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Background: Dual HER2-inhibition combined with neoadjuvant chemotherapy allows increased pathological complete response (pCR) rate. However, with the addition of new agents, there is a growing need to select patients to minimise overtreatment. Herein, we evaluated the 41-gene classifier TRAR to predict pCR to anti-HER2 therapies in the NeoALTTO trial. Patients and methods: Gene expression data were obtained using RNA from 226 pretreatment tumour biopsies. Logistic regression analysis and the area under the receiver operating characteristic (ROC) curve (AUC) were used to evaluate TRAR predictive and discriminatory capabilities. Results: TRAR levels were associated with pCR (odds ratio, OR: 0.25, 95% confidence interval, CI: 0.15–0.42). The ROC analysis showed AUC values of 0.73 (95% CI: 0.67–0.80) overall; 0.70 (0.59–0.81) and 0.71 (0.62–0.80) for positive and negative oestrogen receptor cases and 0.74 (0.60–0.88), 0.76 (0.65–0.87) and 0.71 (0.59–0.83) for trastuzumab, lapatinib and combined treatment arms, respectively. TRAR provided reliable predictive information beyond established clinicopathological variables (OR: 0.26, 95% CI: 0.14–0.47). Furthermore, addition of TRAR to these variables provided greater predictive capability than the addition of PAM50: AUC 0.78 (0.72–0.84) versus 0.74 (0.67–0.81), p = 0.04. Conclusion: TRAR represents a promising tool to refine the ability to identify patients sensitive to anti-HER2 (including trastuzumab-only)-based therapy and eligible for de-escalated treatment strategies., SCOPUS: ar.j, info:eu-repo/semantics/published

Purpose: Little is known about the efficacy of HER2-targeted therapy in patients with breast cancer showing different HER2-pathway dependence and immune phenotypes. Herein, we report a NeoALTTO exploratory analysis evaluating the clinical value of 22 types of tumor-infiltrating immune cells by CIBERSORT and 5 immune-related metagenes in the overall patient population, and in subgroups defined by the TRAR classifier as HER2-addicted (TRAR-low) or not (TRAR-high). Patients and Methods: Association of baseline TRAR, immune-related metagenes, and CIBERSORT data with pathologic complete response (pCR) and event-free survival (EFS) were assessed using logistic and Cox regression models. Corrections for multiple testing were performed by the Bonferroni method. Results: A total of 226 patients were analyzed: 80 (35%) achieved a pCR, and 64 (28%) experienced a relapse with a median follow-up of 6.7 (interquartile range 6.1–6.8) years; 108 cases were classified as TRAR-low, and 118 TRAR-high. Overall, γδ T-cell fraction [OR = 2.69; 95% confidence interval (CI), 1.40–5.18], and no immune-related metagenes were predictive of pCR. Notably, lymphocyte-specific kinase (LCK) predicted pCR to combination (OR = 2.53; 95% CI, 1.12–5.69), but not to single-agent trastuzumab or lapatinib [OR = 0.74; 95% CI, 0.45–1.22 (Pinteraction = 0.01)]. Integrating LCK with γδ T cells in a multivariate model added to the discriminatory capability of clinical and molecular variables with a shift in AUC from 0.80 (95% CI, 0.74–0.86) to 0.83 (95% CI, 0.78–0.89). In TRAR-low cases, activated mast cells, IFN and MHCII were reduced, and STAT1, HCK1, and γδ T cells were associated with pCR. STAT1 was broadly associated with improved EFS regardless of pCR, and nodal status in overall (HR = 0.68; 95% CI, 0.49–0.94) and in TRAR-low cases (HR = 0.50; 95% CI, 0.30–0.86). Conclusions: Immuno-phenotyping holds the promise to complement current predictive models in HER2-positive breast cancer and to assist in new therapeutic development.

Germline pathogenic variants (PVs) in the BRCA1 or BRCA2 genes cause high breast cancer risk. Recurrent or founder PVs have been described worldwide including some in the Bergamo province in Northern Italy. The aim of this study was to compare the BRCA1/2 PV spectra of the Bergamo and of the general Italian populations. We retrospectively identified at five Italian centers 1019 BRCA1/2 PVs carrier individuals affected with breast cancer and representative of the heterogeneous national population. Each individual was assigned to the Bergamo or non-Bergamo cohort based on self-reported birthplace. Our data indicate that the Bergamo BRCA1/2 PV spectrum shows less heterogeneity with fewer different variants and an average higher frequency compared to that of the rest of Italy. Consistently, four PVs explained about 60% of all carriers. The majority of the Bergamo PVs originated locally with only two PVs clearly imported. The Bergamo BRCA1/2 PV spectrum appears to be private. Hence, the Bergamo population would be ideal to study the disease risk associated with local PVs in breast cancer and other disease-causing genes. Finally, our data suggest that the Bergamo population is a genetic isolate and further analyses are warranted to prove this notion.

