Obtaining tissue for pancreatic carcinoma diagnosis and biomarker assessment to aid drug development is challenging. Circulating tumour cells (CTCs) may represent a potential biomarker to address these unmet needs. We compared prospectively the utility of two platforms for CTC enumeration and characterisation in pancreatic cancer patients in a pilot exploratory study.Blood samples were obtained prospectively from 54 consenting patients and analysed by CellSearch and isolation by size of epithelial tumour cells (ISET). CellSearch exploits immunomagnetic capture of CTCs-expressing epithelial markers, whereas ISET is a marker independent, blood filtration device. Circulating tumour cell expression of epithelial and mesenchymal markers was assessed to explore any discrepancy in CTC number between the two platforms.vs 40%) and in higher numbers  vs 0 (range 0–144)). Heterogeneity observed for epithelial cell adhesion molecule, pan-cytokeratin (CK), E-Cadherin, Vimentin and CK 7 expression in CTCs may account for discrepancy in CTC number between platforms.ISET detected CTCs in more patients than CellSearch (93% ISET detects more CTCs than CellSearch and offers flexible CTC characterisation with potential to investigate CTC biology and develop biomarkers for pancreatic cancer patient management. Medical treatment with single-agent gemcitabine or more recently FOLFIRINOX combination chemotherapy confers a survival benefit with improvement in quality of life measures  and can be falsely elevated .per se and could facilitate discovery of new drug targets. If CTCs are demonstrated to be useful as a biomarker(s), then ultimately they could represent a less invasive alternative to tissue for diagnosis and treatment management, particularly where biopsy is considered unsafe or unfeasible.Substantial challenges exist in obtaining tissue from pancreatic cancer patients for histological diagnosis, and acquiring pre- and post-treatment tumour biopsies to monitor pharmacodynamic responses during clinical trials of novel treatments are even more difficult. Circulating tumour cell (CTC) analysis may yield prognostic, predictive and pharmacodynamic biomarker information. Moreover, the molecular characterisation of CTCs may provide insight on the process of metastasis Recent advances in technology have enabled protocol standardisation to enumerate CTCs and allow comparisons between clinical studies  for tumour cell capture and cytokeratin (CK) for tumour cell confirmation.As EpCAM-negative CTCs are not detected using the CellSearch platform, and homogeneity of EpCAM expression on pancreatic CTCs was not established, we reasoned that an alternative approach for CTC isolation and enumeration should be investigated. Isolation by size of epithelial tumour cells (ISET) is a filtration-based, marker-independent method of CTC detection based on cell size and morphology. ISET has been used successfully to study CTCs in patients with melanoma, breast, lung and hepatocellular carcinomas , number of lines of treatment, Ca19-9 levels (at baseline and 8–12 weeks after treatment) and clinical outcomes. Overall survival and progression-free survival (PFS) were calculated from time of consent onto the study. The study was approved by the Local Research Ethics Committee. Peripheral venous blood was taken at study enrolment (before the first planned treatment) for the analysis of CTCs using both CellSearch and ISET platforms. The CellSearch platform was acquired before the ISET device and blood samples obtained from the first 19 patients enrolled were analysed using CellSearch only.Blood samples were collected in CellSave tubes (Veridex), stored at room temperature and processed within 96 h according to manufacturer's instructions. Procedures relating to the use of the CellSearch platform have been previously described in detail  in a 99 °C water bath for 40 min. Membranes were washed briefly in tris-buffered saline (TBS) before placing in 0.2% Triton for 10 min. Membranes were washed in TBS and incubated in a 3% solution of hydrogen peroxide in methanol for 30 min. Membranes were washed again in distilled water before the primary CD45 antibody  in S0809 antibody diluent (Dako) was added and incubated overnight at 4 °C. Envision Liquid DAB + Substrate Chromagen System (Dako) were used according to manufacturer's instructions to visualise CD45 staining. Counterstaining with 1 × Gill's haematoxylin was performed for 3 min. The membrane spots were mounted on glass slides using Fairmount aqueous mounting medium (Dako) and coverslips were applied. The Bioview duet microscope system (Olympus BX52 microscope (Olympus) and image analysis software, Bioview, Rehovot, Israel) was used to scan membranes at × 40 magnification. Manual image review and scoring of tumour cells was performed by one analyst (blinded).μm were designated as CTCs. Four of the 10 membrane spots obtained were used for CTC enumeration. Mean CTC count was extrapolated to 7.5 ml for direct comparison with the standardised CellSearch enumeration procedure. The mean ISET CTC count from four spots was demonstrated to provide a robust CTC count , anti-CK C-11 antibody to CKs 4,5,6,8,10,13,18 , Vimentin  CK 7, as a pancreatic-specific CK marker  and E-Cadherin . EpCAM expression was assessed in 30 patients, Pan CK (C-11) in 19 patients, CK 7 in 25 patients, E-Cadherin in 30 patients and Vimentin in 31 patients.μm) were stained with haematoxcylin and eosin, EpCAM and CK, dewaxed in xylene, and rehydrated using serial washes in 100–70% alcohol. The same IHC protocol and antibodies used for ISET CTC analysis was adopted to determine tumour EpCAM and CK expression in tumour samples. Tumour differentiation and scoring for staining intensity was assessed independently in a blinded fashion, by an experienced histopathologist (LM).Paraffin-embedded tumour blocks were obtained wherever possible to allow correlation of tumour differentiation status with epithelial and mesenchymal marker expression, and to explore which platform might be more suitable for pancreatic CTC detection, that is, whether CellSearch should be able to detect a majority of CTCs that are EpCAM, CK double positive or whether the marker-independent ISET platform would be more appropriate if epithelial markers were likely to be expressed in a minority sub-population of CTCs only. Sections  for Windows where χ2 test. Univariate survival analysis was performed using the Kaplan–Meier method with data categorised into (median) middle quartiles that were compared using the log-rank (Mantel–Cox) test , with the median CTC count chosen as the cut off level for all analysis. Survival was calculated from time of study enrolment to progression (PFS) or death (OS). The Mann–Whitney U test was used to determine whether CellSearch and ISET were significantly different, that is, they detected different numbers of CTCs.Variables were positively skewed and were log transformed before analysis to stabilise the sample variance and nonparametric tests were used to satisfy the assumptions of variance between the sample groups. The association of CTCs with individual clinical characteristics, including presence of a stent, number of metastatic sites, site of primary, and degree of primary tumour differentiation and performance status were compared by Fisher's exact test or Fifty-four patients aged 35–85 years  were enrolled between July 2008 and October 2009. Ten patients had relapsed or progressive disease, 4 patients relapsed following surgery (one of whom had received adjuvant chemotherapy) and 6 patients had progressive metastatic disease having had previous palliative chemotherapy. The remaining patients were newly diagnosed. Fifty-three patients were evaluable by CellSearch, and of these, 31 patients had paired blood samples for ISET assessment and EMT marker expression. Direct comparison of CTC number between the ISET and CellSearch platforms was possible for 27 patients. Patient clinical characteristics are shown in vs ISET for the 27 patients with matched samples revealed that the mean CTC/7.5 ml was 2 vs 26, the median CTC/7.5 ml was 0 vs 9 and that the range was 0–15 vs 0–240, respectively. Direct comparison of CTC numbers per patient is shown in U test showed a P-value 0.000002 indicating that the CTC numbers detected by CellSearch and by ISET were significantly different to each other.Of the 53 patient blood samples assessed using the CellSearch platform, 21 patients had detectable CTCs defined as ⩾1 CTC . The majvs those with CTCs was 140 vs 94 days ) and 164 vs 127 days ) , higher burdens of disease (54% had more than one site of disease) and had primaries situated in the head or tail of the pancreas . However, perhaps due to small sample size none of these variables reached statistical significance , and a l8–1.29)) , respectCirculating tumour cell/7.5 ml measured by ISET  did not correlate with either OS or PFS in this small sample set (data not shown). Only nine patients were recruited onto the study with locally advanced disease, but even when these patients were excluded clinical end points of PFS and OS remained non-statistically significant with regard to CTC/7.5 ml.P=0.2, OS P=0.19; ISET PFS P=0.85, OS P=0.36. The percentage change in CA19-9 levels was statistically significant for both PFS and OS  confirming previous studies . Circulating tumour cell/7.5 ml determined using either CellSearch or ISET platforms was not significant for PFS or OS: CellSearch PFS n=6), 2+ (n=7) or 3+ (n=8)  was also evaluated where sufficient biospy allowed in 13/21 cases. Tumours, reported by a certified pathologist (LM), were graded as either well, moderately or poorly differentiated. All tumours expressed EpCAM; EpCAM expression was graded as 1+ (3+ (n=8) . There wThe low number of CTCs detected by CellSearch could arise due to low or no CTC expression of EpCAM and/or CK. Of the 30 patients’ CTCs analysed for EpCAM expression, 9 had EpCAM-negative CTCs, 3 had CTCs that were all weakly positive for EpCAM and in the remaining 18 patients EpCAM expression was heterogenous, that is, only a proportion of CTCs were EpCAM postive . Of the Tumour cells can circulate in the bloodstream as single cells or in groups termed microemboli. Our previous studies of patients with SCLC showed that circulating tumour microemboli (CTM) were detectable using the ISET but not the CellSearch platform  and 6 whBiomarkers are required to develop better therapies for pancreatic cancer patients where access to tumour biopsies is challenging. We undertook a pilot exploratory study comparing the enumeration of CTCs using two technology platforms (CellSearch and ISET) to assess the feasibility of CTCs as a useful source for biomarker research in this disease.vs without CTCs 110.5 vs 375.8 days, respectively, P<0.001)  (vs 40%) and in higher numbers  vs 0 (range 0–144)), respectively.CellSearch detected low CTC numbers in pancreatic cancer patients in comparison with other epithelial carcinomas such as breast, prostate, colorectal and small cell lung cancer  have been identified in prostate cancer and found to have prognostic significance (The identification of viable CTCs by CellSearch is defined as a (minimum) 4 × 4-ificance . The ideificance  and tumoificance . In futuTo our knowledge, this is the first study to begin to evaluate the ISET platform for pancreatic CTC detection, enumeration and characterisation. Epithelial to mesenchymal transition is thought to occur as cells extravasate into the circulation with the reverse process; mesenchymal to epithelial transition (MET) necessary for cells to intravasate and subsequently form secondary tumours These are most likely to be dynamic events and not necessarily ‘all or nothing’ binary processes. Here, we have begun to reveal EMT marker heterogeneity in CTCs from the pancreatic cancer patients, and a more comprehensive study of their biology with simultaneous assessment of multiple markers is now underway. Epithelial to mesenchymal transition may explain the discrepancies in CTC number between CellSearch and ISET. The finding of both individual CTCs and, more rarely, CTM in pancreatic cancer patients raises questions regarding the mechanisms of cell migration and extravasation, evoking the possibility of collective migration where cells maintain their contacts to invade . It remaThe detection of pancreatic CTCs/CTM on ISET membranes and their downstream characterisation highlight potential for exploiting CTCs/CTM as a surrogate marker of the primary tumour, in a disease where it is notoriously difficult to biopsy tumour diagnostically or to assess tumour evolution or early indication of drug responses. The wider dynamic range of CTC number detected by ISET may be appropriate for pharmacodynamic monitoring to aid dose selection in early clinical trials. The ability to characterise CTCs downstream of ISET filtration in a flexible manner may facilitate measurement of predictive biomarkers allowing stratification of pancreatic cancer patients most likely to benefit from novel targeted therapies, for example, KRAS and EGFR mutation analysis.Although larger studies are now needed to demonstrate whether the number of CTCs is sufficient for in depth and systemic biomarker assessment, and to exploit the potential of pancreatic CTCs/CTM, this study has highlighted several promising avenues for exploration.


