fMRIPrep: a robust preprocessing pipeline for functional MRI.

Preprocessing of functional magnetic resonance imaging (fMRI) involves numerous steps to clean and standardize the data before statistical analysis. Generally, researchers create ad hoc preprocessing workflows for each dataset, building upon a large inventory of available tools. The complexity of these workflows has snowballed with rapid advances in acquisition and processing. We introduce fMRIPrep, an analysis-agnostic tool that addresses the challenge of robust and reproducible preprocessing for fMRI data. fMRIPrep automatically adapts a best-in-breed workflow to the idiosyncrasies of virtually any dataset, ensuring high-quality preprocessing without manual intervention. By introducing visual assessment checkpoints into an iterative integration framework for software testing, we show that fMRIPrep robustly produces high-quality results on a diverse fMRI data collection. Additionally, fMRIPrep introduces less uncontrolled spatial smoothness than observed with commonly used preprocessing tools. fMRIPrep equips neuroscientists with an easy-to-use and transparent preprocessing workflow, which can help ensure the validity of inference and the interpretability of results.

A global analysis of the reconstitution of PTEN function by translational readthrough of PTEN pathogenic premature termination codons.

The PTEN tumor suppressor gene is mutated with high incidence in tumors and in the germline of patients with cancer predisposition or with macrocephaly associated with autism. PTEN nonsense mutations generating premature termination codons (PTC) and producing nonfunctional truncated PTEN proteins are frequent in association with human disease. However, there are no studies addressing the restoration of full-length PTEN proteins from the PTC-mutated PTEN gene by translational readthrough. Here, we have performed a global translational and functional readthrough analysis of the complete collection of PTEN PTC somatic or hereditary mutations found in tumors or in the germline of patients (disease-associated PTEN PTCome), and we set standards for the analysis of the potential of readthrough functional reconstitution in disease-relevant genes. Our analysis indicates that prevalent pathogenic PTEN PTC mutations are susceptible to PTEN functional restoration in response to readthrough-inducing compounds. Comprehensive readthrough analyses of disease-associated PTComes will be valuable tools for the implementation of readthrough-based precision interventions in specific groups of patients.

Structure-Function Connectomics Reveals Aberrant Developmental Trajectory Occurring at Preadolescence in the Autistic Brain.

Accumulating neuroimaging evidence shows that age estimation obtained from brain connectomics reflects the level of brain maturation along with neural development. It is well known that autism spectrum disorder (ASD) alters neurodevelopmental trajectories of brain connectomics, but the precise relationship between chronological age (ChA) and brain connectome age (BCA) during development in ASD has not been addressed. This study uses neuroimaging data collected from 50 individuals with ASD and 47 age- and gender-matched typically developing controls (TDCs; age range: 5-18 years). Both functional and structural connectomics were assessed using resting-state functional magnetic resonance imaging and diffusion tensor imaging data from the Autism Brain Imaging Data Exchange repository. For each participant, BCA was estimated from structure-function connectomics through linear support vector regression. We found that BCA matched well with ChA in TDC children and adolescents, but not in ASD. In particular, our findings revealed that individuals with ASD exhibited accelerated brain maturation in youth, followed by a delay of brain development starting at preadolescence. Our results highlight the critical role of BCA in understanding aberrant developmental trajectories in ASD and provide the new insights into the pathophysiological mechanisms of this disorder.

PTEN Activity Defines an Axis for Plasticity at Cortico-Amygdala Synapses and Influences Social Behavior.

Phosphatase and tensin homolog on chromosome 10 (PTEN) is a tumor suppressor and autism-associated gene that exerts an important influence over neuronal structure and function during development. In addition, it participates in synaptic plasticity processes in adulthood. As an attempt to assess synaptic and developmental mechanisms by which PTEN can modulate cognitive function, we studied the consequences of 2 different genetic manipulations in mice: presence of additional genomic copies of the Pten gene (Ptentg) and knock-in of a truncated Pten gene lacking its PDZ motif (Pten-ΔPDZ), which is required for interaction with synaptic proteins. Ptentg mice exhibit substantial microcephaly, structural hypoconnectivity, enhanced synaptic depression at cortico-amygdala synapses, reduced anxiety, and intensified social interactions. In contrast, Pten-ΔPDZ mice have a much more restricted phenotype, with normal synaptic connectivity, but impaired synaptic depression at cortico-amygdala synapses and virtually abolished social interactions. These results suggest that synaptic actions of PTEN in the amygdala contribute to specific behavioral traits, such as sociability. Also, PTEN appears to function as a bidirectional rheostat in the amygdala: reduction in PTEN activity at synapses is associated with less sociability, whereas enhanced PTEN activity accompanies hypersocial behavior.