Face and body perception is mediated by configural mechanisms, which allow the perception of these stimuli as a whole, rather than the sum of individual parts. Indirect measures of configural processing in visual cognition are the face and body inversion effects (FIE and BIE), which refer to the drop in performance when these stimuli are perceived upside-down. Albeit FIE and BIE have been well characterized at the behavioral level, much still needs to be understood in terms of the neurophysiological correlates of these effects. Thus, in the current study, the brain’s electrical activity has been recorded by a 128 channel electroencephalogram (EEG) in 24 healthy participants while perceiving (upright and inverted) faces, bodies and houses. EEG data were analyzed in both the time domain (i.e., event-related potentials—ERPs) and the frequency domain [i.e., induced theta (5–7 Hz) and gamma (28–45 Hz) oscillations]. ERPs amplitude results showed increased N170 amplitude for inverted faces and bodies (compared to the same stimuli presented in canonical position) but not for houses. ERPs latency results showed delayed N170 components for inverted (vs. upright) faces, houses, but not bodies. Spectral analysis of induced oscillations indicated physiological FIE and BIE; that is decreased gamma-band synchronization over right occipito-temporal electrodes for inverted (vs. upright) faces, and increased bilateral frontoparietal theta-band synchronization for inverted (vs. upright) faces. Furthermore, increased left occipito-temporal and right frontal theta-band synchronization for upright (vs. inverted) bodies was found. Our findings, thus, demonstrate clear differences in the neurophysiological correlates of face and body perception. The neurophysiological FIE suggests disruption of feature binding processes (decrease in occipital gamma oscillations for inverted faces), together with enhanced feature-based attention (increase in frontoparietal theta oscillations for inverted faces). In contrast, the BIE may suggest that structural encoding for bodies is mediated by the first stages of configural processing (decrease in occipital theta oscillations for inverted bodies).

Circulating microRNA (ct-miRNAs) are able to identify patients with differential response to HER2-targeted therapy. However, their dynamics are largely unknown. We assessed 752 miRNAs from 52 NeoALTTO patients with plasma pairs prior and two weeks after trastuzumab. Increased levels of ct-miR-148a-3p and ct-miR-374a-5p were significantly associated with pathological complete response (pCR) (p = 0.008 and 0.048, respectively). At a threshold &ge, the upper limit of the 95%CI of the mean difference, pCR resulted 45% (95%CI 24%&ndash, 68%), and 44% (95%CI 22%&ndash, 69%) for ct-miR-148a-3p and ct-miR-374a-5p, respectively. Notably, ct-miR-148a-3p retained its predictive value (OR 3.42, 95%CI 1.23&ndash, 9.46, p = 0.018) in bivariate analysis along with estrogen receptor status. Combined information from ct-miR-148a-3p and ct-miR140-5p, which we previously reported to identify trastuzumab-responsive patients, resulted in greater predictive capability over each other, with pCR of 54% (95%CI 25%&ndash, 81%) and 0% (95%CI 0%&ndash, 31%) in ct-miR-148a/ct-miR-140-5p high/present and low/absent, respectively. GO and KEGG analyses showed common enriched terms between the targets of these ct-miRNAs, including cell metabolism regulation, AMPK and MAPK signaling, and HCC progression. In conclusion, early modulated ct-miR-148-3p may inform on the functional processes underlying treatment response, integrate the information from already available predictive biomarkers, and identify patients likely to respond to single agent trastuzumab-based neoadjuvant therapy.

Motor deficits are common outcomes of neurological conditions such as stroke. In order to design personalised motor rehabilitation programmes such as robot-assisted therapy, it would be advantageous to predict how a patient might respond to such treatment. Spontaneous neural activity has been observed to predict differences in the ability to learn a new motor behaviour in both healthy and stroke populations. This study investigated whether spontaneous resting-state functional connectivity could predict the degree of motor adaptation of right (dominant) upper limb reaching in response to a robot-mediated force field. Spontaneous neural activity was measured using resting-state electroencephalography (EEG) in healthy adults before a single session of motor adaptation. The degree of beta frequency (β; 15–25 Hz) resting-state functional connectivity between contralateral electrodes overlying the left primary motor cortex (M1) and the anterior prefrontal cortex (aPFC) could predict the subsequent degree of motor adaptation. This result provides novel evidence for the functional significance of resting-state synchronization dynamics in predicting the degree of motor adaptation in a healthy sample. This study constitutes a promising first step towards the identification of patients who will likely gain most from using robot-mediated upper limb rehabilitation training based on simple measures of spontaneous neural activity.

BACKGROUND: As a neoadjuvant regimen for HER2-positive early breast cancer (BC), the use of two HER2-directed agents is more effective in producing pathological complete (pCR) responses than trastuzumab alone. Nevertheless, highly effective dual anti-HER2 combination may be unnecessary in patients who already benefit from a single agent. We previously reported that our 41-gene TRAR score is an accurate predictor of response to trastuzumab, with low scores being predictive of response to trastuzumab and favorable prognosis (Triulzi T. et al., 2015). PATIENTS AND METHODS: Tissue specimens from HER2-positive BC patients of Neo-ALTTO trial who received neoadjuvant trastuzumab and/or lapatinib plus paclitaxel were included in this study. Analysing RNA from fresh tissue using the 41-gene signature test, the area under the ROC curve (AUC) and the corresponding 95% confidence interval was computed to evaluate the predictive ability of TRAR score with respect to pCR, the primary endpoint of Neo-ALTTO. The prognostic role of TRAR score was investigated using a Cox regression model in univariate fashion. The patterns of Event Free Survival (EFS) according to the dichotomized TRAR score were estimated using the Kaplan–Meier method. RESULTS: The TRAR score was assessed for 226 of the 455 (49.7%) patients enrolled in the Neo-ALTTO study: 136 (60%) presented with T2 tumors, 188 (83%) with N0/1 and 128 (56.6%) with estrogen receptor negative BC. In details, basal TRAR score was available for 69, 79 and 78 patients assigned to neoadjuvant trastuzumab, lapatinib, and their combination, respectively. Overall, patients achieving a pCR showed significantly lower levels of TRAR score than those with residual disease (p CONCLUSION: Overall, we show that our 41-gene signature is accurate in predicting patient response to neoadjuvant HER2 targeted therapy in terms of pCR. In particular, low levels of TRAR score can identify a HER2-positive breast cancer subgroup highly responsive to trastuzumab as monotherapy for whom combination with other HER2-targeted drugs does not appear justified and may be one tool used for exploring de-escalating strategies without sacrificing outcomes. Citation Format: Di Cosimo S, Triulzi T, De Cecco L, Pizzamiglio S, de Azambuja E, Fumagalli D, Pusztai L, Harbeck N, Izquierdo M, de la Pena L, Huober J, Baselga J, Piccart M, Verderio P, Tagliabue E. Identifying clinically relevant subgroups of women with HER2-positive breast cancer: An analysis of Neo-ALTTO using the 41-gene TRAR score [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-03.