Dopamine neurons in the ventral tegmental area (VTA) have been traditionally studied for their roles in reward-related motivation or drug addiction. Here we study how the VTA dopamine neuron population may process fearful and negative experiences as well as reward information in freely behaving mice. Using multi-tetrode recording, we find that up to 89% of the putative dopamine neurons in the VTA exhibit significant activation in response to the conditioned tone that predict food reward, while the same dopamine neuron population also respond to the fearful experiences such as free fall and shake events. The majority of these VTA putative dopamine neurons exhibit suppression and offset-rebound excitation, whereas ∼25% of the recorded putative dopamine neurons show excitation by the fearful events. Importantly, VTA putative dopamine neurons exhibit parametric encoding properties: their firing change durations are proportional to the fearful event durations. In addition, we demonstrate that the contextual information is crucial for these neurons to respectively elicit positive or negative motivational responses by the same conditioned tone. Taken together, our findings suggest that VTA dopamine neurons may employ the convergent encoding strategy for processing both positive and negative experiences, intimately integrating with cues and environmental context. Dopamine neurons in the ventral tegmental area (VTA) have been traditionally studied for their roles in reward-related motivation or drug addiction The role of the VTA dopamine neuron in negative motivation has also been noted. A number of studies have found that aversive events  or negative states  can alter dopamine concentrations in brain areas innervated by the VTA dopamine neurons On the other hand, recent studies find that dopamine neurons in the substantia nigra pars compacta (SNc) can respond to both reward  and aversive stimuli  and two populations of SNc dopamine neurons may distinctly convey positive and negative motivational signals To address these important questions, we employed multi-tetrode extracellular recording in freely behaving mice, and used two types of robust fearful events  We implanted movable bundles of 8 tetrodes (32 channels) into the VTA of the right hemisphere of mice, and the recording electrodes' positions were confirmed by histology at the end of our experiment . Data frWe used two types of robust fearful events  for examining how the VTA neurons might respond to negative experiences P<0.05, Wilcoxon signed-rank test), but also a strong offset-rebound excitation at the termination of both events. We defined the rebound excitation as the offset peak firing rate (smoothed with a Gaussian filter) to be at least two times higher than the baseline firing rate and with z-scores larger than 2. Such rebound excitation may signal the safety at the end of fearful events or a motivation by such events. We then asked whether these type-1 dopamine neurons were responsive to reward signals. By repeatedly pairing a neutral tone with the subsequent delivery of a sugar pellet, we found that these neurons also significantly increased their firing to the conditioned tone that reliably predicted reward  during free fall or shake, but they did not have rebound activation after these events were terminated (z-scores <2) (Type-2 VTA putative dopamine neurons showed significant suppression (ores <2) . Thus, bP<0.05, Wilcoxon signed-rank test). Their increased firing was typically followed by an offset suppression. Moreover, these type-3 dopamine neurons can also increase their firing in response to the conditioned tone that predicted reward  increased their firing to both free fall and shake events . This clearly shows that the vast majority of type-1 and type-2 VTA dopamine neurons are capable of bi-directionally responding to both positive and negative events, that is, they show excitation by reward information while suppression by fearful experiences. On the other hand, about 71% of the type-3 putative dopamine neurons (12/17) that were activated by fearful events can also be activated by the reward signals . This strongly suggests that the fearful events, not just reward, can excite some of the VTA putative dopamine neurons.Overall, type-1 and type-2 neurons constitute a majority (72%) of the recorded VTA putative dopamine neuronal population, whereas type-3 neurons constitute about 25%, with the remaining putative dopamine neurons (3%) not responsive to the fearful events . Moreove signals   and/or quinpirole  which have been mainly shown to inhibit the activity of the dopamine neuron P<0.001, Student's t-test). These results suggest that responses of the type-1 putative dopamine neurons correlate with the duration of the fearful events , suggesting that responses of type-1 VTA dopamine neurons to negative may also reflect, to less a degree, the intensity of the free fall events.To further understand the encoding properties of the VTA dopamine neurons for fearful events, we conducted a set of parametric experiments. Different heights of free fall (10 and 30 cm) and different durations of shake  were performed in random orders during the recording experiments. We found that VTA dopamine neurons exhibited temporal dynamic activity changes that were proportional to the durations of the fearful events. As shown in l events . It was P<0.001, one-way ANOVA). Follow-up Student's t-tests showed highly significant differences for each comparison . We also varied the intensity of the shake event: type-1 neurons exhibited slightly higher offset excitation peak by the high-intensity shake events compared with the low-intensity one . In response to 0.2, 0.5 and 1 sec shake events . Follow-up Student's t-test showed highly significant differences for each comparison . We used one neutral tone as the conditioned stimulus (CS) to pair with distinct unconditioned stimuli  in different environments . We subjNeuronal activity recordings in these conditioned mice (after 1-week training) revealed that VTA putative dopamine neurons responded significantly to the conditioned tone that predicted a sugar pellet in the reward chamber . InteresP<0.001, Wilcoxon signed-rank test). In response to the same tone that predicted the free fall event, half of the neurons (8/16) showed a significant decrease in firing rate , while the other half (8/16) showed a brief immediate activation peak (at least two times higher than the baseline firing rate and with z-scores larger than 2) followed by a significant suppression . In response to the same tone represented in a neutral chamber, there was very limited or no changes in firing  dopamine neurons from the mice that were subjected to the bidirectional conditioning protocol. All of these neurons exhibited a significant increase in firing rates after onset of the conditioned tone that reliably predicted the sugar pellet (t-test). Collectively, the above contextual experiments suggest that information represented at the VTA dopamine neuron level is highly processed and richly integrated to encode a given set of positive or negative motivational events associated with environmental contexts.The importance of contexts in producing distinct conditioned responses was also evident in type-3 dopaminergic-like neurons. As an example, the type-3 neuron responded significantly to the conditioned tone that was associated with a sugar pellet in the reward chamber  or free We next set out to examine the response onset latency of the putative dopamine neurons to both the reward and fearful events. Peri-event histograms of 10 and 30 cm free fall events and peri-event histograms of 0.2, 0.5, and 1 sec shake events were combined for individual dopamine neurons for the calculation of the response onset latency. Response onset latency was determined by first obtaining the mean firing rate (mean) and standard deviation (s.d.) from the 1000 bins (bin  = 10 ms) immediately before the stimulus onset. Response latency was taken to be the time corresponding to the first bin of at least three consecutive bins with Z-scores ≥2 after onset of the stimulus. Due to the low baseline firing rate of the dopamine neuron, peri-event histograms (bin  = 10 ms) were smoothed with a Gaussian filter (filter width  = 3 bins) for the calculation of response onset latency of suppression .Our results showed that type-1 and type-2 putative dopamine neurons exhibited similar response onset latencies to free fall and shake events  . Type-3 Since dopamine levels in the target areas have been often linked to various cognitive outcomes, it has long been hypothesized that synchronized firing of dopamine neurons may represent a neural mechanism for implementing this neural chemical strategy In addition, significant synchronization was also observed within the type-3 putative dopamine neuron population . Out of (SNc) for conveying both positive and negative signals Our above ensemble recordings and analyses have provided a set of evidence for the role of the dopamine neurons in processing both positive and negative experiences. We found that VTA dopamine neurons exhibited diverse response properties and the vast majority of the putative dopamine neurons respond to both reward and fearful stimuli. This convergent encoding strategy by the VTA dopamine neurons is interesting in light of a highly cited study in awake monkeys which shows that dopamine neurons preferentially respond to stimuli with appetitive rather than aversive motivational value In the VTA, an earlier study has demonstrated that different populations of VTA putative dopamine neurons were activated or suppressed by differential fear conditioning It is important to note that our current extracellular recording technique does not have the capacity to visualize different types of putative dopamine neurons in our experiment. We estimate that type-3 dopaminergic-like neurons recorded in our experiment seem to be located more dorsally or anteriorly in the VTA area . FurtherOur current findings also provide several novel insights into the role of VTA dopamine neurons in both positive and negative motivation. First, VTA putative dopamine neurons respond to different negative stimuli in similar manners in awake animals. That is, neurons that responded to free fall always responded to shake in a similar manner . The uni-directional responses to the negative events within a given type of VTA dopamine neurons are similar to that of their responses to a wide range of novel and reward-related events The second notable feature is the strong offset rebound excitation of type-1 dopamine neurons at the termination of free fall or shake events. This offset excitation in the freely behaving animals may encode information reflecting not only a relief at the termination of such fearful events Third, VTA putative dopamine neurons exhibit temporal dynamic activities that correlate tightly with the durations of the fearful events. The use of temporal activity change for encoding the fearful event duration seems to make a good sense because that the suppression is very limited due to the low baseline firing rate of most dopamine neurons. This is interesting in compare with of the finding that dopamine neurons exhibit different peak responses to different values of reward boluses Fourth, we further reveal that VTA dopamine neurons can exhibit