential biases consistently suggested that the addition of bevacizumab to chemotherapy alone increases the rate of major histopathologic responses, conflicting results were provided with regard to the comparison of chemotherapy plus either bevacizumab or an anti-EGFR.33 Thirdly, although the homogeneity of chemotherapy backbones definitely represents a strong point of this analysis, 31 patients received capecitabine as part of the chemotherapy regimen, in combination with cetuximab. The association of fluoropyrimidines other than infusional 5-fluoruracil with anti-EGFR monoclonal antibodies is not recommended by current guidelines. Nevertheless, in the multivariable model the impact of the targeted agent on the probability of achieving a major histopathologic response is independent of the associated chemotherapy regimen. Finally, we were not able to provide formal demonstration that regimens able to determine better histopathologic responses favorably affect survival, thus failing to prove the surrogacy of TRG for OS, as other previous series in this field did.Our effort suffers of some limitations. Firstly, trials included in our pooled analysis did not randomise between bevacizumab and cetuximab. However, inclusion criteria of these studies were perfectly superimposable, with the exception of the molecular selection for studies evaluating cetuximab-containing regimens, and the studies were conducted in the same timeframe. As a consequence, characteristics of enrolled patients were highly balanced in the two groups. Secondly, only patients with initially unresectable or borderline resectable but at high risk of recurrence are included, thus preventing from applying present results to easily resectable patients at low risk of recurrence. However, these patients are not candidate to receive biologic agents, and in particular anti-EGFRs since a potential detrimental effect with the addition of cetuximab to perioperative oxaliplatin-based doublets was evidenced.21 The available retrospective literature suggests that replacement HGP may be associated with poorer prognosis, worse histopathologic response to neoadjuvant treatments and lack of survival benefit from the addition of bevacizumab to chemotherapy alone. To the best of our knowledge, this is the first attempt to potentially catch a differential benefit from bevacizumab vs cetuximab according to HGPs. Even if our series cannot provide definitive conclusions, interesting results about the better efficacy of bevacizumab and cetuximab in the pushing and in the desmoplastic patterns, respectively, were found and should be validated through properly designed randomised trials. The lack of prognostic impact of HGPs in the present series, differently from literature data, may be explained by the fact that the adopted highly active pre-operative regimens may have weakened the weight of poor prognostic factors including replacement HPG. A similar effect was previously shown by our group about the lack of negative impact of BRAF mutation in mCRC patients with LLD, resected after FOLFOXIRI plus bevacizumab.7 Moreover, the use of systemic treatments may somehow change the percentage of a specific HGPs component in favor of another thus representing a potential confounding effect in our and previous studies, which mostly included patients treated in the pre-operative setting. Finally, assessing HGPs post-operatively clearly hampers its potential application to the choice of the pre-operative strategy. Therefore, to deepen and hopefully translate to clinical practice the predictive power of HGPs, additional valuable information should be prospectively obtained through liver biopsies performed before starting the conversion/neoadjuvant treatment. To this purpose, the possibility to classify accurately HGPs by means of pre-treatment imaging parameters should be investigated.The evaluation of CRCLM’ HGPs has recently gained attention from the oncology perspective due to its potential prognostic and even predictive meaning, as well as its easy assessment in H&E stained slides.Supplementary figure 1SUPPLEMENTARY table 1SUPPLEMENTARY table 2SUPPLEMENTARY FIGURE 2SUPPLEMENTARY FIGURE 3SUPPLEMENTARY FIGURE 4Color form


Stuttering affects nearly 1% of the population worldwide and often has life-altering negative consequences, including poorer mental health and emotional well-being, and reduced educational and employment achievements. Over two decades of neuroimaging research reveals clear anatomical and physiological differences in the speech neural networks of adults who stutter. However, there have been few neurophysiological investigations of speech production in children who stutter. Using functional near-infrared spectroscopy (fNIRS), we examined hemodynamic responses over neural regions integral to fluent speech production including inferior frontal gyrus, premotor cortex, and superior temporal gyrus during a picture description task. Thirty-two children (16 stuttering and 16 controls) aged 7–11 years participated in the study. We