The majority of the human genome is transcribed, even though only 2% of transcripts encode proteins. Non-coding transcripts were originally dismissed as evolutionary junk or transcriptional noise, but with the development of whole genome technologies, these non-coding RNAs (ncRNAs) are emerging as molecules with vital roles in regulating gene expression. While shorter ncRNAs have been extensively studied, the functional roles of long ncRNAs (lncRNAs) are still being elucidated. Studies over the last decade show that lncRNAs are emerging as new players in a number of diseases including cancer. Potential roles in both oncogenic and tumor suppressive pathways in cancer have been elucidated, but the biological functions of the majority of lncRNAs remain to be identified. Accumulated data are identifying the molecular mechanisms by which lncRNA mediates both structural and functional roles. LncRNA can regulate gene expression at both transcriptional and post-transcriptional levels, including splicing and regulating mRNA processing, transport, and translation. Much current research is aimed at elucidating the function of lncRNAs in breast cancer and mammary gland development, and at identifying the cellular processes influenced by lncRNAs. In this paper we review current knowledge of lncRNAs contributing to these processes and present lncRNA as a new paradigm in breast cancer development. Ribonucleic acids were long thought to act mainly as carriers of genetic information between DNA and the protein-synthesizing machinery of the cell. Whole genome technology, including deep sequencing and microarray analyses, has demonstrated that coding regions constitute only 2% of the human genome. On the other hand, 70–90% of the genome is transcribed . InitialNon-coding RNAs (ncRNAs) are defined as those RNAs which are transcribed from the genome but do not encode proteins. They are divided into two broad classes based on their function: housekeeping and regulatory. Housekeeping ncRNAs include ribosomal, transfer, small nuclear, and small nucleolar RNAs (snoRNAs). These ncRNAs are constitutively expressed and crucial to the operation of vital cellular functions. Regulatory ncRNAs are differentially expressed according to tissue and stage of development or disease process. These regulatory ncRNAs are divided into two broad classes based on their size. The first class of regulatory ncRNAs comprises short transcripts <200 nt long and includes microRNA (miRNA), small interfering RNA (siRNA), piwi interacting RNA (piRNA), transcription initiation RNA (tiRNA), and small cajal body-specific RNA (scaRNA). They control a number of developmental and physiological pathways and have been shown to be deregulated in disease processes . The secde novo from transposable elements or non-exonic sequences . A large number of lncRNAs associate with actively translating ribosomes .Breast development is orchestrated by a series of genes that regulate these different cellular processes. Alterations to genes encoding growth factors, nuclear transcription factors, cell cycle regulatory proteins and tumor suppressor proteins have been suggested to create favorable conditions to allow the outgrowth of cells. A major focus in breast cancer research is to understand how normal breast epithelium is transformed into malignant epithelium. With the advent of genome-wide sequencing, it became apparent that it is not solely genetic changes that contribute to breast cancer. Epigenetic changes also play a major part in altering gene expression in such a way as to contribute to cancer . The rolcis or trans  was identified initially in the rat mammary gland and exhibits conserved synteny in the genomes of other mammals. It is differentially expressed as pregnancy progresses  may exert these effects on the mammary gland by regulating cell cycle progression. In HC11 cells, mPINC relocates from the nucleus to the cytoplasm as the cell cycle progresses (PINC interacts with retinoblastoma associated protein 46 (RbAp46), a component of PRC2, suggesting that PINC may exert its role in differentiation through modification of chromatin and that it acts to repress gene expression. Several potential target genes, known to be involved in mammary gland development, have been identified, and encode members of the Wnt and Notch signaling pathways  is upregulated in a number of different cancers, including breast  and preventing it from interacting with the glucocorticoid response element .The ability of lncRNAs to form complex secondary structures gives them the capacity to present a diversity of interfaces for binding to proteins. LncRNAs may recruit or organize proteins as evidenced by their roles as chromatin modifiers, or act as decoys which inhibit protein functions. One such lncRNA, BC200 is normally selectively expressed in brain tissue and germ cells. It exhibits a high degree of tissue specificity in the brain, showing expression in somatodendritic domains in a subset of neurons but not in other somatic cells. BC200 functions to repress translation through its oligo(A) rich region which is able to bind polyA binding protein (PABP), so that it is no longer able to bind translation initiation factors  acts as a nuclear coactivator for a number of steroid hormone receptors, including the estrogen, progesterone (SRAP) which has unique