Background Gait impairments during real-world locomotion are common in neurological diseases. However, very little is currently known about the neural correlates of walking in the real world and on which regions of the brain are involved in regulating gait stability and performance. As a first step to understanding how neural control of gait may be impaired in neurological conditions such as Parkinson’s disease, we investigated how regional brain activation might predict walking performance in the urban environment and whilst engaging with secondary tasks in healthy subjects. Methods We recorded gait characteristics including trunk acceleration and brain activation in 14 healthy young subjects whilst they walked around the university campus freely (single task), while conversing with the experimenter and while texting with their smartphone. Neural spectral power density (PSD) was evaluated in three brain regions of interest, namely the pre-frontal cortex (PFC) and bilateral posterior parietal cortex (right/left PPC). We hypothesized that specific regional neural activation would predict trunk acceleration data obtained during the different walking conditions. Results Vertical trunk acceleration was predicted by gait velocity and left PPC theta (4–7 Hz) band PSD in single-task walking (R-squared = 0.725, p = 0.001) and by gait velocity and left PPC alpha (8–12 Hz) band PSD in walking while conversing (R-squared = 0.727, p = 0.001). Medio-lateral trunk acceleration was predicted by left PPC beta (15–25 Hz) band PSD when walking while texting (R-squared = 0.434, p = 0.010). Conclusions We suggest that the left PPC may be involved in the processes of sensorimotor integration and gait control during walking in real-world conditions. Frequency-specific coding was operative in different dual tasks and may be developed as biomarkers of gait deficits in neurological conditions during performance of these types of, now commonly undertaken, dual tasks.

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses – [1]; Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience – [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience – [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of pre-analytical phase of blood samples on genomic DNA quality – [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis.Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

Background In order to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time. Methods SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. Results/conclusions 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on pre-analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent pre-analytical conditions.

Purpose: To investigate the potential of circulating-miRNAs (ct-miRNA) as noninvasive biomarkers to predict the efficacy of single/dual HER2-targeted therapy in the NeoALTTO study. Experimental Design: Patients with plasma samples at baseline (T0) and/or after 2 weeks (T1) of treatment were randomized into training (n = 183) and testing (n = 246) sets. RT-PCR–based high-throughput miRNA profiling was employed in the training set. After normalization, ct-miRNAs associated with pathologic complete response (pCR) were identified by univariate analysis. Multivariate logistic regression models were implemented to generate treatment-specific signatures at T0 and T1, which were evaluated by RT-PCR in the testing set. Event-free survival (EFS) according to ct-miRNA signatures was estimated by Kaplan–Meier method and Cox regression model. Results: In the training set, starting from 51 ct-miRNAs associated with pCR, six signatures with statistically significant predictive capability in terms of area under the ROC curve (AUC) were identified. Four signatures were confirmed in the testing set: lapatinib at T0 and T1 [AUC 0.86; 95% confidence interval (CI), 0.73–0.98 and 0.71 (0.55–0.86)], respectively; trastuzumab at T1 (0.81; 0.70–0.92); lapatinib + trastuzumab at T1 (0.67; 0.51–0.83). These signatures were confirmed predictive after adjusting for known variables, including estrogen receptor status. ct-miRNA signatures failed to correlate with EFS. However, the levels of ct-miR-140-5p, included in the trastuzumab signature, were associated with EFS (HR 0.43; 95% CI, 0.22–0.84). Conclusions: ct-miRNAs discriminate patients with and without pCR after neoadjuvant lapatinib- and/or trastuzumab-based therapy. ct-miRNAs at week two could be valuable to identify patients responsive to trastuzumab, to avoid unnecessary combination with other anti-HER2 agents, and finally to assist deescalating treatment strategies.

Background: Patients with extremity and trunk wall soft tissue sarcoma (STS) with high malignancy grade and size >5 cm are at high-risk of death. This risk varies depending also on other patient and tumour features, including histologic subtype. This study investigated whether a prognostic nomogram can improve risk assessment of these patients. Methods: Data from high-risk STS patients enrolled in a randomised controlled trial investigating different perioperative chemotherapy regimens were analysed. Ten-year probability of overall survival (OS) and incidence of distant metastasis (DM) were computed using the prognostic nomogram Sarculator (pr-OS and inc-DM, respectively). Tumour response according to RECIST and Choi criteria was also investigated. Findings: Variation in pr-OS and inc-DM were observed and patients stratified in three prognostic groups. The 10-year OS in the low, intermediate, and high pr-OS categories were 0.42 (95%CI 0.32-0.52), 0.63 (95%CI 0.53-0.72), and 0.78 (95%CI 0.68-0.85), respectively. Patients in the intermediate (HR 0.51, P = 0.002) and high (HR 0.28, P < 0.001) pr-OS categories were at statistically significant lower risk of death compared with those in the low pr-OS category. Higher rate of Choi partial tumour responses were detected in intermediate pr-OS category. Tumour response according to Choi but not to RECIST criteria stratified patient survival of pr-OS categories, particularly for patients with intermediate to low pr-OS. Analyses conducted for 10-year inc-DM were consistent with results for pr-OS for prognostic value of Sarculator predictions and Choi tumour response. Interpretation: Sarculator identifies variations in outcomes of high-risk STS treated with perioperative chemotherapy and improve prognostic classification, which is also associated with different patterns of tumour response, an outcome that further stratifies survival particularly for patients predicted at higher risk. Future trials investigating neoadjuvant chemotherapy should consider prognostic tool for selecting patients to be enrolled. (C) 2018 Elsevier Ltd. All rights reserved.