found distinctly different speech-related hemodynamic responses in the group of children who stutter compared to the control group. Whereas controls showed significant activation over left dorsal inferior frontal gyrus and left premotor cortex, children who stutter exhibited deactivation over these left hemisphere regions. This investigation of neural activation during natural, connected speech production in children who stutter demonstrates that in childhood stuttering, atypical functional organization for speech production is present and suggests promise for the use of fNIRS during natural speech production in future research with typical and atypical child populations. Stuttering emerges in the preschool years as the brain develops the intricate and dynamically interactive networks underlying language formulation and speech production8. Those who stutter know what they wish to communicate; however, involuntary disfluencies impede the forward flow of their speech. Examples of these include sound or syllable repetitions, blocks, and prolongations. Many factors, including heredity, linguistic abilities, speech motor abilities, and temperament, uniquely contribute to the onset of stuttering and in the likelihood of recovery or persistence10. Although most children who begin to stutter will recover, the approximately 25% who persist will struggle with a communication disorder that is resistant to treatment throughout their lives12.Although seemingly effortless, fluent speech production is a remarkably complex process, requiring the functional synergy of multiple neural networks to accomplish language formulation, articulatory planning, motor execution, and auditory and somatosensory integration21. An additional finding in adults who stutter is anomalous degree and symmetry of activation in auditory cortices during speech production14. Gray and white matter anomalies in perisylvian regions are presumed to induce atypical brain activity during speech production in adults who stutter29.An extensive body of neuroimaging research in adults who stutter has provided greater insight into the neurological underpinnings of stuttering, confirming functional differences in speech and language network activation even when they are speaking fluently. Compared to fluent speakers, adults who stutter show heightened activity in right motor cortex and in cerebellar regions, but reduced activation in left anterior speech regions 34. However, there have been few functional neuroimaging investigations undertaken in children who stutter; thus, there are significant gaps in our understanding of speech neurophysiology closer to the onset of the disorder. The paucity of research in this population critically limits our ability to interpret findings from studies of adults who stutter and in turn, to inform developmental theories of stuttering. For example, it is unclear whether atypical lateralization of brain activity during speech production in adults who stutter represents a causal mechanism, or rather, a compensatory response, a supposition supported by studies of adults who stutter35. One magnetoencephalography (MEG) study found left lateralized activation of IFG preceding single-word production in both children who stutter and controls, suggesting that atypical speech lateralization in adults who stutter may not extrapolate to young children who stutter36. Clearly, more research into the neural correlates of speech planning and production in children who stutter is needed.A growing number of anatomical imaging studies in children report decreases in gray matter volume in IFG and temporal regions bilaterally in children who stutter compared to controls, and reduced integrity of white matter tracts interconnecting auditory and motor speech areas37). Neural models of speech production posit that learned articulatory programs for commonly produced speech sounds are stored in left IFG and premotor cortex. Projections from these neurons to primary orofacial motor cortex encode the motor commands to articulate specific speech sounds. Bi-directional projections from the same neurons to superior temporal gyrus (STG) and lateral parietal cortex encode the expected sensory consequences of the articulatory movements. Any discrepancy between the expected and actual feedback is relayed back to motor cortex where it is used to correct and update the articulatory programs. This integration of sensory and motor information is key to the development and maintenance of fluent speech41.The neural regions implicated in stuttering are key components of a speech production network responsible for sequencing phonological codes to articulating sounds to communicate our message 46. The increased rCBF exceeds the oxygen metabolic rate of a region resulting in a peak in Oxy-Hb and a decrease or dip in Deoxy-Hb47. The Oxy- and Deoxy-Hb response from fNIRS measurements is spatially and temporally correlated to the blood oxygen level-dependent (BOLD) response from fMRI49. fNIRS is a safe and more child-friendly neuroimaging