functions as well as overlapping functions to SRA1 in modulating gene expression. SRA1 forms a stem-loop structure, which enables it to bind to multiple proteins including steroid receptors, transcription factors, RNA helicases, and other molecules involved in adipogenesis, myogenesis, and adrenal gland gene function . MiRNAs are regulated transcriptionally and also post-transcriptionally by a class of lncRNAs known as competitive endogenous RNAs (ceRNAs) that act as sponges or decoys to titrate miRNAs away from their target mRNAs and buffer their activity . Several miR-372  and PTENer cells . Non-proer cells . Alignmeer cells . MiRNAs er cells .Long ncRNAs, ceRNAs, and miRNAs form regulatory networks across the transcriptome, communicating with each other to regulate gene expression. This includes short motifs known as pyknons, which are a subset of variable length patterns. Pyknons are conserved in the genome, and are found in coding and non-coding regions, but mostly in 3′UTRs. Pyknon sequences contain 40% of known miRNA sequences, suggesting that they regulate miRNA function . DiffereWhile some established miRNA–lncRNA interaction sites are conserved to various extents, others lack conservation despite showing confirmed functions . It may Small nucleolar RNAs are a well characterized class of small ncRNAs that predominantly localize to the nucleolus and act there as guides for post transcriptional modification of ribosomal RNAs. These snoRNAs bind modifying proteins, then hybridize with specific rRNA sequences, facilitating rRNA modification. Two major classes of snoRNAs are involved in two different types of post-transcriptional modification: the C/D box snoRNAs which define sites for 2′O ribose methylation, and the H/ACA box snoRNAs which define the sites for pseudo-uridylation .Genetic searches for cancer genes have revealed that aside from their housekeeping function for rRNA modifications, a number of snoRNAs are also deregulated in cancer. An example is the snoRNA U50, a candidate tumor suppressor gene. It is downregulated and frequently mutated in prostate and breast cancer .Small nucleolar RNAs are often located within the introns of protein coding and lncRNA genes. The latter may have independent functions, such as involvement in protein synthesis, and may contribute to breast cancer progression.(ZFAS1) is an antisense lncRNA that overlaps the promoter region of the gene ZNFX1, which encodes a zinc finger protein whose exact role remains to be elucidated. ZFAS1 is host to three snoRNAs in three consecutive introns. ZFAS1 is expressed in a wide variety of tissues, showing highest expression in the mammary gland and lung. It contributes to mammary gland development, showing enrichment in the alveolar structures and ducts and differential expression during different stages of pregnancy in mouse mammary glands. As well as acting as a snoRNA host, Zfas1 has independent regulatory functions. Zfas1 is expressed in both the nucleus and cytoplasm, and knockdown of cytoplasmic Zfas1 by siRNA in mouse mammary epithelial cell lines increases proliferation. This suggests that Zfas1 has dual functions: after splicing in the nucleus to generate snoRNAs, Zfas1 is transported to the cytoplasm where it regulates cellular proliferation. In addition to regulating mammary gland development, ZFAS1 contributes to breast cancer, as its expression is reduced in human ductal carcinoma compared to normal breast epithelium. ZFAS1 plays a role in proliferation as well as differentiation, and its down regulation may promote cancer progression, indicating that it may have tumor suppressor functions (ZNFX1 antisense RNA 1 unctions .GAS5) containing up to 12 exons was first isolated from growth arrested NIH-3T3 cells, induced upon serum starvation of cells  is a nuclear localized lncRNA that was discovered in screens to identify lncRNAs upregulated upon treatment with stress inducing chemicals. In normal tissues, LSINCT5 is highly expressed in proliferative and stress responsive tissues such as the colon and spleen compared to non-proliferative tissues such as brain. Expression of LSINCT5 is increased in several cancers, including breast and ovarian cancer. Because of its high expression in proliferative tissues and cancer, LSINCT5 could potentially function as a regulator of proliferation. This is supported by LSINCT5 knockdown in breast and ovarian cancer cell lines, which impairs proliferation  is a highly conserved, abundant lncRNA whose expression is localized to the nucleus. It was discovered in non-small cell lung cancer as a prognostic marker of later metastasis  in response to growth signals, shifting its location from repressive polycomb bodies on growth control gene loci to active interchromatin granules, activating their expression. MALAT1 acts as a switch for polycomb target genes from repressive nuclear domains to active nuclear domains, influencing gene expression  was identified through genome wide computational analysis  and β-catenin, and increases expression of fibronectin and vimentin  is a transcription factor that acts a key regulator of pluripotency and lies within the intron of a lncRNA, called SOX2OT. SOX2 is required for embryonic stem cell self-renewal and pluripotency and is often deregulated in cancer -box 2  was originally identified as being dramatically