completely opposite change in their firings by the conditioned stimulus for signaling either reward or fearful events which occurred under distinct contexts . This stFinally, our simultaneous recording techniques have allowed us to demonstrate a significant correlation among the type-1 and type-2 putative dopamine neurons, as well as among the type-3 neurons. The specificity of such firing synchrony is highly interesting, given the consideration of the possible VTA network arrangement. This suggests that VTA putative dopamine neurons may employ two highly specific synchronized strategies for optimizing and dopamine transmission efficacy and thereby providing a coordinated modulation of downstream structures such as nucleus accumbens. Lack of synchronized activity between type-3 and type-1/2 neurons is consistent with many other differences among them, both electro-physiologically and pharmacologically . ParticuIn summary, we show that the vast majority of VTA putative dopamine neurons are capable of responding to both reward and fear-driven aversive information. These putative dopamine neurons respond to different negative events in a similar manner and more importantly, their temporal durations of dynamic firing changes are proportional to the durations of the fearful events. VTA putative dopamine neurons also integrate cues and contextual information for distinction between reward and fearful events. Taken together, we suggest that VTA dopamine neurons may employ the convergent encoding strategy at the network population level for processing both positive and negative experiences. Such convergent encoding of experiences is also highly integrated with cues and environmental contexts to further enhance behavioral specificity.All animals used in this study were according to procedures approved by the Institutional Animal Care and Use Committee, Georgia Health Sciences University and covered under protocol number BR-07-11-001.A total of 71 male C57BL/6J mice were used for recording and individually housed on a 12-h light/12-h dark cycle. Only data from 24 mice from which we recorded putative dopamine neurons were used in the current analyses.A 32-channel (a bundle of 8 tetrodes), ultra-light (weight <1 g), movable (screw-driven) electrode array was constructed similar to that described previously Two or three days after surgery, electrodes were screened daily for neural activity. If no dopamine neurons were detected, the electrode array was advanced 40∼100 µm daily, until we could record from a putative dopamine neuron. Multi-channel extracellular recording was similar to that described previously Two fearful events, free fall (from 10 and 30 cm) and shake , were randomly performed in our experiments with an interval of typically 1–2 hours between sessions. We used either a square (10×10×15 cm) or round chamber  for the free fall event. We used a round chamber  for the shake events. In each free fall or shake event session, a mouse was placed into the free fall or shake chamber (mouse could move freely inside the chambers). After 3 min habituation, about 20 trials of free fall (or shake) events were given with an interval of 1–2 min between trials. The free fall chamber was lifted up (either 10 cm or 30 cm height) and tied to a solenoid system  before each free fall event. The free fall event was then delivered by providing precise mechanical control  of the solenoid system to release the suspension rope. The free fall chamber then landed on a soft pad that greatly reduced the bounces and prevented potential damages to the stability of the recording  and S3. We always monitored the stability of recorded units by examining the spike waveforms, baseline firing status, and spike cluster distributions before and after the events as well as through the entire experiments. We only included the datasets from the animals that met these recording criteria for further data analyses. As shown in In particular, we took three steps to ensure that spikes were not contaminated by any artifacts: 1) We reduced the interference for the recording by grounding the whole experimental apparatus. We found that the electrical artifacts generated during the free fall and shake events were at the similar level of those during locomotor exploration. 2) We further cancelled out the remaining artifacts by the Plexon Referencing Client which allowed us to choose a channel with no visibly good units as a reference channel. This greatly eliminated background noises and artifacts. 3) If any possible artifact waveforms were still left, we then removed them during the preprocessing of spike waveforms using Plexon Offline Sorter because artifact waveforms were highly distinct from neuronal spike waveforms.Mice were slightly food restricted before reward association training. In reward conditioning, mice were placed in the reward chamber . Mice were trained to pair a tone  with subsequent sugar pellet delivery for at least two days . The tone was generated by the A12-33 audio signal generator  (Coulbourn Instruments). A sugar pellet (14 mg) was delivered by a food dispenser  and dropped into one of two receptacles (12×7×3 cm) at the termination of the tone .In a separate set of experiments, mice were trained for bi-directional conditioning (both reward and aversive conditioning). The conditioned tone  used was identical, but in different contexts: during reward conditioning , the tone was paired with sugar pellet delivery; during aversive conditioning , the same tone was paired with a free fall event (30 cm high). Mice were trained for one week or more and were counterbalanced: half of the mice received reward conditioning on days 1 and 2, followed by aversive conditioning on days 3 and 4 ; the other half of the mice received aversive conditioning on days 1 and 2, followed by reward conditioning on days 3 and 4 . On days 5 and later, three sessions  were given each day in a random order, including reward conditioning, aversive conditioning, and in a third neutral chamber (55×30×30 cm that was enriched with toys) where the tone did not predict anything. The interval between sessions was 1–2 hours; the interval between trials was 1–2 min. The latency of sugar/control receptacle approach after onset the conditioned tone was examined on day 7. Latencies longer than 60 sec were considered as 60 sec; in the case that the mouse was inside the receptacle during the conditioned tone, the latency was not used for calculation. The backward movement behavior (head and/or limbs moving backward) after onset the conditioned tone was also examined on day 7.On completion of the experiments, the final electrode position was marked by passing a 10-sec, 20-µA current  through two electrodes. Mice were deep anesthetized and perfused with 0.9% saline followed by 4% paraformaldehyde. Brains were then removed and post-fixed in paraformaldehyde for at least 24 h. Brains were rapidly frozen and sliced on a cryostat  and stained with cresyl violet. The histological experiments were performed on 21 mice (in another 3 mice the brain sections were unfortunately not well prepared). Our histology results confirmed that dopamine neurons were recorded from the VTA area in 17 mice and from the VTA-SNc border area in 4 mice .Sorted neural spikes were processed and analyzed in NeuroExplorer (Nex Technologies) and Matlab. Dopamine neurons were classified based on the following three criteria: 1) low baseline firing rate (0.5–10 Hz); 2) relatively long inter-spike interval . The shortest ISI we recorded was 4.1 ms under any conditions in our experiment . The averaged shortest ISIs was 6.8±2.2 ms . In contrast, the ISI for non-dopamine neurons can be as short as 1.1 ms; 3) regular firing pattern when mice were freely behaving (fluctuation <3 Hz). Here, fluctuation represents the standard deviation (s.d.) of the firing rate histogram bar values . In addition, it was noted that the vast majority  of the classified dopamine neurons tested showed significant activation in response to the reward predicting tone . It was Neuronal activity changes to the conditioned and unconditioned stimuli were compared against a 10-sec control period before the onset of the stimulus in each trial with a chosen time window (depending on the durations of the stimuli) using a Wilcoxon signed-rank test. For 10 and 30 cm free fall events, the time windows were 100–230 and 100–340 ms after onset of the free fall event, respectively; for 0.2, 0.5 and 1 sec shake events, the time windows were 100–200, 100–500, and 100–1000 ms after onset of the shake event, respectively . For reward conditioning, the time window was 50–600 ms after onset of the conditioned tone; for aversive conditioning, the time window was 200–600 ms after onset of the conditioned tone.Peri-event rasters  and histograms were conducted in NeuroExplorer (Nex Technologies). All smoothings were conducted in the NeuroExplorer using a Gaussian filter (filter width  = 3 bins). Cross-correlations were conducted between simultaneously recorded dopamine neuron pairs when mice were freely behaving  or sleeping in the homecage. For z-score calculation of the cross-correlation peak value, the cross-correlation histograms were smoothed to get the peak value; mean and standard deviations were obtained from shuffled (randomized) spikes in Matlab Figure S1VTA dopamine neurons are stably recorded and well isolated. (A) An example of a well-isolated type-1 dopamine neuron (blue dots) in a 2-dimensional Principal Component Analysis and its representative waveforms (recorded by tetrode) on day 1 (upper panel) and day 2 (lower panel). Spike isolation was performed using Plexon OfflineSorter . PC1 and PC2 represent the first and second principal components, respectively. Blue dots represent individual spikes for the isolated dopamine neuron; black dots indicate individual spikes for other VTA neurons. (B) An example of a well-isolated type-2 dopamine neuron (blue dots) and its representative waveforms on day 1 (upper panel) and day 2 (lower panel). (C) An example of a well-isolated type-3 dopamine neuron (blue dots) and its representative waveforms on day 1 (upper panel) and day 2 (lower panel).(TIF)Click here for additional data file.Figure S2No temporary loss of unit during free fall and shake events. (A) Responses of four simultaneously recorded VTA dopamine and non-dopamine neurons during free fall events. Note that units recorded from the same tetrode may exhibit opposite responses , suggesting that the recording was stable without any temporary losses of units. (B) Responses of the same four VTA neurons during shake events. (C) Representative waveforms for the same four VTA neurons 1 h before, during free fall and shake event session, and 1 h after.(TIF)Click here for additional data file.Figure S3No noise/artifact contamination during free fall and shake events. (A) Responses of an example putative dopamine neuron (type-1) and its waveforms before (1 sec), during (1 sec), and after (1 sec) the free fall and shake events. Note that the waveforms did not show significant change after free fall and shake event, suggesting that there was no noise/artifact contamination. (B) Responses of another putative dopamine neuron (type-3) and its waveforms before (1 sec), during (1 sec), and after (1 sec) free fall and shake events.(TIF)Click here for additional data file.