approach51 that allows silent, continuous recording of cortical hemodynamic responses during speech production54. fNIRS has been used to examine the regional activation, timing, and lateralization of brain activity for diverse perceptual, language, and cognitive functions (for review55). As such, fNIRS offers an unprecedented, practical means to examine the neurophysiology of speech production in typical and atypical child populations56.Functional near-infrared spectroscopy (fNIRS) is a non-invasive, optical neuroimaging method that measures the relative concentration changes in oxygenated (Oxy-Hb) and deoxygenated (Deoxy-Hb) hemoglobin, two absorbing chromophores in cerebral capillary bloodAs reviewed, there is strong evidence in both adults and children who stutter of atypical neuroanatomical development; however, we know comparatively little about the neurophysiology of sensory and motor regions underlying efficient timing and control of speech movements in children who stutter. At its core, stuttering manifests as a breakdown in speech production and is most likely to occur during connected, extemporaneous speech. Thus, it is critical to assess neural activation during more ecologically valid speaking conditions that place the greatest demands on the speech production networks of children who stutter.58 and from a recent fNIRS verbal fluency investigation with children and adults59. Based on earlier findings in stuttering adults (reviewed above), hemodynamic responses from children who stutter may show atypical symmetry and amplitude during speech production. We expect reduced responses, especially over left hemisphere IFG/PMC areas, and augmented responses over the right hemisphere homologous areas to compensate for left hemisphere neurophysiological deficits. Another possibility is that atypical neural activation patterns in adults who stutter result from years of compensating for their speech disorder, and thus, may not be present in children who stutter.The goal of this study is to examine hemodynamic responses from children who do and do not stutter over neural regions integral to fluent speech production and implicated in the pathophysiology of stuttering. These regions include the left and right IFG, the left and right precentral gyrus (premotor cortex-PMC), and the left and right STG. We hypothesize that connected speech, which maximally engages speech and language networks, will elicit a left-lateralized hemodynamic response over IFG for speech planning and production in the cohort of controls. There is evidence to support this from fMRI studies of picture description and narrative production in adult speakersF = 0.11, p = 0.74 and socioeconomic status (SES) F = 1.4, p = 0.24. Unusable trials  made up 4% and 3% of the total number of trials in the group of children who do and do not stutter, respectively. The between-group difference in the number of unusable trials was not significant  = 0.09, p = 0.77. The two groups had a similar number of trials containing normal or typical speech disfluencies STUTT  and CONT  F = 1.31, p = 0.26. As expected, the children who stutter produced a greater number of stuttered trials compared to controls . As a result, the group of children who stutter had significantly fewer fluent trials  than the controls  F = 19.53, p < 0.01. Finally, the two groups of children produced a comparable number of spoken syllables to describe each picture STUTT  and CONT , F = 0.41, p = 0.53. Examples of typical responses include, “A girl is watering the flower pots”. “There’s a tiger at the zoo with big teeth”. The correlation between age and number of syllables produced was significant . On average, the older participants produced longer responses to each picture.One-way ANOVAs confirmed that there were no differences between the two participant groups in age t-tests were calculated to identify amplitude differences in Oxy-Hb and Deoxy-Hb responses from the group of children who stutter and controls for each channel. These results, along with means and standard errors for each channel are listed in Table Table r = −0.30–0.38; all p > 0.05).Finally, the correlations between age or number of syllables and Oxy-Hb amplitudes for channels collapsed across hemisphere and region of interest in either group were not significant. The correlation between stuttering severity and average Oxy-Hb amplitudes in the group of children who stutter was also not significant  and Deoxy-Hb (blue) hemodynamic responses were grand-averaged across the 16 children in this group and presented on the left side of Fig. t-test confirmed significantly reduced amplitudes of Oxy-Hb responses in left compared to right IFG for the group of children who stutter  and left PMC (channels 8 and 9) compared to the controls  over these left hemisphere regions.62. Although we may only speculate at this point, one explanation is that the deactivation is compensatory and indicates a shift in blood flow to regions outside of the probe boundaries to compensate for functional deficits in