upregulated in bladder cancer, and is known as a highly specific and sensitive biomarker for diagnosing bladder carcinoma . Its expression is mainly restricted to the nucleus, where it plays a key role in the splicing of pre-mRNA , but lowhe other .As discussed in this review, several lncRNAs have been implicated in cancer and modulation of these lncRNAs could lead to new therapeutic advances for cancer treatment.Long ncRNAs can be targeted by RNA interference technologies such as siRNAs and shRNAs. These molecules show a high degree of selectivity and knockdown efficiency and could therefore be used to silence lncRNAs for treatment of disease. Although the use of siRNA and shRNA for therapeutic use is still in development, a few siRNAs have been used in clinical trials for a number of diseases, including cancer . VariousMALAT1 in cancer cells and subcutaneous tumors in nude mice (Other strategies that could be used to target lncRNAs include antisense oligonucleotides (ASO), single stranded DNA or RNA molecules with a specific sequence designed to target a particular RNA. ASOs bind to their target RNAs with high sequence specificity, and induce their degradation by RNAse H1. They have been used experimentally to inhibit ude mice  with sucHOTAIR and BC200 are upregulated in breast cancer as well as in other cancers. High expression of HOTAIR is correlated with poor survival in breast cancer, increased recurrence of hepatocellular carcinoma following surgical hepatectomy, and high grades of gastrointestinal stromal tumor . Their role as therapeutic agents is currently being explored, and highly specific lncRNA expression signatures make them attractive markers for accurate disease diagnostics. As well as their use for diagnosis and prognosis, the advancement of RNA-based therapeutics opens up lncRNAs as new targets for therapy. The functional role of the plethora of lncRNAs is only beginning to be elucidated, and provides an untapped resource for the development of cancer therapies and biomarkers.Long ncRNAs are important regulators of gene expression and have a wide variety of functions that enable them to interact with multiple pathways that modulate breast cancer progressions. These lncRNAs have been shown to act as oncoRNAs, often upregulated in breast cancer and facilitating tumor growth and progression, or acting as tumor suppressors, showing downregulation in breast cancer and inhibiting tumorigenesis (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Social cognition – the ability to identify, perceive, and interpret socially relevant information – is an important skill that plays a significant role in successful interpersonal functioning. Social cognitive performance is recognized to be impaired in several psychiatric conditions, but the relationship with major depressive disorder is less well understood. The aim of this review is to characterize the current understanding of: (i) the different domains of social cognition and a possible relationship with major depressive disorder, (ii) the clinical presentation of social cognition in acute and remitted depressive states, and (iii) the effect of severity of depression on social cognitive performance.Methods: Electronic databases were searched to identify clinical studies investigating social cognition in a major depressive disorder population, yielding 31 studies for this review.Results: Patients with major depressive disorder appear to interpret social cognitive stimuli differently to healthy controls: depressed individuals may interpret emotion through a mood-congruent bias and have difficulty with cognitive theory of mind tasks requiring interpretation of complex mental states. Social cognitive performance appears to be inversely associated with severity of depression, whilst the bias toward negative emotions persists even in remission. Some deficits may normalize following effective pharmacotherapy.Conclusions: The difficulties with social interaction observed in major depressive disorder may, at least in part, be due to an altered ability to correctly interpret emotional stimuli and mental states. These features seem to persist even in remission, although some may respond to intervention. Further research is required in this area to better understand the functional impact of these findings and the way in which targeted therapy could aid depressed individuals with social interactions. Correctly interpreting social information is a crucial part of successful interpersonal interaction. This requires synthesis of a broad range of verbal and non-verbal cues, including facial expressions, prosody in speech, body language, and the mental states of others (theory of mind). Together, these skills are referred to as social cognition and are an important component of cognitive functioning. Social cognition encompasses the identification, perception, and interpretation of socially important information , whilst Social cognitive impairment is widely recognized to be a key feature of several psychiatric diseases, such as schizophrenia  and autiThe majority of data from previous reviews investigating the relationship between depression and social cognition relate primarily to the emotional domain, particularly through facial expressions or affective theory of mind. One meta-analysis of eight studies found major