The effectiveness of nucleoside analogue on patients with chronic hepatitis B-associated liver failure is still controversial. To address this issue, we did a review of the literatures and analyzed the data with emphasis on the survival and reduction in serum HBV DNA level.We searched 11 randomized controlled trials that included 654 patients with chronic hepatitis B-associated liver failure. 340 patients adopted nucleoside analogue, such as lamivudine (LAM), entecavir (ETV), telbivudine (LdT), or tenofovir disoproxil fumarate (TDF), and the remaining 314 patients adopted no nucleoside analogue or placebo. A meta-analysis was carried out to examine the survival, HBV e antigen serologic conversion, and reduction in serum HBV DNA level. The pooled odds ratio (OR) was used to reflect the treatment effects.The overall analysis revealed nucleoside analogue significantly improved 1-month, 3-month , 12-month survival . Comparison of 3-month HBV DNA showed significant reduction for adoptive nucleoside analogue patients . Comparison of 3-month HBV e antigen serologic conversion showed a highly significant improvement of HBV e antigen lost for patients received adoptive antiviral therapy .The benefits of nucleoside analogue on patients with chronic hepatitis B-associated liver failure is significant for improving patient survival, HBV e antigen serologic conversion, and rapid reduction of HBV DNA levels. Chronic hepatitis B(CHB),caused by the hepatitis B virus (HBV), is a serious health problem worldwide, especially in China and other parts of AsiaAs oral antiviral agents, nucleoside analogue, including lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), and tenofovir disoproxil fumarate (TDF), have activities conferring biochemical, virological, and serological improvement in CHB patientsRecently nucleoside analogue have been proved efficacious in improving the status of patients with severe decompensated chronic liver disease and acute-on-chronic liver failure (ACLF) related with CHBIn the meta-analysis, we included studies from randomized controlled trials that compared the antiviral therapy adopted nucleoside analogue  with no antiviral treatment for patients who were undergoing chronic hepatitis B-associated liver failure.Though the concept of acute-on-chronic liver failure (ACLF) is well identified and the diagnostic criteria are unifiedpreviously diagnosed CHB or HBV induced cirrhosis;progressively raising in serum bilirubin level (>85 µmol/L or >5 mg/dL);prothrombin activity<40% or international normalized ratio (INR)≥1.5;3 copies/ml;HBV DNA level >10And the exclusion criteria were:superinfection with human immunodeficiency virus(HIV) and/or other hepatitis viruses ;other causes of chronic liver failure, like drug hepatitis, autoimmune hepatitis, alcoholic liver disease, et al;patients were receiving antiviral therapy at the moment when they were recruited or had received antiviral therapy in 6 months before the studies;evidence of hepatocellular carcinoma (HCC);patients had received artificial liver treatment during the studieshttp://www.ClinicalTrials.gov website for the information of prospective and ongoing trials. Through the searching task, we used the terms ‘nucleoside analog’,’ nucleoside analogue’,’ nucleotide analog’,’ nucleotide analogue’,’ lamivudine’,’ adefovir dipivoxil’,’ entecavir’,’ telbivudine’,’ tenofovir disoproxil fumarate’,’ liver failure’,’ hepatic failure’,’ chronic hepatitis B’, and ‘severe chronic hepatitis B’.The investigators wrote a protocol and carried out a comprehensive search of Medline, PubMed, Embase, the Cochrane Center Register of Controlled Trials, Biological Abstracts, China National Knowledge Infrastructure, and the Chinese BioMedical Literature Database without language, publication, or date restrictions. However, when the Chinese literatures have no English abstracts, they would be excluded. In addition, reference lists of the trials selected before and relevant reviews were examined for other eligible trials. We also searched Data were extracted independently by two authors, Feng Xie and Long Yan. We screened abstracts of all retrieved articles and then matched the full texts of all articles selected during screening against the inclusion criteria. Disagreements on which articles met the inclusion criteria were resolved by discussion until a consensus was reached. Feng Xie and Long Yan completed the data extraction using a standardized approach that gathered the publication details and study characteristics: year of publication, the first author, number of patients, sex, methods and design, serum HBV DNA level and HBV DNA negative patients, seropositivity and seronegativity for HBV e antigen (HBeAg), alanine transarninase, albumin, serum bilirubin, prothrombin activity or international normalized ratio (INR), number of patients assessable for 1-, 3- and 12-month overall survival.We used the Jadad score (maximum number of points is 5)The analyses were carried out using STATA 11.0 and Review Manager 5. P values at<0.05 were regarded as statistically significant. Since the study carried out by Yao-Li Cui had a three-arm design: it separately compared two regimens (ETV and LAM) to placebo, each antiviral arm was paired to the control arm independentlyThe data of 1-, 3- and 12-months survival, patients with negative HBV DNA level, HBV e antigen serologic conversion in each arm were extracted from each study and combined using analysis method named Mantel and Haenszel 2 test) and I2 test were used for across studies to assess the variation across study results that is due to heterogeneity rather than chance. I2 can be readily calculated from a typical meta-analysis as I2 = 100%×(Q−df)/Q, where Q is Cochran’s heterogeneity statistic and df is the degrees of freedom. Negative values of I2 are equal to zero. A value of 0% indicates no observed heterogeneity, and larger values show increasing heterogeneity2<25% would be considered as there was no or low-level heterogeneity, and the fixed-effects model was adopted when the data across studies was pooled. Otherwise, the random-effects model was used. I2>50% was considered as significant heterogeneity. In addition to the use of subgroup and the random-effects model, some explanation would be made. Publication bias was assessed visually using a funnel plot.Cochran’s Q test, and the P = 0.69 in Cochran’s Q test and I2  = 0% when evaluated the heterogeneity.Four studies2 = 15%, meaning there was no significant variability of these studies. The subgroup of ETV and TDF had individual OR value of 1.38  and 7.33 . The OR value was 2.24  in the LAM subgroup, and it had no evidence of significant heterogeneity (P = 0.34 in Cochran’s Q test and I2 = 7%).Five studies2  = 24%).There were only two studies3 copies/mL (about 178 IU/mL). The pooled OR for these two studies of negative HBV DNA patients showed significant reduction in HBV DNA for adoptive nucleoside analogue patients .Four studies2 = 12%).Three studies None of these studies reported patients developed significant adverse reaction, or need for dose modification, early discontinuation. Only one study observed two patients had mild gastrointestinal reaction after they took ETVA funnel plot of the studies used in the meta-analysis reporting on 1-month survival is shown in The result clearly indicated nucleoside analogue significant improve survival, HBV e antigen serologic conversion, and rapid reduction of HBV DNA levels. The effects of nucleoside analogue for severe liver disease have been demonstrated by plenty of previous trails. But most of them were not RCTs, and the conclusions of many articles, even guidelines, originated from them. What we wanted to do was to get a stricter conclusion. Now that liver failure is considered as a complication of chronic hepatitis B infection, and of course it would provoke concern in recruiting a sufficient number of patients to conduct a controlled trial using no antiviral treatment (even in areas of high endemicity). What’s more, for patients with chronic hepatitis B-associated liver failure, liver transplantation is currently regarded as a primary and complementary measureFor patients with severe liver disease associated with CHB, the severity of liver disease at the time of initiating antiviral therapy, like elevated base-line serum bilirubin and creatinine levels and detectable baseline serum HBV DNA level, is a more relevant determinant of short-term mortality than the virological responseIn our selected RCTs, eight of the eleven searched studies published the survival data after using nucleoside analogue for a short-term time. All of the eight studies showed positive results. Nucleoside analogue can markedly suppress HBV replication by suppression of HBV-polymerase activity, leading to improvement of liver function and reduced incidence of fibrosis, cirrhosis in CHB patientsData regarding the efficacy of long-term treatment with nucleoside analogue for those patients with liver failure was lack in previous literatures. The severity of liver disease, the poor prognosis of patients and the high mortality rateWhen we compared the effects of nucleoside analogues, the strategy of subgroup was adopted in our statistical analysis. But the selected studies can only provide the data of two or three nucleoside analogues during the same period of time, the comparision of effects brought by each drugs could not obtain a definite conclusion. Previously some nonRCT studies had provided information about the effects of nucleoside analogue drugs for patients with HBV-related decompensated liver diseaseWhen initiating antiviral therapy, the severity of liver disease, like elevated base-line serum bilirubin and creatinine levels and detectable baseline serum HBV DNA level could affect the results of patientsIn conclusion, though liver transplantation is currently regarded as a primary measure for patients with chronic hepatitis B-associated liver failure, the early initiation of adaptive nucleoside analogue drugs for antiviral therapy is also necessary.Finally, we had to mention the weaknesses of these selected studies. The low quality and the conducted areas must be the main shortages. When they were assessed using the Jadad score, the score was 4 for one study, 3 for one study and 2 for nine studies. And all the studies belonged to Asia . Obviously these weaknesses could bring some limitations. Under ideal conditions, more double blinding, and large scale randomised control trials should be carried out.