the primary areas. People who stutter report the ability to anticipate moments of stuttering63 and may use idiosyncratic coping mechanisms to avoid or mask their disfluencies. What the listener perceives as fluent speech may in fact be “covert stuttering” or other compensatory strategy requiring significant cognitive effort to circumvent overt breakdowns in speech production. The neurophysiological correlates of increased cognitive effort during speech production have not been measured; however, future fNIRS investigations with expanded probe coverage could assess whether greater blood flow is detected in areas adjacent to IFG/PMC, for example, to prefrontal areas that may mediate the conscious avoidance of speech breakdowns.The differences in hemodynamic profiles that we detected may indicate deficient planning and production processes related to stuttering. Insufficient engagement of left hemisphere IFG and PMC is a reasonable explanation for the reduced or missing early peaks in Oxy-Hb in the stuttering group; however, it cannot explain the abrupt decreases in Oxy-Hb that immediately followed within the window of analysis. The interpretation of negative hemodynamic responses and whether they signify neuronal deactivation, or alternatively, a shift in blood flow to supply activation of adjacent neural regions provokes much debate65. Using the GODIVA/DIVA computational modeling framework to simulate production of multisyllabic speech production, Civier and colleagues demonstrated stuttering-like disfluencies (syllable onset delays) when dopamine levels were atypically high in model’s putamen analog66. The high dopamine levels also resulted in a clear net reduction in the activity in the component of the model that stores speech motor programs hypothetically located in left ventral premotor and adjacent posterior inferior frontal cortex. In the present study, the deactivation of left inferior frontal and premotor cortex in children who stutter occurred during fluent speech production. Thus, it may reflect atypical processes that underlie stuttering, for example, disrupted cortico-basal ganglia circuits as suggested above, that have been corrected or that fail to reach a threshold that results in a disfluency.An alternative explanation for deactivation of left IFG/PMC in the stuttering group is a disruption of cortical-subcortical interactions resulting in a net inhibition of these regions. Dopamine hyperactivity in the basal ganglia has been associated with stuttering67. Deactivation of frontal and temporal speech regions was reported by an earlier PET study in adults who stutter, although this anomaly was reversed when fluency was induced through choral reading16. Finally, decreased frontal and temporoparietal activation for the planning and production of both speech and nonspeech stimuli in adults who stutter has been shown with fMRI14. According to a meta-analysis of PET and fMRI studies with adults who stutter, neural activation for fluent speech in adults who stutter is characterized by underactivity in left motor regions accompanied by a right hemisphere shift in activity in the stuttering group68. Although not specifically delineated, right hemisphere compensation for left hemisphere deficits has been proposed in some capacity to explain findings of reversed laterality of neural activation in adults who stutter21. We did not detect “overactivation” of right hemisphere channels in the children who stutter; right hemisphere responses did not significantly differ between the groups. Rather, our findings suggest that atypical left, rather than right IFG/PMC hemodynamic responses, motivated the significant differences between the two groups of children . A follow-up study in children who stutter examining hemodynamic responses from both types of trials  is underway that may help clarify current findings. For example, if hemodynamic response patterns that we found for fluent speech are wholly different from hemodynamic responses recorded during stuttered speech, then this may provide support for compensatory mechanisms.Earlier functional imaging studies in adults who stutter relied upon less natural speaking tasks such as choral speech, word generation, oral reading, speaking in time with a metronome, or producing overlearned content. We recorded fluent and disfluent speech tokens during a more natural speaking task. The present study focused exclusively on fluent responses, allowing us to compare hemodynamic responses between groups of children who do or do not stutter  within the group of children who stutter. We will examine whether the correlation between severity and Oxy-Hb amplitude for stuttered speech in a follow-up study with children who stutter. It may be the case, however, that average hemoglobin response amplitude is too coarse a metric to differentiate between a mild or moderate stuttering problem. A focus for future research, in our lab and others, is to explore the use of pattern recognition or feature selection algorithms to classify participants into