depressive disorder to be significantly associated with impaired recognition of emotional facial expressions with a moderate overall effect size . A largeTo our knowledge, there is a lack of similar comprehensive reviews of behavioral studies in the domains of prosody, body language, and theory of mind to determine if these findings are consistent across all components of social cognition in major depressive disorder. Evidence from individual studies indicates that patients with major depressive disorder may also struggle with verbal cues, with two studies reporting a negative bias when depressed patients interpret prosodic stimuli , 15. SevIn addition to the available behavioral data, there is also a significant body of literature that exists on functional neuroimaging in patients with depression undergoing social cognitive testing. Much of this literature indicates a neural basis for the observed phenomenology, primarily centering around dysregulation of both the amygdala and ventromedial frontal cortex –19. In lBetter understanding of the nature of social cognition in major depressive disorder is important, as it will help to further characterize the phenotype of the disease and may offer explanation for the poor social functioning observed in depression. It is also important to consider the potential therapeutic implications, as previous reviews have suggested that impairments in social cognition may be reversible , 13. ImpThe present review of the existing literature on social cognition and major depressive disorder aims to increase the understanding of: (i) the impact of depressed mood across different domains of social cognition including facial affect, prosody, body language, and theory of mind, (ii) the clinical presentation of social cognition in acute and remitted depressive states, and (iii) the effect of severity of depression on social cognitive performance.An electronic search strategy was employed to identify published studies investigating the behavioral relationship between social cognition and depression. Pertinent original research articles were identified and retrieved via the Medline, Embase, and PsycINFO databases, as well as the reference lists of included articles. The search was limited to only the most recent English literature, taking into account the 10-year period from June 2003 to June 2013. The subject headings used across the different databases were “social cognition,” “social perception,” “depression,” “major depression,” “mood disorders,” “depressive disorder,” and “affective disorders.” This was combined with the key word search terms of “social cognition,” “social perception,” “social interaction,” “depression,” “mood disorder,” “affective disorder,” “melancholia,” and “dysthymia.” Truncated search terms were included where possible to maximize results, while inclusion of broad key words such as “mood disorder,” “melancholia,” or “dysthymia” was intended to enhance the sensitivity of the search.The titles and abstracts of the results generated from the three databases were screened to exclude irrelevant articles. The full articles of the remaining studies were downloaded and the methods inspected in detail to determine eligibility Figure . The ideStudies were excluded if the patient group was co-morbid with a separate psychiatric disorder, such as schizophrenia, dementia, a learning disorder, eating disorder, or pervasive developmental disorder. Studies were also excluded if the patient group demonstrated bipolar features. Functional MRI studies with no reported behavioral results were excluded. Once selected, the article’s reference list was then scoured for further relevant articles missed in the initial database searches.Over the past decade, much has been published to further characterize the relationship between social cognition and major depressive disorder. Following a thorough search of the literature, a total of 31 articles were included in this review  incorporated variations of intensity into the assessment of facial affect recognition. Patients with major depressive disorder were identified as more likely to misattribute the expression as being of a higher intensity . FurtherMany studies incorporated assessment of reaction time, in addition to accuracy, for facial affect recognition. Several case-control studies showed individuals with major depressive disorder to have slower recognition of facial expressions than healthy controls , 31 and There were little data available investigating discrepancies in the way depressed and non-depressed subjects react to emotional stimuli. One group of researchers observed that depressed individuals demonstrate more avoidant behavior on an implicit joystick task when confronted by both negative and positive emotional stimuli, even when this information was correctly interpreted by the participant , 45. HowReading the Mind in the Eyes assessment task (There was greater consistency in the literature regarding affective theory of mind in major depressive disorder. Almost all studies employed the ent task , which rent task , 29, 32.The only two studies that did not reveal a difference between groups on theory of mind performance indicated instead that the depressed group showed less perceptual reasoning than controls for the affective rather than cognitive components of theory of mind , 