Most gait training systems are designed for acute and subacute neurological inpatients. Many systems are used for relearning gait movements (nonfunctional training) or gait cycle training (functional gait training). Each system presents its own advantages and disadvantages in terms of functional outcomes. However, training gait cycle movements is not sufficient for the rehabilitation of ambulation. There is a need for new solutions to overcome the limitations of existing systems in order to ensure individually tailored training conditions for each of the potential users, no matter the complexity of his or her condition. There is also a need for a new, integrative approach in gait rehabilitation, one that encompasses and addresses all aspects of physical as well as psychological aspects of ambulation in real-life multitasking situations. In this respect, a multidisciplinary multinational team performed an overview of the current technology for gait rehabilitation and reviewed the principles of ambulation training.

Recent developments in mobile brain-body imaging (MoBI) technologies have enabled studies of human locomotion where subjects are able to move freely in more ecologically valid scenarios. In this study, MoBI was employed to describe the behavioral and neurophysiological aspects of three different commonly occurring walking conditions in healthy adults. The experimental conditions were self-paced walking, walking while conversing with a friend and lastly walking while texting with a smartphone. We hypothesized that gait performance would decrease with increased cognitive demands and that condition-specific neural activation would involve condition-specific brain areas. Gait kinematics and high density electroencephalography (EEG) were recorded whilst walking around a university campus. Conditions with dual tasks were accompanied by decreased gait performance. Walking while conversing was associated with an increase of theta (θ) and beta (β) neural power in electrodes located over left-frontal and right parietal regions, whereas walking while texting was associated with a decrease of β neural power in a cluster of electrodes over the frontal-premotor and sensorimotor cortices when compared to walking whilst conversing. In conclusion, the behavioral "signatures" of common real-life activities performed outside the laboratory environment were accompanied by differing frequency-specific neural "biomarkers". The current findings encourage the study of the neural biomarkers of disrupted gait control in neurologically impaired patients.

BACKGROUND: Muscle co-contraction is a strategy of increasing movement accuracy and stability employed in dealing with perturbation of movement. It is often seen in neuropathological populations. The direction of movement influences the pattern of co-contraction, but not all movements are easily achievable for populations with motor deficits. Manipulating the direction of the force instead, may be a promising rehabilitation protocol to train movement with use of a co-contraction reduction strategy. Force field learning paradigms provide a well described procedure to evoke and test muscle co-contraction. OBJECTIVE: The aim of this study was to test the muscle co-contraction pattern in a wide range of arm muscles in different force-field directions utilising a robot-assisted force field learning paradigm of motor adaptation. METHOD: 42 participants volunteered to participate in a study utilising robot-assisted motor adaptation paradigm with clockwise or counter-clockwise force field. Kinematics and surface electromyography (EMG) of eight arm muscles has been measured. RESULTS: Both muscle activation and co-contraction was earlier and stronger in flexors in clockwise condition and in extensors in the counter-clockwise condition. CONCLUSIONS: Manipulating the force field direction leads to changes in the pattern of muscle co-contraction.

The peculiar ability of humans to recognize hundreds of faces at a glance has been attributed to face-specific perceptual mechanisms known as holistic processing. Holistic processing includes the ability to discriminate individual facial features (i.e., featural processing) and their spatial relationships (i.e., spacing processing). Here, we aimed to characterize the spatio-temporal dynamics of featural- and spacing-processing of faces and objects. Nineteen healthy volunteers completed a newly created perceptual discrimination task for faces and objects (i.e., the "University of East London Face Task") while their brain activity was recorded with a high-density (128 electrodes) electroencephalogram. Our results showed that early event related potentials at around 100 ms post-stimulus onset (i.e., P100) are sensitive to both facial features and spacing between the features. Spacing and features discriminability for objects occurred at circa 200 ms post-stimulus onset (P200). These findings indicate the existence of neurophysiological correlates of spacing vs. features processing in both face and objects, and demonstrate faster brain processing for faces.

Circulating tumour cells (CTC) are identified exploiting their protein/gene expression patterns or distinct size compared to blood cells. Data on CTC in bladder cancer (BC) are still scarce. We comparatively analyzed CTC enrichment by AdnaTest ProstateCancerSelect (AT) and ScreenCell®Cyto (SC) kits, combined with identification by EPCAM, MUC1, and ERBB2 expression and by cytological criteria, respectively, in 19 nonmetastatic (M0) and 47 metastatic (M+) BC patients, at baseline (T0) and during treatment (T1). At T0, CTC positivity rates by AT were higher in M+ compared to M0 cases (57.4% versus 25%, p = 0.041). EPCAM was detected in 75% of CTC-positive samples by AT, showing increasing expression levels from T0 to T1 (median (interquartile range, IQR): 0.18 (0.07–0.42) versus 0.84 (0.33–1.84), p=0.005) in M+ cases. Overall, CTC positivity by SC was around 80% regardless of clinical setting and time point of analysis, except for a lower occurrence at T1 in M0 cases. At T0, circulating tumour microemboli were more frequently (25% versus 8%) detected and more numerous in M+ compared to M0 patients. The approach used for CTC detection impacts the outcome of CTC studies. Further investigations are required to clarify the clinical validity of AT and SC in specific BC clinical contexts.