Structure-function multi-scale connectomics reveals a major role of the fronto-striato-thalamic circuit in brain aging.

Physiological aging affects brain structure and function impacting morphology, connectivity, and performance. However, whether some brain connectivity metrics might reflect the age of an individual is still unclear. Here, we collected brain images from healthy participants (N = 155) ranging from 10 to 80 years to build functional (resting state) and structural (tractography) connectivity matrices, both data sets combined to obtain different connectivity features. We then calculated the brain connectome age-an age estimator resulting from a multi-scale methodology applied to the structure-function connectome, and compared it to the chronological age (ChA). Our results were twofold. First, we found that aging widely affects the connectivity of multiple structures, such as anterior cingulate and medial prefrontal cortices, basal ganglia, thalamus, insula, cingulum, hippocampus, parahippocampus, occipital cortex, fusiform, precuneus, and temporal pole. Second, we found that the connectivity between basal ganglia and thalamus to frontal areas, also known as the fronto-striato-thalamic (FST) circuit, makes the major contribution to age estimation. In conclusion, our results highlight the key role played by the FST circuit in the process of healthy aging. Notably, the same methodology can be generally applied to identify the structural-functional connectivity patterns correlating to other biomarkers than ChA.

DUSP5 expression associates with poor prognosis in human neuroblastoma.

Regulation of growth and differentiation of neuroblastoma (NB) cells is the rational of some maintenance therapies for high-risk NB. MAP kinase phosphatases (MKPs) are potential physiologic regulators of neuronal differentiation and survival, but their expression patterns in NB are scarcely known. Here, an expression analysis of the MKP family has been performed using human NB tumor samples and human NB cell lines (SH-SY5Y, SMS-KCNR, and IMR-32) undergoing retinoic acid (RA)-induced differentiation or subjected to stimuli that activate the MAPK ERK1/2 pathway. We have identified candidate MKPs that could modulate differentiation and growth of NB cells. pERK1/2 high expression correlated with high expression of the MKP DUSP5 in NB tumors, and was associated with poor prognosis. ERK1/2 activation on SH-SY5Y cells was accompanied by increased cell proliferation, and correlated with the expression levels of DUSP5. Accordingly, siRNA knock-down of DUSP5 augmented proliferation of SH-SY5Y cells. Our findings provide insights into the dynamic expression of MKPs in NB cells, disclose DUSP5 as a potential marker of NB poor prognosis, and suggest a role for DUSP5 in limiting ERK1/2-mediated NB proliferation.

Proper name retrieval and structural integrity of cerebral cortex in midlife: A cross-sectional study.

There is currently little understanding on whether retrieval of proper names differs in midlife compared to young adulthood and if so, whether the age differences in this ability are associated with differences in structural integrity of the cerebral cortex. To answer these questions, we studied retrieval of proper names in 115 cognitively healthy middle-aged persons (49.7, ±3.2), comparing their performance on a tip-of-the-tongue (TOT) task with that of 68 young persons (25.4, ±3.5) from the Cam-Can data repository (http://www.mrc-cbu.cam.ac.uk/datasets/camcan/). Grey matter (GM) density and cortical thickness were used as indices of structural integrity of the cerebral cortex. The middle-aged (MA) group experienced more TOTs during proper names retrieval than young adults (YA), (t = 3.789, p < .005) and had considerably less GM density and cortical thickness across a range of brain areas bilaterally. Small clusters in left BA 45 and right BA 44 (cortical thickness) and in right BA 40 (volumetry) revealed group differences when accounting for TOTs. However, we observed no correlations between MA's TOT scores and GM volumes or cortical thickness of the brain regions typically reported as implicated in retrieval of proper names: left anterior temporal lobe, left insula, and left superior and middle temporal gyri.

Interaction Information Along Lifespan of the Resting Brain Dynamics Reveals a Major Redundant Role of the Default Mode Network.