EGFR, EML4-ALK, and KRAS, some of which have been translated into targeted therapies. However, the molecular alterations identified thus far cannot explain all of the clinical and biological disparities observed in lung tumors of NS and smokers. To this end, we performed an unbiased genome-wide, comparative study to identify novel genomic aberrations that differ between smokers and NS.Recent evidence suggests that the observed clinical distinctions between lung tumors in smokers and never smokers (NS) extend beyond specific gene mutations, such as High resolution whole genome DNA copy number profiling of 69 lung adenocarcinomas from smokers (n = 39) and NS (n = 30) revealed both global and regional disparities in the tumor genomes of these two groups. We found that NS lung tumors had a greater proportion of their genomes altered than those of smokers. Moreover, copy number gains on chromosomes 5q, 7p, and 16p occurred more frequently in NS. We validated our findings in two independently generated public datasets. Our findings provide a novel line of evidence distinguishing genetic differences between smoker and NS lung tumors, namely, that the extent of segmental genomic alterations is greater in NS tumors. Collectively, our findings provide evidence that these lung tumors are globally and genetically different, which implies they are likely driven by distinct molecular mechanisms. EGFR mutations and EML4-ALK translocations, findings that have been clinically translated for selection of targeted therapies While the majority of lung cancer cases can be attributed to tobacco smoking, up to one quarter of lung cancers arise in never smokers (NS) Previous studies have focused on locus specific genetic features or have been limited by the use of low resolution technologies lacking whole genome coverage, comparison between NS and smokers, and/or validation in external cohorts Copy number alterations (CNAs) are known contributors to tumorigenesis, and different cancer subtypes display distinct copy number profiles which are associated with distinct phenotypic and clinical features All tissues were collected from the Tumor Tissue Repository of the British Columbia Cancer Agency under informed, written patient consent and with approval from the University of British Columbia - BC Cancer Agency Research Ethics Board.Both tumor and adjacent non-malignant lung tissues were accrued for 69 treatment naive lung adenocarcinoma (AC) patients undergoing surgical resection with curative intent (BCCA tumors). These included 39 smokers (current smokers (CS) at the time of diagnosis) and 30 NS (patients who had smoked fewer than 100 cigarettes in their lifetime). Tissues were fresh-frozen and underwent pathological review to confirm AC histology and absence of cancer cells in the adjacent non-malignant lung tissue. DNA was extracted using standard phenol:chloroform procedures. Each tumor was microdissected with guidance of a pathologist (JCE or AFG) to ensure >70% tumor cell content.KRAS and EGFR mutation status by PCR amplification and product sequencing. Exons 19 and 21 and exon 2 were screened in EGFR and KRAS, respectively. PCR was performed on 50–100 ng of DNA in 25 µL reactions using the Applied Biosystems GeneAmp PCR System 9700. Applied Biosystems BigDye Terminator v3.1 cycle sequencing kit and capillary instrumentation were used to sequence PCR products. Primer sequences and PCR conditions are supplied in EGFR and KRAS mutation with smoking status. A p-value<0.05 was considered significant. The relationships between clinical variables  and EGFR and KRAS mutation status were assessed using Pearson correlation.Genomic DNA from each tumor was sequenced to determine Partek Genomics Suite Software . Probe sequence, fragment length, GC content and background adjustments were applied to correct for biases in signal intensities. Copy number profiles were generated following the Copy Number, Paired Analysis Workflow such that matched non-malignant profiles were used as a copy number baseline for each respective tumor. Genomic segmentation was applied with stringent significance thresholds to identify segmental regions of CNA (gain and loss) using the following parameters: signal to noise >0.3, minimum of 50 markers per segment, p-value threshold of 10−7 for the statistical difference between intensities of adjacent segments, and p-value threshold of 10−7 for significance of deviation of intensities in tumor tissue from intensities in non-malignant lung. This stringency enabled us to confidently distinguish altered genomic segments from technical noise. Identified segments were merged using a 1 Mbp window to combine adjacent regions of copy gain or loss. All genomic mapping was based on the March 2006, hg18 genome build. The genomic coordinates for RefSeq genes (hg18) were obtained from the UCSC genome browser Genomic DNA from tumor and matched non-malignant lung tissues were hybridized to Affymetrix SNP 6.0 arrays according to the manufacturer's instructions. Raw CEL probe intensity files were processed using th percentiles were excluded to reduce the effects of outliers. A multifactor ANOVA was performed in R to assess the contributions of clinical  and genetic (EGFR and KRAS mutation status) variables to the observed PGA.For each tumor the sum of base pairs encompassed by CNAs (both gains and losses) was used to calculate the proportion of genome altered (PGA) for each tumor. Differences in PGA between tumor genomes were investigated using a two-tailed, Student's t-test assuming unequal variance. P-values<0.05 were considered significant. Tumors with PGAs in the fifth and 95R using a 3×2 contingency table generating a p-value for each genomic region ResCalc was used to determine the functional resolution of the SNP array data given the segmentation parameters we applied (139 kbp). Thus, we only considered differentially altered regions satisfying this size threshold Segmental alterations identified in each tumor were parsed into typed copy numbers for each SNP array element, such that every array probe was scored as 1 (copy gain), 0 , or −1 (copy loss). Probes with similar copy number states within individual tumors were then collapsed into genomic regions across all tumor samples. The frequency of DNA gains, DNA losses, and neutral copy number were compared in smoker and NS tumor genomes using a Fisher's Exact test with a p-value<0.05 considered significant. The Fisher's Exact test was performed in GISTIC (Genomic Identification of Significant Targets in Cancer) algorithm was used to investigate high-level DNA alterations  in smokers and NS in the BCCA cohort as it had the highest resolution copy number data for mapping focal genomic events GISTIC analysis on the segmental alteration data with the following parameters: amplification threshold = 0.848, deletion threshold = 0.737, join segment size = 50, q-value threshold of 0.05, and hg18 genome build. Regions identified in smokers and NS were then compared to determine regions of overlap and regions of difference based on the region limit boundaries defined by GISTIC.The http://cbio.mskcc.org/Public/lung_array_data/FACADE with default parameters and a baseline distribution of 10 kbp EGFR and KRAS mutation data were also available for these tumors from the same source. Affymetrix SNP 250 K array data generated by the Database of Genotypes and Phenotypes from the Tumor Sequencing Project (TSP) were also accessed (Study Accession: Study Accession: phs000144.v1.p1). Array data for 72 current smoker and 37 NS lung adenocarcinoma and matched non-malignant tissues were processed in Partek with the same normalization and segmentation parameters as the BCCA tumors, except for using a 20 marker minimum for defining segments due to lower array density. PGA was calculated for each tumor in the MSKCC and TSP datasets. A Student's t-test was used to compare PGA in smoker and NS tumors with a p-value<0.05 considered significant.Publically available microarray data with accompanying smoking status annotation was accessed to compare findings from the BCCA tumors with external cohorts. Normalized, lung adenocarcinoma Agilent 4×44 k array comparative genomic hybridization (aCGH) data generated by the Memorial Sloan Kettering Cancer Centre (MSKCC) was obtained from EGFR and KRAS mutation status).Differentially altered regions in smokers and NS were identified in the MSKCC and TSP datasets applying the same strategy as that for the BCCA tumors. Minimal common regions (MCRs) of overlap for regions differentially altered in the same direction in all three datasets were mapped. Correlations between the MCRs identified were investigated in the BCCA tumors using a Pearson correlation. A multifactor ANOVA was also performed to assess relationships between each of the six MCRs discovered and the clinical and genetic variables discussed above in the BCCA tumors , while our smoker cohort was enriched for Caucasian patients. One smoker sample was from a Native American patient.Lung adenocarcinoma and matched non-malignant tissue specimens were collected from 69 patients, including 39 smokers and 30 NS . CollectEGFR and KRAS were associated with smoking status, confirming the accuracy of smoking history classifications. EGFR mutations (exons 19 and 21) were more frequent in NS  while KRAS mutations were more frequent in smokers   while Caucasians had significantly more KRAS mutations than Asians . There was no difference in mutation rates in females compared to males. A Pearson correlation analysis confirmed these associations (EGFR mutations were negatively correlated with smokers (Pearson's r = −0.61) while KRAS mutations were positively correlated with smokers (Pearson's r = 0.52). EGFR mutations were also positively correlated with Asian ethnicity (Pearson's r = 0.63) and negatively correlated with KRAS mutations (Pearson's r = −0.48). Smoking status was also correlated with race (Pearson's r = −0.68) as our NS cohort was predominantly comprised of Asians.Consistent with reported literature, mutation rates for .4×10−5) . Tumors ciations . EGFR muEGFR and KRAS mutation data was also available for the MSKCC lung adenocarcinoma tumors; consistent with the BCCA tumors, EGFR mutations were more prevalent in NS  and KRAS mutations were more prevalent in smokers   . There wEGFR mutants (n = 17) versus EGFR wild-types (n = 13) and Asians (n = 21) versus non-Asians (n = 9) in NS tumors only were compared. There was no significant difference in PGA between mutant and wild-type EGFR NS tumors . There was also no significant difference in PGA in Asian versus non-Asian NS tumors .Copy number profiles were generated for each BCCA lung tumor by performing genomic segmentation with stringent significance thresholds to ensure alterations called were non-random genetic events. All