22. ThiA number of authors considered the effect of certain clinical features of depression on affective theory of mind performance, linking suicidal behaviors , excessiA number of researchers also chose to use tools based on cognitive theory of mind ability in their assessment of possible social cognition impairments in depressed patients, with some evidence to suggest that patients with major depressive disorder have difficulties with these skills in comparison to healthy controls. In a case-control study involving 60 patients, Zobel et al.  found thIn contrast, there were also a number of studies that found little difference in cognitive theory of mind performance between the depressed and non-depressed. Wolkenstein et al.  found deSome authors also examined whether acutely depressed patients performed differently on social cognitive tasks compared to those with remitted major depressive disorder. A very small sample base was retrieved, with the three relevant studies being detailed in Table The relationship between acute and remitted major depressive disorder can also be considered in terms of treatment. A number of studies have considered the role of intervention, with one randomized controlled trial finding that acute administration of citalopram improved facial affect recognition accuracy of fear compared to placebo . EmotionTwo longitudinal studies of serially assessed patients with no therapeutic intervention have found that response accuracy did not change over time , 42. IndSocial cognitive performance in major depressive disorder is linked to the severity of depressive symptoms, as described in Table These findings remain consistent with previous evidence suggesting a negative interpretative bias in emotional recognition. Theory of mind ability is also affected, as one study showed that increasing depressive severity was associated with responses that showed poor understanding of mental states .In addition to severity of symptoms overall, the presence of certain severe symptoms also has an impact. Suicidal behavior and psychotic features are features that indicate a severe depressive illness and, as reported previously, both strongly correlate with the impaired interpretation of social stimuli , 21, 47.The link observed between social cognitive performance and severity of major depressive disorder could also help explain the discrepancies observed in the group difference results, as studies that have a high proportion of milder depression cases may not achieve significant results. Nevertheless, there were also some studies that found no association with symptom severity , while aThis review of the literature demonstrates a number of emerging trends in the way that individuals with major depressive disorder process and interpret socially salient information. Importantly, a negative bias may exist in the way that depressed subjects recognize emotion, which manifests as performance differences according to valence of emotion. Additionally, the review indicates that social cognitive performance may be related to severity of the depressive illness, with more pronounced deficits being associated with increased scores on depressive symptom scales. Some authors have suggested that depressed subjects may require greater intensity of emotion and a longer reaction time when interpreting emotional stimuli compared to controls.The literature, however, remains equivocal regarding many aspects of the relationship of social cognition and major depressive disorder, with a paucity of evidence in the domains of prosody and body language as well as limited data investigating patients in clinical remission. A noteworthy number of studies do not support these trends with some even finding no observed group differences between the depressed and controls in overall performance on tests of social cognitive function. This would suggest that any deficits in depression are subtler than in other disorders and may not be identified by broad measures of functioning.One explanation for this variation in findings could be the predisposition of depressed patients to negatively interpret social stimuli. This bias would appear to improve performance for negatively valenced stimuli whilst simultaneously impairing performance for positively or neutrally valenced stimuli. The net effect of this bias could reduce statistical significance for overall performance on a task. Thus, the true differences in facial affect recognition may be masked if valence is not taken into account.This observed negative interpretive bias on social cognitive tasks is mood-congruent and consistent with the cognitive paradigm of depression, as proposed by Beck , 50. ThiFew authors have addressed the important conceptual aspect of general cognition. Several cognitive domains, such as attention, memory, and executive functioning, have previously been found to be impaired in major depressive disorder , 52, impIndeed, difficulties with social interaction observed in patients with major depressive disorder may, at least in part, be due to an impaired ability to interpret emotional stimuli and mental states. In fact, social cognitive performance in depression may impact on the development of the disorder through impairing social functioning. Inaccurate assessments of feedback from interpersonal interactions exacerbate maladaptive