Interaction Information (II) generalizes the univariate Shannon entropy to triplets of variables, allowing the detection of redundant (R) or synergetic (S) interactions in dynamical networks. Here, we calculated II from functional magnetic resonance imaging data and asked whether R or S vary across brain regions and along lifespan. Preserved along lifespan, we found high overlapping between the pattern of high R and the default mode network, whereas high values of S were overlapping with different cognitive domains, such as spatial and temporal memory, emotion processing and motor skills. Moreover, we have found a robust balance between R and S among different age intervals, indicating informational compensatory mechanisms in brain networks.

Vitronectin and dermcidin serum levels predict the metastatic progression of AJCC I-II early-stage melanoma.

Like many cancers, an early diagnosis of melanoma is fundamental to ensure a good prognosis, although an important proportion of stage I-II patients may still develop metastasis during follow-up. The aim of this work was to discover serum biomarkers in patients diagnosed with primary melanoma that identify those at a high risk of developing metastasis during the follow-up period. Proteomic and mass spectrophotometry analysis was performed on serum obtained from patients who developed metastasis during the first years after surgery for primary tumors and compared with that from patients who remained disease-free for more than 10 years after surgery. Five proteins were selected for validation as prognostic factors in 348 melanoma patients and 100 controls by ELISA: serum amyloid A and clusterin; immune system proteins; the cell adhesion molecules plakoglobin and vitronectin and the antimicrobial protein dermcidin. Compared to healthy controls, melanoma patients have high serum levels of these proteins at the moment of melanoma diagnosis, although the specific values were not related to the histopathological stage of the tumors. However, an analysis based on classification together with multivariate statistics showed that tumor stage, vitronectin and dermcidin levels were associated with the metastatic progression of patients with early-stage melanoma. Although melanoma patients have increased serum dermcidin levels, the REPTree classifier showed that levels of dermcidin <2.98 µg/ml predict metastasis in AJCC stage II patients. These data suggest that vitronectin and dermcidin are potent biomarkers of prognosis, which may help to improve the personalized medical care of melanoma patients and their survival.

Snail heterogeneity in clear cell renal cell carcinoma.

BACKGROUND: Intratumor heterogeneity may be responsible of the unpredictable aggressive clinical behavior that some clear cell renal cell carcinomas display. This clinical uncertainty may be caused by insufficient sampling, leaving out of histological analysis foci of high grade tumor areas. Although molecular approaches are providing important information on renal intratumor heterogeneity, a focus on this topic from the practicing pathologist' perspective is still pending. METHODS: Four distant tumor areas of 40 organ-confined clear cell renal cell carcinomas were selected for histopathological and immunohistochemical evaluation. Tumor size, cell type (clear/granular), Fuhrman's grade, Staging, as well as immunostaining with Snail, ZEB1, Twist, Vimentin, E-cadherin, ß-catenin, PTEN, p-Akt, p110α, and SETD2, were analyzed for intratumor heterogeneity using a classification and regression tree algorithm. RESULTS: Cell type and Fuhrman's grade were heterogeneous in 12.5 and 60 % of the tumors, respectively. If cell type was homogeneous (clear cell) then the tumors were low-grade in 88.57 % of cases. Immunostaining heterogeneity was significant in the series and oscillated between 15 % for p110α and 80 % for Snail. When Snail immunostaining was homogeneous the tumor was histologically homogeneous in 100 % of cases. If Snail was heterogeneous, the tumor was heterogeneous in 75 % of the cases. Average tumor diameter was 4.3 cm. Tumors larger than 3.7 cm were heterogeneous for Vimentin immunostaining in 72.5 % of cases. Tumors displaying negative immunostaining for both ZEB1 and Twist were low grade in 100 % of the cases. CONCLUSIONS: Intratumor heterogeneity is a common event in clear cell renal cell carcinoma, which can be monitored by immunohistochemistry in routine practice. Snail seems to be particularly useful in the identification of intratumor heterogeneity. The suitability of current sampling protocols in renal cancer is discussed.

Induction of Translational Readthrough on Protein Tyrosine Phosphatases Targeted by Premature Termination Codon Mutations in Human Disease.