CNAs identified were somatic events as opposed to germline variants, as each profile was generated using matched non-malignant lung tissue as a reference. The frequency of CNAs throughout the genome  for the 39 smokers and 30 NS is depicted in EGFR and KRAS mutation status). A multivariate analysis revealed that smoking status explained the greatest amount of variance observed in PGA  compared to all other factors, followed by EGFR mutation    . Since N = 0.09) , althougEGFR and KRAS mutation frequencies were consistent with smoking status classifications, we observed the same global pattern of CNAs; NS had greater PGA than smokers , validating our findings , consistent with our observations in the BCCA tumors.To verify our observations were not limited to the BCCA tumors alone, we investigated publically available data for smoker and NS lung adenocarcinomas from the MSKCC and lung TSP, respectively findings . NS tumoEGFR mutations were also associated with higher PGA  . The repgher PGA , which wARNT, TERT, EGFR, MYC, and ERBB2 . Common regions of copy number loss (frequency >20% in both groups) included chromosomes 3p, 6q, 8p, 9p, 17p and 19p, and encompassed known tumor suppressors including FHIT, CDKN2A, TP53, and LKB1  of differential alteration shared by the BCCA and MSKCC lung tumors is provided in EGFR did not map to the MCRs on chromosome 7p; however, we observed a significant association between both 7p12.3 DNA gains and EGFR gains and mutations . This finding is consistent with the fact that EGFR function can be influenced by multiple genetic mechanisms.We also applied this comparison to an additional cohort (TSP) to identify the most prominent differentially altered regions between NS and smokers in the combined datasets acknowledging that this stringent criteria may increase the number of false-negative regions. This analysis revealed six MCRs concordant in all three independent datasets. All of these regions were copy number gains specific to NS, and included two regions on chromosome 5q, three regions on chromosome 7p, and one region on chromosome 16p, which encompassed a total of 13 genes . The smaTo assess whether the MCRs identified were independent events within our tumors, we sought to investigate whether any of the MCRs we identified were correlated with one another in the BCCA tumors . EmployiEGFR mutations and smoking status, we expected to see that smoking history and EGFR mutation would account for the greatest amount of variance observed for each region. Indeed, smoking status and EGFR were most strongly associated with all of the regions we discovered, except for 7p14.1, for which the factor explaining the greatest amount of variance was age (followed closely by EGFR mutation).We next asked whether any of the MCRs we identified could explain the variance in PGA that we observed. None of the six MCRs contributed significantly to the observed PGA across the BCCA tumor dataset. Lastly, although we identified these regions by comparing smokers and NS, we performed a multivariate analysis to confirm that smoking status was the factor most strongly linked with each of the MCRs we identified. As discussed, given the highly pronounced association between Given the enormous efforts put forth to promote smoking cessation and prevention initiatives, in the next few decades NS (and former smokers) will constitute a larger proportion of the lung cancer population EGFR and KRAS mutations in the BCCA and MSKCC tumors segregated with NS and smokers, respectively, consistent with the reported literature. This confirmed the accuracy of our smoking status classifications and validated that these tumors were appropriate to perform a smoker versus NS genome comparison. Genes and regions known to be frequently disrupted in lung adenocarcinoma were not preferentially disrupted in smokers or NS, and the recurrent alterations we observed in both groups were highly concordant with recent reports TERT. Gain of 5p was also the most common genomic alteration observed by Weir et al. in a collection of over 350 lung adenocarcinoma tumors EGFR or KRAS mutation. A clean comparison would require large numbers of patients in each smoking/mutation/race combination, at least 300 subjects for each, in order to achieve 80% power to detect a 10% difference in PGA at a significance level of 0.05.Intriguingly, our comparative study revealed that NS lung tumors have a greater fraction of the genome encompassed by genomic alterations. Despite the caveat that our smoker and NS groups were not balanced for ethnicity or mutation  our multivariate analysis suggested smoking history was the clinical variable most strongly associated with this observed difference. However, since an earlier study that sampled a small fraction of the genome had suggested a greater degree of alterations in smoker tumors EGFR mutation) was the strongest factor associated with genomic regions of difference identified. Broet et al. recently reported regions differentially altered in East-Asian and Western European lung adenocarcinoma tumors, none of which overlapped with the smoking-related regions we identified, indicating that our regions are not ethnic specific Amidst the genomic instability observed in the lung adenocarcinoma tumors, we identified frequent genomic alterations whose recurrent nature signifies their selection in tumor genomes. After cataloguing numerous differentially altered regions in our dataset, we interrogated two independent cohorts to validate our findings and to reveal the most robust and pronounced regional differences in smokers and NS. We identified six MCRs of copy number gain on chromosomes 5q, 7p and 16p. It is possible that additional, less prominent MCRs, may have been identified had we used less stringent concordance criteria between the three datasets. The regions we have reported are the most robust as they are present in multiple independent cohorts. Again, we performed a multivariate analysis to confirm that smoking status , albeit without a comparison against tumors from smokers, reporting several MCRs of copy number gain and loss Early profiling studies led to the discovery that gains of chromosome 16p are more common in NS than smokers, and this remains one of the few consistently replicated NS-specific genetic alterations discovered to date, which implicates the importance of this region in NS tumor biology EGFR, located on chromosome 7p11.2. The closest region (7p12.3) was situated 5.4 Mbp telomeric of the EGFR locus. The presence of these MCRs could imply that additional oncogenes are responsible for 7p gains, as previously suggested DUSP4, a gene located on chromosome 8p12, was down regulated and associated with EGFR mutation DUSP4 in the BCCA dataset and confirmed this association . Interestingly, we also mapped an MCR of loss on 8p in the BCCA and MSKCC datasets which encompassed DUSP4 and found that this region was more frequently lost in NS tumors. Although 8p was not one of our most robust differentially altered regions, it appears genomic loss of DUSP4 is associated with EGFR mutation and NS.Our analysis revealed three distinct MCRs of gain on 7p, however none encompassed the lung cancer oncogene, RAS wild-type and exhibit widespread copy number aberrations While many known regions of copy number alteration in lung adenocarcinoma were present in both our smoker and NS cohorts, our results, along with the well established differences in mutational profiles and clinical features, suggest lung tumors of smokers and NS develop through different molecular mechanisms. This may be similar to what has been observed in ovarian cancer, where Type I serous ovarian cancers are typically chromosomally stable and harbor mutations in the Ras signaling pathway, while high-grade serous ovarian cancers (Type II) are EGFR and KRAS mutations It is well known that the mutation profile of NS lung adenocarcinoma is distinct from that of smokers Figure S1PGA in smokers and NS in the TSP dataset. Although it did not meet statistical significance, NS lung tumors have greater PGA than smoker lung tumors on average.(TIF)Click here for additional data file.Figure S2Six minimal common regions (MCRs) of difference between smokers and NS. The six regions described in (TIF)Click here for additional data file.Table S1Genomic DNA PCR primers. PCR on genomic DNA was performed to determine EGFR and KRAS mutation status in the BCCA lung tumor cohort. Mutations and primer sequences are shown.(DOC)Click here for additional data file.Table S2Matrix summarizing correlations between clinical and genetic variables in the 69 BCCA tumors. A correlation analysis was performed to determine the associations between different clinical and molecular factors in the BCCA lung tumor cohort. Correlation coefficients for each pair of variables are shown.(DOC)Click here for additional data file.Table S3Summary of alteration frequencies in BCCA smoker (n = 39) and NS (n = 30) lung tumors. The frequency of copy number alterations throughout the genome  was determined and summarized for each group . The minimum (Min), maximum (Max), median, and average frequencies for each group are indicated. Total frequency (Gain & Loss), frequency of gain, and frequency of loss are reported.(DOC)Click here for additional data file.Table S4Multifactor ANOVA test results for assessing the effects of clinical and genetic factors on observed PGA in 69 BCCA tumors. A multifactor ANOVA was performed to investigate the effects of multiple factors on PGA in the BCCA lung tumor cohort (n = 69). The ANOVA test statistics are shown.(DOC)Click here for additional data file.Table S5Multifactor ANOVA test results for assessing the effects of clinical and genetic factors on observed PGA in NS tumors only. A multifactor ANOVA was performed to investigate the effects of multiple factors on PGA in never smokers only (n = 30). The ANOVA test statistics are shown.(DOC)Click here for additional data file.Table S6High-level DNA changes in smoker lung tumors.GISTIC was used to reveal high level DNA alterations in 39 smoker lung tumors from the BCCA. Copy number status, genomic location, and frequency of alteration are indicated.(DOC)Click here for additional data file.Table S7High-level DNA changes in NS lung tumors.GISTIC was used to reveal high level DNA alterations in 30 never smoker lung tumors from the BCCA. Copy number status, genomic location, and frequency of alteration are indicated.(DOC)Click here for additional data file.Table S8Matrix summarizing correlations between each minimal common region identified in the 69 BCCA tumors. The six minimal common regions identified in the BCCA tumor cohort were assessed for their correlations with one another. The correlation coefficients for each pair of regions are shown.(DOC)Click here for additional data file.