Background Currently used CA15–3 and CEA have found their clinical application particularly in the follow-up of patients with advanced disease. Novel biomarkers are urgent, especially for improving early diagnosis as well as for discriminating between benign and malignant disease. Patients and methods In the present study, we used a proteomic approach based on surface-enhanced laser desorption/ionization–time of flight–mass spectrometry screening with the aim of identifying differentially expressed 2–30 kDa proteins in plasma of patients with malignant (65 cases) and benign (88 cases) breast lesions with respect to 121 healthy controls. Results We found that the most promising SELDI peaks were those corresponding to hepcidin-25 and ferritin light chain. We evaluated the capability of these peaks in predicting malignant and benign breast lesions using the area under the receiver operating characteristic curve (AUC). The results showed a good capacity to predict malignant breast lesions for hepcidin-25 [AUC: 0.82; 95% confidence interval (CI) 0.75–0.90] and ferritin light chain (AUC: 0.86; 95% CI 0.79–0.92). Conversely, a weak and satisfactory capability to predict benign breast lesion was observed for hepcidin-25 (AUC: 0.63; 95% CI 0.41–0.85) and ferritin light chain (AUC: 0.73; 95% CI 0.49–0.97). A significant association between HER2 status and hepcidin-25 was observed and the distribution of transferrin and ferritin were found significantly different in patients with breast cancer when compared with that of controls. Conclusions This study provides evidence that hepcidin and ferritin light chain level in plasma may be of clinical usefulness to predict malignant and benign disease with respect to healthy controls.

Background: Plasma circulating tumour-specific microRNAs (miRNAs) are promising biomarkers of tumour presence and recurrence, especially for diseases whose best chance of successful treatment requires early diagnosis and timely surgery of an already malignant but not yet invasive tumour, such as colorectal cancer (CRC). Methods: Expression levels of miRNAs previously found to be differently expressed in tumour vs normal colon tissues were investigated by quantitative real-time PCR in plasma from CRC patients and from healthy donors and confirmed in independent case control series. The validated miRNAs were also measured after surgery. Analyses were repeated on the subsets of haemolysis-free samples. Results: We identified four miRNAs differently expressed between the compared groups, two (miR-21 and miR-378) of which were validated. miR-378 expression decreased in non-relapsed patients 4–6 months after surgery and miR-378 ability to discriminate CRC patients from healthy individuals was not influenced by haemolysis levels of plasma samples. Conclusion: The miRNA analysis on plasma samples represents a useful non-invasive tool to assess CRC presence as well as tumour-free status at follow-up. Plasma levels of miR-378 could be used to discriminate CRC patients from healthy individuals, irrespective of the level of haemoglobin of plasma samples.

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.

Early age at onset of breast cancer (eoBC) is suggestive of an increased genetic risk. Although genetic testing is offered to all eoBC-affected women, in isolated cases the detection rate of pathogenic variants is &lt, 10%. This study aimed at assessing the role of constitutive promoter methylation at BC-associated loci as an underlying predisposing event in women with eoBC and negative family history. Promoter methylation at 12 loci was assessed by the MassARRAY technology in blood from 154 BRCA1/2 negative patients with eoBC and negative family history, and 60 healthy controls. Hypermethylation was determined, within each promoter, by comparing the patient&rsquo, s mean methylation value with thresholds based on one-sided 95% bootstrap confidence interval of the controls&rsquo, mean. Three patients had hypermethylated results, two at BRCA1 and one at RAD51C. Analyses on tumor tissue from the patient exceeding the highest threshold at BRCA1 revealed a mean methylation &gt, 60% and loss of heterozygosity at chromosome 17q. The patient hypermethylated at RAD51C showed low methylation in the tumor sample, ruling out a role for methylation-induced silencing in tumor development. In isolated eoBC patients, BRCA1 constitutive promoter methylation may be a predisposing event. Further studies are required to define the impact of methylation changes occurring at BC-predisposing genes and their role in tumorigenesis.

Circulating microRNAs (miRNAs) are promising non-invasive biomarkers whose expression may be affected by confounding factors, including hemolysis, that should be considered in studies of miRNA discovery. The present study proposes a methodology for evaluating the impact of hemolysis on the expression of miRNAs. An experiment of in vitro controlled hemolysis was designed for assessing if changes in the expression of eight miRNAs observed to be circulating in plasma may be associated with hemolysis, and also to estimate the level of red blood cell (RBC) contamination in plasma samples where the expression of these miRNAs will be measured. It was confirmed that four miRNAs, miR-16, miR-92a, miR-451 and miR-486, known to be present in blood cells, were influenced by contamination of RBCs. Furthermore, it was demonstrated that miR-378 and miR-30c are hemolysis-independent and that the expression of miR-320 and miR-324-3p was associated with the level of RBC contamination. This procedure is proposed as a tool for the evaluation of the influence of hemolysis on candidate circulating miRNA biomarkers prior to their analysis in plasma samples.