Nonsense mutations generating premature termination codons (PTCs) in various genes are frequently associated with somatic cancer and hereditary human diseases since PTCs commonly generate truncated proteins with defective or altered function. Induced translational readthrough during protein biosynthesis facilitates the incorporation of an amino acid at the position of a PTC, allowing the synthesis of a complete protein. This may evade the pathological effect of the PTC mutation and provide new therapeutic opportunities. Several protein tyrosine phosphatases (PTPs) genes are targeted by PTC in human disease, the tumor suppressor PTEN being the more prominent paradigm. Here, using PTEN and laforin as examples, two PTPs from the dual-specificity phosphatase subfamily, we describe methodologies to analyze in silico the distribution and frequency of pathogenic PTC in PTP genes. We also summarize laboratory protocols and technical notes to study the induced translational readthrough reconstitution of the synthesis of PTP targeted by PTC in association with disease in cellular models.

Open datasets and code for multi-scale relations on structure, function and neuro-genetics in the human brain.

The human brain is an extremely complex network of structural and functional connections that operate at multiple spatial and temporal scales. Investigating the relationship between these multi-scale connections is critical to advancing our comprehension of brain function and disorders. However, accurately predicting structural connectivity from its functional counterpart remains a challenging pursuit. One of the major impediments is the lack of public repositories that integrate structural and functional networks at diverse resolutions, in conjunction with modular transcriptomic profiles, which are essential for comprehensive biological interpretation. To mitigate this limitation, our contribution encompasses the provision of an open-access dataset consisting of derivative matrices of functional and structural connectivity across multiple scales, accompanied by code that facilitates the investigation of their interrelations. We also provide additional resources focused on neuro-genetic associations of module-level network metrics, which present promising opportunities to further advance research in the field of network neuroscience, particularly concerning brain disorders.

Heart-brain synchronization breakdown in Parkinson's disease.

Heart rate variability (HRV) abnormalities are potential early biomarkers in Parkinson's disease (PD) but their relationship with central autonomic network (CAN) activity is not fully understood. We analyzed the synchronization between HRV and brain activity in 31 PD patients and 21 age-matched healthy controls using blood-oxygen-level-dependent (BOLD) signals from resting-state functional brain MRI and HRV metrics from finger plethysmography recorded for 7.40 min. We additionally quantified autonomic symptoms (SCOPA-AUT) and objective autonomic cardiovascular parameters (blood pressure and heart rate) during deep breathing, Valsalva, and head-up tilt, which were used to classify the clinical severity of dysautonomia. We evaluated HRV and BOLD signals synchronization (HRV-BOLD-sync) with Pearson lagged cross-correlations and Fisher's statistics for combining window-length-dependent HRV-BOLD-Sync Maps and assessed their association with clinical dysautonomia. HRV-BOLD-sync was lower significantly in PD than in controls in various brain regions within CAN or in networks involved in autonomic modulation. Moreover, heart-brain synchronization index (HBSI), which quantifies heart-brain synchronization at a single-subject level, showed an inverse exposure-response relationship with dysautonomia severity, finding the lowest HBSI in patients with severe dysautonomia, followed by moderate, mild, and, lastly, controls. Importantly, HBSI was associated in PD, but not in controls, with Valsalva pressure recovery time (sympathetic), deep breathing E/I ratio (cardiovagal), and SCOPA-AUT. Our findings support the existence of heart-brain de-synchronization in PD with an impact on clinically relevant autonomic outcomes.

Spatial characterization of the effect of age and sex on macular layer thicknesses and foveal pit morphology.

Characterizing the effect of age and sex on macular retinal layer thicknesses and foveal pit morphology is crucial to differentiating between natural and disease-related changes. We applied advanced image analysis techniques to optical coherence tomography (OCT) to: 1) enhance the spatial description of age and sex effects, and 2) create a detailed open database of normative retinal layer thickness maps and foveal pit shapes. The maculae of 444 healthy subjects (age range 21-88) were imaged with OCT. Using computational spatial data analysis, thickness maps were obtained for retinal layers and averaged into 400 (20 x 20) sectors. Additionally, the geometry of the foveal pit was radially analyzed by computing the central foveal thickness, rim height, rim radius, and mean slope. The effect of age and sex on these parameters was analyzed with multiple regression mixed-effects models. We observed that the overall age-related decrease of the total retinal thickness (TRT) (-1.1% per 10 years) was mainly driven by the ganglion cell-inner plexiform layer (GCIPL) (-2.4% per 10 years). Both TRT and GCIPL thinning patterns were homogeneous across the macula when using percentual measurements. Although the male retina was 4.1 µm thicker on average, the greatest differences were mainly present for the inner retinal layers in the inner macular ring (up to 4% higher TRT than in the central macula). There was an age-related decrease in the rim height (1.0% per 10 years) and males had a higher rim height, shorter rim radius, and steeper mean slope. Importantly, the radial analysis revealed that these changes are present and relatively uniform across angular directions. These findings demonstrate the capacity of advanced analysis of OCT images to enhance the description of the macula. This, together with the created dataset, could aid the development of more accurate diagnosis models for macular pathologies.