thought processes, ultimately affecting mood. This could then diminish motivation to engage in social interaction, which would reduce reward from socializing and further intensify isolation. Awareness of social cognitive ability could be a beneficial component in cognitive-based psychotherapies.The discrepancies in results between some studies may be due to the variety of different tools employed to measure social cognition. Multiple measures exist for each component of social cognition and the tools are often tailored to the specific hypotheses of the investigation. This prevents accurate comparison between results, as well as limiting the reproducibility. Additionally, the majority of studies on social cognition use a unimodal design that exclusively focuses on one domain of social cognition, most commonly emotion recognition. There would be benefit in employing a study design that tests multiple domains in the same population. Some components of social cognition, such as prosody or body language, would also benefit from further directed research in major depressive disorder. It is difficult to make confident conclusions from only a small collection of studies.Even the term “social cognition” is not universally used throughout the literature, with some authors preferring “social perception” , 4. ThisA clear need exists for further research, particularly in domains such as prosody and body language interpretation, which have not yet been well-described in a major depressive disorder population. In general, larger longitudinal studies of major depressive disorder would be helpful in furthering understanding of the relationship between acute and remitted depression, as well as response to treatment. Agreement on or standardization of both terminology and testing in the area of social cognition would be a helpful advent and allow for greater reproducibility and ease for comparison of results.Understanding the relationship between major depressive disorder and social cognitive performance is important in characterizing both the etiology and phenotypology of major depressive disorder. In terms of potential implications for therapy, the results of two of the interventional studies included in this review indicated that treatment with anti-depressants, in particular citalopram, had a normalizing effect for a number of the observed social cognition defects of facial affect recognition. These findings suggest that the changes to social cognitive skills in depression are reversible. Despite being amenable to pharmacotherapy, it has not yet been established whether the observed findings impact on daily functioning and quality of life independent of that from depression. It is therefore important to quantify the functional burden, if any, of impaired social cognitive performance in major depressive disorder to determine its specific clinical relevance. Further research in this area would also be pertinent.Patients with major depressive disorder appear to interpret emotional stimuli differently to healthy controls, although this is not a consistent finding throughout the recent literature. The difficulties with social interaction and functioning observed in depressed patients may, at least in part, be due to an altered ability to correctly interpret emotions or mental states. It appears that depressed patients may struggle more with subtle or nuanced expressions of emotion, as well as demonstrating a mood-congruent bias for interpreting stimuli more negatively. These features are linked with severity of the illness and may persist even in the remitted state. However, it appears that some impairments are at least partially reversible using anti-depressants or psychotherapy. Further research is required in this area to better understand the functional impact of these findings and the role of targeted therapy in improving how depressed individuals interact socially.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The interocular suppression technique termed continuous flash suppression (CFS) has become an immensely popular tool for investigating visual processing outside of awareness. The emerging picture from studies using CFS is that extensive processing of a visual stimulus, including its semantic and affective content, occurs despite suppression from awareness of that stimulus by CFS. However, the current implementation of CFS in many studies examining processing outside of awareness has several drawbacks that may be improved upon for future studies using CFS. In this paper, we address some of those shortcomings, particularly ones that affect the assessment of unawareness during CFS, and ones to do with the use of “visible” conditions that are often included as a comparison to a CFS condition. We also discuss potential biases in stimulus processing as a result of spatial attention and feature-selective suppression. We suggest practical guidelines that minimize the effects of those limitations in using CFS to study visual processing outside of awareness. During our waking hours our eyes provide us with more sensory information than we can possibly process in detail, and only a small proportion of this information reaches awareness. At the same time, it would be adaptive for our brains to continue monitoring potentially relevant sensory signals, even those that do not