Previous studies suggest that the responsiveness of TrkB receptor to BDNF is developmentally regulated in rats. Antidepressant drugs (AD) have been shown to increase TrkB signalling in the adult rodent brain, and recent findings implicate a BDNF-independent mechanism behind this phenomenon. When administered during early postnatal life, ADs produce long-lasting biochemical and behavioural alterations that are observed in adult animals.We have here examined the responsiveness of brain TrkB receptors to BDNF and ADs during early postnatal life of mouse, measured as autophosphorylation of TrkB (pTrkB).ex vivo. Basal p-TrkB status in the brain of BDNF deficient mice was significantly reduced only during early postnatal period. Enhancing cAMP (cyclic adenosine monophosphate) signalling failed to facilitate TrkB responsiveness to BDNF. Reduced responsiveness of TrkB to BDNF was not produced by the developmental increase in the expression of dominant-negative truncated TrkB.T1 because this reduction was similarly observed in the brain microslices of trkB.T1−/− mice. Moreover, postnatal AD administration produced long-lasting behavioural alterations observable in adult mice, but the responses were different when mice were treated during the time when ADs did not (P4-9) or did (P16-21) activate TrkB.We found that ADs fail to induce TrkB signalling before postnatal day 12 (P12) after which an adult response of TrkB to ADs was observed. Interestingly, there was a temporally inverse correlation between the appearance of the responsiveness of TrkB to systemic ADs and the marked developmental reduction of BDNF-induced TrkB in brain microslices We have found that ADs induce the activation of TrkB only in mice older than 2 weeks and that responsiveness of brain microslices to BDNF is reduced during the same time period. Exposure to ADs before and after the age when ADs activate TrkB produces differential long-term behavioural responses in adult mice. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are critical regulators of neuronal survival, differentiation, axonal and dendritic growth, and synapse formation BDNF and TrkB play a central role in the mechanism of action of antidepressant drugs (AD) Previous finding suggests that the responsiveness of TrkB to BDNF is regulated during development; BDNF readily activates TrkB in early postnatal rat brain microslices, but this effect of BDNF is strongly blunted after the second week postnatally bdnf and trkB mRNA expression in mice Exposure to ADs during early life has been shown to produce long-lasting behavioural changes in rodents that are evident even in adulthood. Specifically, depressive and anxiety-like, motor and sexual behaviours are modified in adulthood ex vivo BDNF at different stages of mouse postnatal development. Our results suggest the intriguing hypothesis that during the postnatal development, there is a shift in TrkB responsiveness from a receptor that is readily activated by BDNF but refractory to ADs to a TrkB clearly phosphorylated by ADs but only weakly activated by BDNF. This shift may play a role in the development of certain adult emotional phenotypes.We have here studied the phosphorylation response of TrkB to systemic ADs and We have previously shown that a single intraperitoneal (i.p.) injection of AD induces phosphorylation of TrkB within an hour in the prefrontal cortex (PFC) and hippocampus (HC) of adult rodent brain Our results showed that, along postnatal period, TrkB response to ADs is age-dependently regulated. In the PFC, acute IMI treatment of mice between P5 and P11 failed to increase TrkB phosphorylation at the PLCγ1 site (pY816) . Furthermore, as already reported in adult brain data not shown).We have previously shown that ADs specifically induce phosphorylation of TrkB at the PLCγ1 site (Y816) and the phosphorylation of CREB (cAMP response element binding protein), the downstream target of this pathway, whereas no increase in phosphorylation levels is observed at the Shc site (pY515) or its downstream target Akt et alFigure S2).Knüsel et aldata not shown).The studies by Knüsel The developmentally reduced ability of BDNF to induce TrkB phosphorylation in the mouse brain microslices is probably not due to reduced penetrance of BDNF into mature brain tissue since a highly related neurotrophin, NGF (nerve growth factor), readily induced TrkA phosphorylation (Y674/5) in hippocampal microslices obtained from P24 mice  Bdnf+/− mice  and/or serotonin (5-HT) that subsequently can activate Gs –linked postsynaptic receptors. Consequently, we next tested whether acute treatment with IMI might restore the responsiveness of TrkB to BDNF ex vivo in adult hippocampus. As expected, IMI treatment increased the pTrkB levels in the hippocampus but no further phosphorylation was observed when BDNF was applied .Cyclic adenosine monophosphate (cAMP) signalling has been shown to facilitate or “gate” BDNF-induced TrkB signalling in mature hippocampal neurons ex vivo assay in brain microslices obtained from adult mice pre-treated with fluoxetine (FLX) via drinking water (0.08 mg/ml) for 21 days Long-term systemic treatment with antidepressant fluoxetine has been shown to reopen developmental-like plasticity in the adult rodent brain in vitro cell-free kinase assay to examine whether there are any structural modifications in the TrkB receptor protein itself that might prevent BDNF from binding to and activating TrkB in the mature brain tissue. When the adult PFC and HC homogenates were subjected to the kinase assay in the presence of adenosine triphosphate , a clear increase in TrkB phosphorylation in response to BDNF was observed (ex vivo assay (.We used an data not shown), suggesting that long-lasting alterations of the levels of BDNF or TrkB do not explain the behavioural phenotype observed in adult animals.Exposure of rodents to ADs during postnatal life has been shown to elicit alterations in emotional behaviour that are evident in adulthood, long after the cessation of the treatment Table S1 and post hoc test showed that mice exposed to L-PS CLO show a significantly increased Time in the light .When we tested behaviour of adult animals at 3 months of age, we found that animals exposed to CLO during E-PS or L-PS, long before the behavioural testing, showed differential alterations in exploratory locomotion and anxiety-related behaviour that correlated with the age of AD treatment. Specifically, exposure to CLO treatment produced strong long-term behavioural effects in the Light-Dark test when the drug was given during the L-PS, but not when it was administered during the E-PS (Table S1 and post hoc test showed a reduced latency to feed in mice treated with CLO in the E-PS when compared to control mice at the same age (p<0.05) (Table S1 and Table S1), but the post hoc test did not show any difference among controls and CLO treated animals. No differences were found between the groups for weight loss before the test for postnatal age and treatment (Table S1).In the novelty-suppressed feeding (NSF) test, only the administration of CLO during the E-PS significantly influenced adult behaviour. In this paradigm, food-deprived mice are presented a food pellet in a novel environment and the latency to approach the food is measured ex vivo BDNF stimulation in late postnatal stage and adulthood. AD exposure before or after this developmental switch produces differential long-lasting effects on motor and anxiety-like behaviours in adult animals. Although an inverse temporal correlation between the appearance of the responsiveness of TrkB to systemic ADs and the robust reduction of BDNF-induced TrkB phosphorylation in ex vivo microslices cannot be taken as any evidence of causality, it is tempting to speculate that the same maturation processes that bring about the AD responsiveness, lead to developmental changes that restrict the effects of BDNF on TrkB.In the present study, we observed an age-dependent switch from a TrkB receptor readily activated by BDNF but not by ADs during early postnatal stage to a receptor activated by ADs but less sensitive to direct Pharmacologically, ADs such as the drugs used in the present study bind to and inhibit the monoamine reuptake pumps which lead to enhanced monoamine receptor signalling. While monoaminergic neurons are among the first neurons to be generated during early embryonic development, full maturation of these cells extends beyond postnatal life in rodents Ser133 is induced by a variety of signalling pathways, including PKA (protein kinase A), Ca2+-calmodulin kinase II and Mitogen-activated protein kinase pathways AD-induced TrkB phosphorylation during late postnatal period takes place specifically at the Y816 (binding site of PLCγ1), but not at the Y515 (binding site of