Altered expression of miRNAs is associated with development and progression of various human cancers by regulating the translation of oncogenes and tumor suppressor genes. In colorectal cancer, these regulators complement the Vogelstein multistep model of pathogenesis and have the potential of becoming a novel class of tumor biomarkers and therapeutic targets. Using quantitative real-time PCR, we measured the expression of 621 mature miRNAs in 40 colorectal cancers and their paired normal tissues and identified 23 significantly deregulated miRNAs. We subsequently evaluated their association with clinical characteristics of the samples and presence of alterations in the molecular markers of colorectal cancer progression. Expression levels of miR-31 were correlated with CA19-9 and miR-18a, miR-21, and miR-31 were associated with mutations in APC gene. To investigate the downstream regulation of the differentially expressed miRNAs identified, we integrated putative mRNA target predictions with the results of a meta-analysis of seven public gene expression datasets of normal and tumor samples of colorectal cancer patients. Many of the colorectal cancer deregulated miRNAs computationally mapped to targets involved in pathways related to progression. Here one promising candidate pair (miR-1 and MET) was studied and functionally validated. We show that miR-1 can have a tumor suppressor function in colorectal cancer by directly downregulating MET oncogene both at RNA and protein level and that reexpression of miR-1 leads to MET-driven reduction of cell proliferation and motility, identifying the miR-1 downmodulation as one of the events that could enhance colorectal cancer progression. Mol Cancer Res; 10(4); 504–15. ©2012 AACR.

article i nfo Background: Although important improvements of downstream molecular in vitro diagnostics assays based on RNA from blood were made, the pre-analytical workflow is still poorly defined. Methods: We performed a multicenter study within the EU-granted SPIDIA project to investigate blood col- lection and shipping influence on the following RNA quality parameters: yield, purity, integrity, RT-qPCR in- terference and IL1B, IL8, FOS and GAPDH gene expression. Two models were designed: Exp A. Ten laboratories collected blood from an own donor into two different tubes (with or without stabilizer) and extracted RNA at two different times; Exp B. Blood was drawn from a single donor and shipped to ten labo- ratories in two different tubes (with or without stabilizer) for RNA extraction. Results: In both models and collection tubes, reliable results were obtained for purity, yield, GAPDH expres- sion, and interferences. A substantial variation in RIN (Exp A) and in transcription levels of IL1B, IL8 and FOS (Exp B) was observed for blood collected in tube without stabilizer tubes. Overall the variability was higher among data obtained from unstabilized blood samples. Conclusions: We defined the experimental setup for a larger ring trial throughout Europe. The chosen down- stream analyses verified their potential, serving as adequate markers to test the quality of blood RNA.

11558Background: We already reported (Cancer 2012;118:5857) the correlation of Choi criteria (Choi) and RECIST with outcome of pts affected by high-risk STS entering a multicentric Italian/Spanish ...

11518Background: To determine whether high-risk STS patients identified using individual patient data and the nomogram Sarculator, benefitted from adjuvant chemotherapy in a RCT which failed to det...

Zito F A, Verderio P, Simone G, Angione V, Apicella P, Bianchi S, Conde A F, Hameed O, Ibarra J, Leong A, Pennelli N, Pezzica E, Vezzosi V, Ventrella V, Pizzamiglio S, Paradiso A & Ellis I (2010) Histopathology 56, 720–726 Reproducibility in the diagnosis of needle core biopsies of non-palpable breast lesions: an international study using virtual slides published on the world-wide web Aims: To conduct an internet-based study using virtual slides (VS) of sterotactic core biopsy specimens of non-palpable breast lesions in order to evaluate interobserver reproducibility between pathologists. Methods and results: A total of 18 breast lesions, determined to be histologically complex by two pathologists, were selected. Digitized VSs were then created using QuickTime Virtual Reality technology (Apple, Cupertino, CA, USA) and posted on the world-wide web. In all, 10 pathologists completed the evaluations of 18 VSs using the five diagnostic categories (B1–B5) from the European guidelines for quality assurance in breast cancer screening and diagnosis. Their results were compared with those of every other participating pathologist, and were then individually compared with the results of a highly experienced breast pathologist (referee). Of the 18 cases, 10 (56%) were classified by the referee as borderline (B3 and B4). Comparisons with reference values showed a less than satisfactory level of reproducibility (median κw = 0.60). As regards interobserver reproducibility, results showed that, in general, the level of agreement was not satisfactory (median κw = 0.53). Conclusions: Overall, the findings are comparable to those quality control studies using circulating slides when analysis is done on borderline cases.

Objective: Hepcidin-25 production is stimulated by systemic inflammation, and it interferes with iron utilization, leading to anemia. This study aimed to investigate the relationships between the plasma levels of hepcidin, interleukin-6 (IL-6), erythropoietin (EPO) and erythroferrone (ERFE) in patients with benign breast disease or cancer. Methods: Plasma samples from a cohort of 131 patients (47 with benign breast disease and 84 with breast cancer) were subjected to the evaluation of hepcidin, IL-6, EPO and ERFE using SELDI-TOF-MS or immunoassays. Results: An elevated hepcidin was observed in malignant breast tumors compared to benign ones. No correlation was observed between hepcidin and IL-6, EPO or ERFE. Conclusion: Since the study included a cohort of patients (87%) with breast cancers smaller than 2 cm, these results may support our previous evidence about the potential role of hepcidin in breast cancer disease.