A Controlled Thermoalgesic Stimulation Device for Exploring Novel Pain Perception Biomarkers.

OBJECTIVE: To develop a new device for identifying physiological markers of pain perception by reading the brain's electrical activity and hemodynamic interactions while applying thermoalgesic stimulation. METHODS: We designed a compact prototype that generates well-controlled thermal stimuli using a computer-driven Peltier cell while simultaneously capturing electroencephalography (EEG) and photoplethysmography (PPG) signals. The study was performed on 35 healthy subjects (mean age 30.46 years, SD 4.93 years; 20 males, 15 females). We first determined the heat pain threshold (HPT) for each subject, defined as the maximum temperature that the subject can withstand when the Peltier cell gradually increased the temperature. Next, we defined the painful condition as the one occurring at temperature equal to 90% of the HPT, comparing this to the no-pain state (control) in the absence of thermoalgesic stimulation. RESULTS: Both the one-dimensional and the two-dimensional spectral entropy (SE) obtained from both the EEG and PPG signals differentiated the condition of pain. In particular, the SE for PPG was significantly reduced in association with pain, while the SE for EEG increased slightly. Moreover, significant discrimination occurred within a specific range of frequencies, 26-30 Hz for EEG and about 5-10 Hz for PPG. CONCLUSION: Hemodynamics, brain dynamics and their interactions can discriminate thermal pain perception. SIGNIFICANCE: The possibility of monitoring on-line variations in thermal pain perception using a similar device and algorithms may be of interest to study different pathologies that affect the peripheral nervous system, such as small fiber neuropathies, fibromyalgia or painful diabetic neuropathy.

Foveal Remodeling of Retinal Microvasculature in Parkinson's Disease.

BACKGROUND: Retinal microvascular alterations have been previously described in Parkinson's disease (PD) patients using optical coherence tomography angiography (OCT-A). However, an extensive description of retinal vascular morphological features, their association with PD-related clinical variables and their potential use as diagnostic biomarkers has not been explored. METHODS: We performed a cross-sectional study including 49 PD patients (87 eyes) and 40 controls (73 eyes). Retinal microvasculature was evaluated with Spectralis OCT-A and cognitive status with Montreal Cognitive Assessment. Unified PD Rating Scale and disease duration were recorded in patients. We extracted microvascular parameters from superficial and deep vascular plexuses of the macula, including the area and circularity of foveal avascular zone (FAZ), skeleton density, perfusion density, vessel perimeter index, vessel mean diameter, fractal dimension (FD) and lacunarity using Python and MATLAB. We compared the microvascular parameters between groups and explored their association with thickness of macular layers and clinical outcomes. Data were analyzed with General Estimating Equations (GEE) and adjusted for age, sex, and hypertension. Logistic regression GEE models were fitted to predict diagnosis of PD versus controls from microvascular, demographic, and clinical data. The discrimination ability of models was tested with receiver operating characteristic curves. RESULTS: FAZ area was significantly smaller in patients compared to controls in superficial and deep plexuses, whereas perfusion density, skeleton density, FD and lacunarity of capillaries were increased in the foveal zone of PD. In the parafovea, microvascular parameters of superficial plexus were associated with ganglion cell-inner plexiform layer thickness, but this was mainly driven by PD with mild cognitive impairment. No such associations were observed in controls. FAZ area was negatively associated with cognition in PD (non-adjusted models). Foveal lacunarity, combined with demographic and clinical confounding factors, yielded an outstanding diagnostic accuracy for discriminating PD patients from controls. CONCLUSION: Parkinson's disease patients displayed foveal microvascular alterations causing an enlargement of the vascular bed surrounding FAZ. Parafoveal microvascular alterations were less pronounced but were related to inner retinal layer thinning. Retinal microvascular abnormalities helped discriminating PD from controls. All this supports OCT-A as a potential non-invasive biomarker to reveal vascular pathophysiology and improve diagnostic accuracy in PD.