Shc), which is consistent to our previous findings in adult brain et alWe have here confirmed and extended the unexpected observations previously reported by Knüsel trkB.T1−/− mice. Another possible explanation could be that the ability of BDNF to induce TrkB activation in the mature brain depends on additional gating mechanisms such as cAMP or adenosine i signalling, might facilitate or restore the ability of BDNF to activate TrkB, but this did not turn out to be the case. Moreover, direct up-regulation of [cAMP]i signalling in the ex vivo assay failed to facilitate BDNF-induced TrkB phosphorylation in mature microslices. It is possible that functional maturation of neuronal networks and closure of sensitive periods may modify the responsiveness of TrkB to BDNF (and ADs). Chronic treatment with AD fluoxetine has been shown to reopen developmental-like plasticity in the adult rodent cortex ex vivo BDNF in the adult mouse brain. However, the cell-free TrkB kinase assay suggests that there does not appear to be any structural modifications in the TrkB protein itself that would prevent the receptor from binding to and being activated by BDNF.It was previously suggested that the developmentally regulated increase in the truncated TrkB.T1 receptor that can act as dominant-negative partner for full-length TrkB, might explain the specific reduction of the responsiveness of TrkB to BDNF. We ruled out this possibility, since hippocampal TrkB receptors responded similarly to BDNF and ADs in the tissue samples prepared from WT and ex vivo assay after early postnatal life remains unclear. Although recent evidence suggest that lack of BDNF has only minor effects on the survival and structure of cortical neurons in the CNS in vivo infusion of BDNF into the adult brain increases TrkB phosphorylation, albeit only at relatively high concentrations via TrkB bdnf+/− mice and in mice over-expressing an inhibitory form of trkB (TrkB.T1) in brain The reason for and significance of the apparently complete loss of TrkB responsiveness to BDNF , 2, the brains quickly removed and bilateral hippocampus and prefrontal cortex were dissected out on a dish cooled on dry-ice. Samples were homogenized in a NP++ buffer  and 2 mM Na3VO4). After incubation on ice for 15 min, samples were centrifuged  and the supernatant collected for further analysis.Dams with their litters were housed individually. For postnatal acute antidepressant treatments, age-matched litters (P5-21) were randomly assigned to receive i.p. injection of saline (SAL) , imipramine (IMI) , or clomipramine  and following indicated lag-time (30–60 min) hippocampus and medial prefrontal cortex were collected as described in Figure S4).Dams with their litters were housed individually. The litters were randomly assigned to 4 groups: saline injected (SAL) , and clomipramine injected (CLO)  Mice were weaned on P22 and males were housed together (4–6 mice/cage) until the behavioural experiments. For biochemical analyses the animals were killed after 2 weeks of the last behavioural test and their tissues were collected and processed as described before.For the acute AD treatments, adult (∼P90) mice received a single i.p. injection of imipramine  or SAL and were killed 30 minutes after. For chronic AD treatment, adult (∼P90) mice had free access to either tap water or to 0.08 mg/ml solution of fluoxetine  for 21 days ex vivo BDNF stimulation assay was performed according to Knüsel et alThe et al2, 0.05% BSA, 2% DMSO with or without 100 µM ATP and/or 50 ng/mL BDNF. The reaction was initiated adding 40 µg of NP++ lyzed protein extract to the mix and the incubation was allowed to proceed for 10 min at 37°C. For a subset of samples, the reaction was quenched by adding an equal volume of 4× Laemmli sample buffer and proteins were separated by SDS-PAGE and TrkB phosphorylation status analyzed with western blotting as described below. Rest of the samples we immediately transferred to Trk antibody containing ELISA (enzyme-linked immunosorbent assay) plates, 3% BSA/PBS-T (+2 mM Na3VO4) added ad 200 µl, and phospho-TrkB ELISA proceeded as previously described Assays were performed according to the procedures described by Angeles Y816 , anti-p-TrkA/BY490/Y515 , MA, USA), anti-p-TrkA/BY674-5/Y705-6 , anti-TrkBout , anti-p-CREBS133 , anti-CREB , CA, USA), anti-p-AktThr308 , anti-AKT , anti-p-PLCγ1Y783  or anti-Trk (#sc-11 (rabbit), 1∶1000, SCB). Further the membranes were washed with TBS/0.01% Tween® (TBST) and incubated with horseradish peroxidase conjugated secondary antibodies . After subsequent TBST washes, secondary antibodies were visualized using electrochemiluminescence kits (Amersham Biosciences) followed by an exposure to Fuji LAS-3000 Camera  for ECL detection.Sample protein concentrations were measured using the Lowry Method (Bio-Rad DC protein assay). Lectin precipitation was carried out essentially as described Battery of behavioural tests Testing was performed for 10 min in an acrylic cage (28.5×28.5×20 cm)  divided into two equal sized compartments: one part with transparent walls, open topped and brightly illuminated (∼450 lx by a 40 W light bulb fixed 55 cm above the floor), the other part made from black plastic (passing infrared light) and covered by a lid. The two compartments were separated by a partition containing an opening (7×5 cm) in its centre at floor level. The animal was placed in the centre of the light compartment facing away from the opening, and the latency to enter dark area, time spent in the compartments, total distance travelled, immobility time and the number of entries to dark were measured over 10 min. In addition, rearing time was also calculated. Testing apparatus was thoroughly cleaned before each animal using 70% ethanol.ad libitum. On testing day each animal was removed from its home cage and placed in a holding cage for 30 min before the test and then put in one corner of the arena. The latency to beginning of a feeding episode was measured (maximum over a period of 5 min). Immediately, after starting of feeding the mouse was removed from the arena and placed with the food pellet in its home cage and allowed to feed ad libitum over a period of 5 min. The amount of food consumed was quantified by weighting the pellet.Novelty-Suppressed Feeding: The Novelty-Suppressed Feeding (NSF) was performed as previously described post hoc test. The criterion for significance was set to p<0.05.Immunoblot bands were quantified using NIH ImageJ. All the data are expressed as mean ± SEM (Standard Error of Mean) and as percentage of control. Statistical analyses were performed using GraphPad Prism 4.0 for Windows . For comparison between two groups Two-way Student's t-test was used. Two-way ANOVA or two-way ANOVA for repeated measures was used to reveal main effect and interaction between the factors followed by Bonferroni Figure S1Effect of imipramine on the phosphorylations of TrkB Shc binding site and Akt during development. (a) Phosphorylation of TrkB Shc binding site (Y515) after acute imipramine treatment  in prefrontal cortex (PFC) and hippocampus (HC). (b) Phosphorylation of Akt (Thr308) after acute imipramine treatment  in prefrontal cortex (PFC) and hippocampus (HC). Phospho-protein values are normalized against corresponding total protein levels. (TIF)Click here for additional data file.Figure S2Acute BDNF stimulations does not regulate total TrkB protein levels in brain microslices. Representative blots showing the levels of full-length TrkB after control or BDNF stimulation in cortical (PFC) or hippocampal (HC) microslices prepare from P5-P60 old mice.(TIF)Click here for additional data file.Figure S3Imipramine and fluoxetine does not directly induce TrkB phosphorylation in hippocampal microslices ex vivo. P20 hippocampal microslices were incubate with vehicle or different concentrations of imipramine  (a) or fluoxetine  (b) for 15 min at 37°C and TrkB phosphorylation (Y705/6) analyzed with western blotting. n = 3 per group.(TIF)Click here for additional data file.Figure S4The effect of postnatal clomipramine on weight gain of mouse. A daily dose of clomipramine  or saline during early  or late  postnatal period produced no changes on weight gain during (P4-21) (a) or after the treatments (P28-120) (b). n = 10–15 per group.(TIF)Click here for additional data file.Table S1Postnatal treatments with clomipramine lead to long-lasting and distinct behaviours depending on early exposure period. The effect of early  and late  postnatal clomipramine treatment  on behaviour in light-dark box test and novelty suppressed feeding test in adult (P90) animals. Two-Way ANOVA followed with Bonferroni post hoc test was performed for statistical analysis; *P<0.05, ** P<0.01. n = 10–15 per group.(DOCX)Click here for additional data file.