© The Author 2015. Published by Oxford University Press. All rights reserved. Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p. Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p. Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Background MicroRNAs (miRNAs), small noncoding RNAs, are involved in tumorigenesis and in the development of various cancers. Quantitative real-time polymerase chain reaction (qPCR) is the most commonly used tool to investigate miRNA expression, and qPCR low-density arrays are increasingly being used as an experimental technique for both the identification of potentially relevant miRNAs and their subsequent validation. Due to the reduced number of microRNAs to be validated, this phase is generally performed on ad hoc customized cards for which a technical robustness is assumed similar to that of the high-throughput cards used during the identification phase. Methods With the aim of investigating the degree of reproducibility between the 2 types of cards, we analyzed plasma-circulating miRNAs evaluated in 60 subjects enrolled in a colorectal cancer screening program. Results Our results showed a reproducibility between the 2 methods that was not fully satisfactory, with a concordance correlation coefficient equal to 0.69 (95% confidence interval, 0.12-0.92). Conclusions This report highlights the need to add a technical validation step to the high-throughput-based miRNA identification workflow, after their discovery and before the validation step in an independent series.

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (k w) = 0.73 (95 % confidence interval (CI), 0.41–0.94)), with a higher agreement between the reference evaluation and the PFPE samples (k w = 0.86 (95 % CI, 0.67–1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis.

1 Unit of Medical Statistics, Biometry and Bioinformatics, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2 Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3 Molecular Genetics of Cancer, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy 4 Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5 Scientific Directorate, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

Despite the excellent survival rate of Wilms tumor (WT) patients, only approximately one-half of children who suffer tumor recurrence reach second durable remission. This underlines the need for novel markers to optimize initial treatment. We investigated 77 tumors using Illumina 370CNV-QUAD genotyping BeadChip arrays and compared their genomic profiles to detect copy number (CN) abnormalities and allelic ratio anomalies associated with the following clinicopathological variables: relapse (yes vs. no), age at diagnosis (≤24 months vs. >24 months), and disease stage (low stage, I and II, vs. high stage, III and IV). We found that CN gains at chromosome region 1q21.1-q31.3 were significantly associated with relapse. Additional genetic events, including allelic imbalances at chromosome arms 1p, 1q, 3p, 3q, and 14q were also found to occur at higher frequency in relapsing tumors. Interestingly, allelic imbalances at 1p and 14q also showed a borderline association with higher tumor stages. No genetic events were found to be associated with age at diagnosis. This is the first genome wide analysis with single nucleotide polymorphism (SNP) arrays specifically investigating the role of genetic anomalies in predicting WT relapse on cases prospectively enrolled in the same clinical trial. Our study, besides confirming the role of 1q gains, identified a number of additional candidate genetic markers, warranting further molecular investigations. © 2012 Wiley Periodicals, Inc.

Background A significant portion of breast cancer patients treated in the neoadjuvant setting does not achieve a pathological complete response (pCR) and has an increased risk of relapse after surgery. The current lack of reliable predictors of response to guide patient therapy in clinical practice requires the identification of predictive biomarkers that could discriminate between responders and non-responders. The purpose of this study was to investigate the potential of circulating microRNAs (miRNAs) as novel non-invasive predictive biomarkers of response to neoadjuvant treatment in HER2-positive breast cancer patients enrolled in the Neo-ALTTO study. Methods We performed a longitudinal miRNA monitoring of the well-annotated and high-quality plasma samples of patients treated with neoadjuvant lapatinib, trastuzumab or their combination. Before profiling, the overall plasma samples were randomly splitted in two sets, namely the training set and the testing set, with distribution of timing of plasma collection, arm of treatment, rate of pCR, and tumor characteristics resembling that of the entire Neo-ALTTO patient population. To this end a PCR-based high-throughput approach was firstly employed in the training set. After a proper normalization step, for each treatment arm and time-point, a panel of potential miRNAs associated to pCR was identified by resorting to univariate approaches. Multivariate penalized logistic regression models were implemented thereafter, in order to identify specific signatures for each time-point and treatment arm. Finally, the predictive capability of the aforementioned signatures was evaluated in the testing set. Results Plasma miRNA profiles were available for 435 of the 455 (96%) patients enrolled in the Neo-ALTTO study. In details, miRNA levels were analyzed in 141, 151 and 143 patients assigned to neoadjuvant trastuzumab, lapatinib, and their combination, respectively, at baseline and after 2 weeks of treatment. A total of 30 miRNAs (including both normalizators and specific miRNAs) were identified and 6 signatures were found predictive of pCR in terms of Area Under the ROC Curve (AUC) in the training population. The predictive capability of 4 of 6 of the identified signatures was confirmed in the testing set, specifically: lapatinib at baseline AUC (95%CI) = 0.86 (0.73-0.98) and after two weeks AUC (95%CI) = 0.71 (0.55-0.86), trastuzumab after two weeks AUC (95%CI) = 0.81 (0.70-0.92), and lapatinib + trastuzumab after two weeks AUC (95%CI) = 0.67 (0.51-0.83). Of note, the predictive value of the signature of trastuzumab after two weeks was confirmed after adjustment for hormonal receptor status - training set AUC (95%CI) = 0.90 (0.80-0.99), testing set, AUC (95%CI) = 0.84 (0.74-0.94). Conclusion These findings provide the first evidence of the potential of circulating miRNAs to predict treatment response in HER2-positive breast cancer patients treated with neoadjuvant therapy. Results obtained in the trastuzumab arms are of special value as in women with unfavorable miRNA signature it is anticipated an unfavorable response to paclitaxel plus trastuzumab after just 2 weeks of treatment. Data on correlation with elapse free survival will be presented at the meeting. Citation Format: Di Cosimo S, Appierto V, Tiberio P, Verderio P, Pizzamiglio S, Bottelli S, Iorio M, Baselga J, Piccart M, Huober J, Brase J, de la Pena L, Fumagalli D, de Azambuja E, de Braud F, Daidone MG. Plasma microRNA levels for predicting therapeutic response to neoadjuvant treatment in HER2-positive breast cancer: Results from Neo-ALTTO [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S3-02.