their respective groups by comparing, for example, temporal features from an individual’s data with group averages70. Ichikawa and colleagues utilized responses from a subset of fNIRS channels to classify children with 84% accuracy into either attention-deficit/hyperactivity-ADHD or autism spectrum disorder-ASD groups71.Finally, we did not find significant correlations between Oxy-Hb response amplitude and stuttering severity . Moreover, 10 out of the 15 studies elicited covert responses. As this review clearly highlights, there is a need for more ecologically valid investigations of language development. Although fNIRS has coarser spatial resolution compared to fMRI, it is well suited to examine the activation of broad neural regions and to investigate the functional lateralization of brain activity55. This study represents a vital step toward taking this research into younger populations of children who stutter near onset to determine whether the aberrant hemodynamic responses in school-aged children are indicative of a higher risk for developing a persistent stuttering problem.The results from this study advance the potential of using fNIRS to examine neural activation during natural speech production and indicate its promise as an innovative research tool to study speech perception and production deficits associated with other speech and language disorders. Although the neural substrates of speech and language processes are well-defined in adults, a recent review of the fMRI literature from the past two decades reveals few investigations into the functional development of these networks in childrenM = 9.1 yrs, SD = 1.5 years); . Per parent report, all children were native speakers of North American English with no history of cognitive, developmental, neurological, or speech and language disorders other than stuttering in the group of children who stutter. Children were screened for medications affecting the central nervous system . The participants scored within normal limits on the Clinical Evaluation of Language Fundamentals Screening Test (CELF-IV75)and passed a bilateral pure-tone hearing screening at 500, 1000, 2000, 4000, and 6000 Hz at 20 dB. All participants were right-handed, assessed with the Handedness Questionnaire76. Finally, the two groups had comparable SES determined by the parents’ level of education77. SES was evaluated on a 7-point scale . Criteria for the diagnosis of stuttering and general characteristics of the children who stutter are provided in Appendix Thirty-two children participated in the study, 16 children who stutter (13 boys) and 16 SES and age-matched controls (11 boys). The participants were between the ages of 7 and 11 years  on a PC synchronized with the data acquisition unit to insert precise onset markers into the continuous fNIRS recording at the beginning of each trial. We employed a slow event-related design in which each trial began with an activation interval to allow the hemodynamic response to reach its maximum followed by a rest interval for recovery79. The null trials were included to reduce habituation to the talk trials, and critically, to establish a baseline condition in which no language formulation and speech production occurred to contrast with the talk trials in subsequent analyses. This approach, used in earlier language-related studies with children, allowed us to target speech-related activation evoked by the picture description task /legal guardian and written informed assent from the children, the participants were seated approximately 50 in. in front of a 24 in. computer monitor. The chair height was adjusted so the monitor was at the child’s eye-level. As shown in Fig. 87, channels 6–7 recorded over STG89, and channels 8–9 over precentral gyrus/PMC91. For consistent probe placement, we made markings on each participant’s scalp denoting left and right probe boundaries using customized 10–20 EEG caps . After the EEG caps were removed, the probes were placed symmetrically over each hemisphere utilizing these markings92.The participants’ cerebral hemodynamic changes were recorded during the experiment with a continuous-wave fNIRS system . The system uses near-infrared lasers at 690 and 830 nm as light sources, and avalanche photodiodes (APDs) as detectors for measuring intensity changes in the diffused light at a 25-Hz sampling rate. To ensure reliable placement of the optodes over regions of interest, two customized probes (one for each hemisphere) were created. First, we designed a template of the left and right hemisphere layout based upon International 10–20 system coordinates  representing the age range of the participants95. This procedure converted the real-world stereotaxic coordinates of the optodes from the digitizing stylus to stereotaxic coordinates in a 10-year-old brain template. The template  was generated as an average of whole-head images from 72 children  scanned by 1.5 T or 3.0 T MRI72. Age-specific MRI brain templates created from typically developing participants provided an approximation