Dorsal White Matter Integrity and Name Retrieval in Midlife.

BACKGROUND: Recent findings on retrieval of proper names in cognitively healthy middle- aged persons indicate that Tip-Of-The-Tongue (TOT) states occurring during proper name retrieval implicate inferior frontal (BA 44) and parietal (BA 40) cortical areas. Such findings give rise to the possibility that anatomical connectivity via dorsal white matter may be associated with difficulties in name retrieval in midlife. OBJECTIVES & METHOD: Using Diffusion Tensor Imaging, we examined in vivo microstructural properties of white matter in 72 cognitively healthy Middle-Aged (MA) and 59 Young Adults (YA), comparing their naming abilities as well as testing, for possible associations between dorsal white matter integrity and naming abilities in the MA group. RESULTS: The MA group was better in retrieving correct names (U = 1525.5, p = .006), but they also retrieved more incorrect names than YA believing they had retrieved the correct ones (U = 1265.5, p < .001). Furthermore, despite being more familiar with the tested names than YA (U = 930, p < .001), MA experienced significantly more TOTs relative to YA (U = 1498.5, p = .004). Tract-based spatial statistics showed significant group differences in values of fractional anisotropy (FA), mean diffusivity, axial diffusivity, radial diffusivity, and mode of anisotropy in a range of white matter tracts. In the MA group, FA values in the right Superior Longitudinal Fasciculus (SLF) were positively correlated with "don't know" scores (rs = .287, p = .014). CONCLUSION: The association of SLF integrity and name retrieval ability in midlife indicates a need to revisit the models of name retrieval that posit no role for dorsal white matter in proper name retrieval.

Protein tyrosine phosphatase PTPN1 modulates cell growth and associates with poor outcome in human neuroblastoma.

BACKGROUND: Protein tyrosine phosphatases (PTPs) regulate neuronal differentiation and survival, but their expression patterns and functions in human neuroblastoma (NB) are scarcely known. Here, we have investigated the function and expression of the non-receptor PTPN1 on human NB cell lines and human NB tumor samples. MATERIAL/METHODS: NB tumor samples from 44 patients were analysed by immunohistochemistry using specific antibodies against PTPN1, PTPRH, PTPRZ1, and PTEN. PTPN1 knock-down, cell proliferation and tyrosine phosphorylation analyses, and RT-qPCR mRNA expression was assessed on SH-SY5Y, SMS-KCNR, and IMR-32 human NB cell lines. RESULTS: Knock-down of PTPN1 in SH-SY5Y NB cells resulted in increased tyrosine phosphorylation and cell proliferation. Retinoic acid-mediated differentiation of NB cell lines did not affect PTPN1 mRNA expression, as compared with other PTPs. Importantly, PTPN1 displayed high expression on NB tumors in association with metastasis and poor prognosis. CONCLUSIONS: Our results identify PTPN1 as a candidate regulator of NB cell growth and a potential NB prognostic biomarker.

Multisite tumor sampling enhances the detection of intratumor heterogeneity at all different temporal stages of tumor evolution.

Intratumor heterogeneity (ITH) is an inherent process of tumor development that has received much attention in previous years, as it has become a major obstacle for the success of targeted therapies. ITH is also temporally unpredictable across tumor evolution, which makes its precise characterization even more problematic since detection success depends on the precise temporal snapshot at which ITH is analyzed. New and more efficient strategies for tumor sampling are needed to overcome these difficulties which currently rely entirely on the pathologist's interpretation. Recently, we showed that a new strategy, the multisite tumor sampling, works better than the routine sampling protocol for the ITH detection when the tumor time evolution was not taken into consideration. Here, we extend this work and compare the ITH detections of multisite tumor sampling and routine sampling protocols across tumor time evolution, and in particular, we provide in silico analyses of both strategies at early and late temporal stages for four different models of tumor evolution (linear, branched, neutral, and punctuated). Our results indicate that multisite tumor sampling outperforms routine protocols in detecting ITH at all different temporal stages of tumor evolution. We conclude that multisite tumor sampling is more advantageous than routine protocols in detecting intratumor heterogeneity.