culminate in a conscious experience. Indeed, several lines of research suggest that unperceived visual information can influence perceptual and cognitive operations, without our awareness  being presented to the two eyes, CFS critically involves a much less balanced design. During CFS one eye views rapidly flashing contour-rich patterns of high contrast (sometimes referred to as dynamic Mondrians), while the other views a stimulus that is typically stationary and of moderate contrast. The ever-changing patterns viewed by one eye cause periods of invisibility of the unchanging stimulus viewed by the other eye, and these periods can last for dozens of seconds, about 10 times longer than suppression produced with traditional binocular rivalry . CFS hasWhen looking at findings from studies using CFS, the evidence for stimulus processing outside of awareness seems compelling. As reviewed below, CFS suppression does not appear to preclude neural processing, either of low-level stimulus features, or of abstract stimulus properties with dedicated representations at more advanced stages of the visual system. In the case of low-level features, the notion that these are registered outside of awareness has also been confirmed using a number of other perceptual suppression techniques, including traditional binocular rivalry. With regard to more advanced stages of analysis, however, the picture from the literature as a whole is worth revisiting, given that there is little evidence for high-level processing during suppression phases of traditional binocular rivalry which, ironically, is reputed to create weaker interocular suppression than does CFS .In addition to this apparent discrepancy between CFS and traditional binocular rivalry, another motivation behind this review is our conviction that CFS experiments into processing outside of awareness, although often straightforward in their basic idea, are surprisingly complicated, and their design and interpretation are fraught with subtleties. Careful consideration of these subtleties is particularly important because work that uses CFS for this purpose may have a significant impact on current theories of neural information processing, emotional processing, and psychopathology. Indeed, the utilization of CFS has already found its way into clinical research , and so The objective of our paper is to recommend practical guidelines for researchers interested in exploring this technique as a means of investigating stimulus processing outside of awareness. Our recommendations are centered on answering four primary questions:(1) What are suitable paradigms to use with CFS to study processing outside of awareness?(2) What are the optimal ways to determine whether a stimulus is genuinely invisible?(3) What are effective methods for gaging the specificity and strength of stimulus processing outside of awareness?(4) What are factors that influence the robustness of stimulus-driven effects under CFS?We draw attention to several considerations that should be made when answering each of these questions. For the first question, we review the common approaches used to study processing outside of awareness with CFS. For the second question, we reexamine the methods used to assess observers’ perception during CFS suppression, since evidence for processing outside of awareness rests critically on demonstrating absence of awareness. This is particularly pertinent to behavioral priming and adaptation paradigms as well as neurophysiological studies that use CFS to examine stimulus processing outside of awareness. For the third question, we discuss the application of “visible” conditions used to provide a comparison for CFS conditions in order to determine the specificity and strength of stimulus-driven effects during CFS. For the fourth question, we discuss mechanisms that can potentially modulate stimulus processing under CFS such as those engaged in attention and feature-selective suppression. Following the discussion of each issue, we propose strategies for effectively resolving these questions and for minimizing methodological confounds in using CFS to study processing outside of awareness.From the outset we acknowledge that the matter of “processing outside of awareness” is fraught with controversy, with sharp points of disagreement within the field. Our views are unlikely to be received without question by all who are interested in this issue. We do hope that our views can advance the conversation in a constructive way.Figure 1). We focus on three types of behavioral effects that have been investigated to tackle this question: (1) adaptation aftereffects of suppressed stimuli, (2) priming effects evoked by suppressed stimuli, and (3) dependence of suppression duration on the nature of the suppressed stimulus. In each case we will also briefly address how recent CFS findings compare to previous findings obtained using traditional binocular rivalry.In this section, we review research that has used CFS to ask whether visual processing can transpire outside of awareness . A variety of different aftereffects exist, each specifically affecting detection or appearance of particular stimulus attributes, ranging from low-level properties such as orientation  to high-level features such as face identity and facial expression. Aftereffects have been widely used