of head and brain structures of participants in this study.After the probe was secured on each participant, his/her five fiducial points  and the location of each optode were recorded with a digitizing stylus . In order to estimate the cortical regions that the fNIRS probe covered, a spatial registration procedureSpeech output was recorded with a TASCAM linear PCM digital recorder and video (with accompanying audio) was captured with a Toshiba HD Camcorder fitted with a preamplifier. The participants’ utterances were transcribed by one experimenter online during the experiment, and confirmed with video and audio during offline analysis. A syllable count was obtained for each picture.96. Only channels showing clear, real-time cardiac pulses were used in subsequent analyses. Data corrupted by large movement artifact  appearing as large shifts from baseline were manually removed from the record. Trials in which the participant failed to respond, responded before the go sign, continued speaking after the stop sign, or produced less than three syllables were not included in subsequent analyses. The remaining trials were classified as null, talk (i.e. fluent speech), or stutter (disfluent speech). The aim of the present study is to compare hemodynamic responses elicited from children who do and do not stutter. Therefore, only fluent speech trials were considered in the analyses.The fNIRS data was preprocessed using Homer2 software97. In order to correct for speech-related motion artifacts due to jaw movement, we employed a correlation-based signal improvement (CBSI) approach98. CBSI is a channel-by-channel subtraction procedure applied to Oxy-Hb and Deoxy-Hb data, and is a valid method for reduction of speech-related motion artifacts99.The raw data were band-pass filtered (0.03–0.5 Hz) to remove slow drift and high frequency noise and converted into changes in optical density. Next, the concentration changes in Oxy-Hb and Deoxy-Hb were calculated based on the Modified Beer-Lambert Law which accounts for scattering properties of tissue with a differential pathlength factor of 6.0100. Next, the average concentrations of Oxy-Hb and Deoxy-Hb of these responses were calculated within a 3–8 s post-stimulus onset window for each channel. This window was specifically targeted to accommodate individual differences in task-evoked activation peaks while considering the temporal lag of the hemodynamic response101.After motion artifact correction, we derived each participant’s averaged Oxy-Hb and Deoxy-Hb hemodynamic response from each channel from 0 s (stimulus onset) to 21 s post-stimulus onset across trials of the same condition . We used the 2 seconds prior to stimulus onset to set the baseline of each hemodynamic response, which is a standard option in Homer2. In order to specify neural activation exclusively associated with speech production, we subtracted the averaged hemodynamic response from a child’s null trials from the averaged talk response to derive a “differential” hemodynamic response for each channel94, based on the 10-year-old brain template72 and corresponding stereotaxic coordinates obtained from the spatial registration procedure. This toolbox implemented 2D angular interpolation to generate topographic images of brain activation by approximating the cortex as a hemispherical surface. In this study, the channel-wise amplitudes of Oxy-Hb and Deoxy-Hb responses within the 3–8 s window were used to generate activation maps for the talk condition for each group, and the channel-wise t-statistic values reflecting the statistical power of difference between the two groups were used to generate the t-maps for group comparison.Topographic images were generated using a Matlab-based optical topography toolbox, EasyTopop-values are reported. If interactions between group and within-subject factors reached significance, independent sample t-tests (two-tailed) were calculated employing the false discovery rate (FDR) correction to correct for multiple comparisons (p < 0.05)102. To examine the laterality of activation, we calculated paired-samples t-tests comparing the amplitudes of Oxy-Hb responses for left and right regions of interest (collapsed across channels) for each group. Finally, we calculated Pearson bivariate correlation analyses to examine potential relationships between amplitudes of Oxy-Hb responses from channels collapsed across hemisphere and region of interest and behavioral variables .One-way analyses of variance (ANOVAs) were calculated to ensure that the groups were matched for age and SES and to compare behavioral performance during the experimental task. To assess the amplitudes of Oxy-Hb and Deoxy-Hb responses during the null trials and the talk trials, we computed separate repeated-measures ANOVAs with group (controls and children who stutter) as a between-subjects factor, and hemisphere (left and right), and channel (9) as within-subject factors. In cases where the assumption of sphericity was violated, Huynh-Feldt corrected degrees of freedom and Appendices