Purpose:Monoallelic germ-line deleterious mutations of PALB2 (partner and localizer of BRCA2) are associated with breast cancer risk and have been found in several populations, with carrier frequencies of ∼1-2%. Initially, these mutations were considered to have moderate penetrance, but accumulating evidence now indicates that they are associated with much higher risk.Methods:In this study, we sequenced the PALB2 coding regions unlinked to BRCA (breast cancer) genes in 575 probands from Italian breast cancer families recruited in Milan.Results:We found 12 carriers (2.1%) of deleterious mutations, and none of the mutations was found in 784 controls collected in Milan. One of these mutations, the c.1027C>T (p.Gln343X), was found to be recurrent in the province of Bergamo in northern Italy, being detected in 6/113 (5.3%) familial breast cancer cases and 2/477 (0.4%) controls recruited in this area (Fisher's exact test: P < 0.01).Conclusions:Our data provide confirmatory findings that, in the Italian population also, deleterious mutations of PALB2 are relatively frequent predisposing factors for breast cancer and may be associated with high risk of the disease.Genet Med 16 9, 688-694.

Background An External Quality Assessment (EQA) program was developed to investigate the state of the art of HER2 immunohistochemical determination in breast cancer (BC) in 16 Pathology Departments in the Lazio Region (Italy). This program was implemented through two specific steps to evaluate HER2 staining (step 1) and interpretation (step 2) reproducibility among participants. Methods The management activities of this EQA program were assigned to the Coordinating Center (CC), the Revising Centers (RCs) and the Participating Centers (PCs). In step 1, 4 BC sections, selected by RCs, were stained by each PC using their own procedures. In step 2, each PC interpreted HER2 score in 10 BC sections stained by the CC. The concordance pattern was evaluated by using the kappa category-specific statistic and/or the weighted kappa statistic with the corresponding 95% Jackknife confidence interval. Results In step 1, a substantial/almost perfect agreement was reached between the PCs for scores 0 and 3+ whereas a moderate and fair agreement was observed for scores 1+ and 2+, respectively. In step 2, a fully satisfactory agreement was observed for 6 out of the 16 PCs and a quite satisfactory agreement was obtained for the remaining 10 PCs. Conclusions Our findings highlight that in the whole HER2 evaluation process the two intermediate categories, scores 1+ and 2+, are less reproducible than scores 0 and 3+. These findings are relevant in clinical practice where the choice of treatment is based on HER2 positivity, suggesting the need to share evaluation procedures within laboratories and implement educational programs.

An association of preferential X chromosome inactivation (XCI) with BRCA gene status and breast/ovarian cancer risk has been reported. We evaluated XCI in a large group of BRCA mutation carriers compared to non-carriers and investigated associations between preferential XCI (≥90:10) and age, mutated gene, cancer development and chemotherapy. XCI was analysed by human androgen receptor (HUMARA) assay and pyrosequencing in 437 BRCA1 or BRCA2 mutation carriers and 445 age-matched controls. The distribution of XCI patterns in the two groups was compared by logistic regression analysis. The association between preferential XCI and selected variables was investigated in both univariate and multivariate fashion. In univariate analyses preferential XCI was not significantly associated with the probability of being a BRCA mutation carrier, nor with cancer status, whereas chemotherapeutic regime and age both showed a significant association. In multivariate analysis only age maintained significance (odds ratio, 1.056; 95% confidence interval, 1.016-1.096). Our findings do not support the usefulness of XCI analysis for the identification of BRCA mutation carriers and cancer risk assessment. The increasing preferential XCI frequency with ageing and the association with chemotherapy justify extending the investigation to other categories of female cancer patients to identify possible X-linked loci implicated in cell survival. © 2012 Elsevier Ltd. All rights reserved.

BackgroundGenome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.MethodsRNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.ResultsThirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.ConclusionsOur results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.

Background To explore correlation between the quality of surgery and outcome in high-risk soft tissue sarcoma (STS) patients treated within a phase III randomized trial. Patients and Methods In the trial, all patients received three cycles of preoperative chemotherapy (CT) with epirubicin 120 mg/m2 and ifosfamide 9 g/m2 and were randomly assigned to receive two further postoperative cycles. Radiotherapy (RT) could be delivered in the preoperative or postoperative setting. The association between surgical margins and overall survival (OS) was studied in a univariate and multivariate fashion. Results Two hundred and fifty-two patients completed the whole treatment and were operated conservatively. At a median follow-up of 60 months (IQR, 45–74 months), the 5-year OS was 0.73, even in patients with positive and negative margins. The 5-year cumulative incidence (CI) of local recurrence (LR) in patients with positive and negative microscopic margins was 0.17 (standard error, SE, 0.08) and 0.03 (SE, 0.01), respectively. In the subgroup of patients receiving combined preoperative CT–RT and with positive surgical margins, the CI of LR was 0. Conclusions In this setting of high-risk STS treated by preoperative CT or CT–RT, the negative impact of positive margins on the outcome was limited. When close margins can be anticipated preoperative CT–RT may be a reasonable option to maximize the chance of cure.