psychophysically to isolate and probe neural mechanisms involved in processing particular stimulus attributes . These procedures build on traditional priming paradigms that demonstrate improved performance on tasks that involve a target stimulus, when presentation of that stimulus is preceded by presentation of a (prime) stimulus that shares physical or conceptual (semantic) characteristics of the target stimulus . The assumption in these studies is that particular stimuli or categories of stimuli that emerge more quickly from suppression, relative to other stimuli, are being registered despite being suppressed owing to CFS. Unlike adaptation and priming paradigms in which stimulus awareness and behavioral effects driven by processes outside of awareness are measured independently, the “breaking continuous flash suppression” (b-CFS) technique provides a measure of stimulus awareness with which one may be able to infer processing outside of awareness. As a result of this property of b-CFS, there exists disagreement in the literature regarding the extent to which b-CFS actually provides a valid measure of unconscious processing  remain suppressed for shorter periods of time . Using tBreaking continuous flash suppression has also been used to examine whether other high-level properties, including lexical and semantic information, are processed outside of awareness. For instance, images of morphemes that are part of one’s native language tend to emerge from suppression faster than images of unfamiliar, foreign words . SimilarAgain we can compare these results obtained using b-CFS with those found using traditional binocular rivalry. Binocular rivalry findings are similar in showing that cognitively salient  stimuli exhibit predominance over less meaningful stimuli . For exaAdaptation, priming, and b-CFS are all adapted from well-established techniques in studying stimulus processing. Priming and b-CFS techniques may be suitable for investigating both perceptual and higher-level cognitive processes outside of awareness, whereas visual adaptation may be optimal for examining predominantly low-level visual processes and some complex ones as well . Before deciding which technique to use, experimenters should also consider the shortcomings of the current implementation of each technique, which are discussed throughout the remaining parts of this paper.As with any technique used to study subliminal perception, CFS studies that report performance or physiological measures indicative of stimulus processing outside of awareness must demonstrate that the stimuli were genuinely suppressed from awareness. In priming and adaptation paradigms, measures of awareness are assessed independently of the measure of processing outside of awareness . In contrast, b-CFS provides an index of awareness to infer stimulus processing outside of awareness, and so this section does not pertain to the b-CFS paradigm. In establishing the absence of awareness, some researchers advocate subjective measures e.g.,  whereas A very popular way of obtaining a subjective measure of awareness in the context of CFS experiments is to ask participants to report any occasion when they perceive another image besides the CFS suppressor. These subjective reports are then used to discard trials where suppression fails e.g., . Rather In other published studies, a 2-alternative categorization task has been used to infer the extent to which an observer is aware of a stimulus viewed together with CFS. In two versions of this approach, observers are instructed to either classify the suppressed stimulus into one of two object categories , which are more evident in the longer RTs measured in the invisible condition.Another aspect of the relatively small RT variability in the no-suppression condition is related to the fact that the ramping rate typically remains fixed across trials. This constant rate means that observers can develop strong expectancy effects for this condition, effects that are precluded in the invisible conditions by the temporal uncertainty engendered by the more variable durations of suppression. Any differences in expectation between the no-suppression condition and the invisible condition may also lead to differences in observer’s response strategies, and these differences are further exacerbated when no-suppression and invisible trials are presented in separate blocks, as often done in CFS studies .For certain CFS designs involving measures such as adaptation and priming, the concerns expressed above make it unwise to compare CFS conditions to conditions without any mask whatsoever. If the CFS masks are truly potent, only a small percentage of trials will fail to produce reliable suppression. It is then possible, with some adaptation paradigms, to compare trials with CFS masks in which suppression succeeded to those in which suppression failed . One potAs for the b-CFS paradigm, there are several improvements in the no-suppression condition that could be implemented. To minimize differences in RT distributions across no-suppression and invisible conditions, the rate at which a stimulus increases in luminance or contrast during a no-suppression trial can be varied to produce RT distributions similar to those measured in the invisible condition e.g., , 2012b. To circumvent effects attributed to differences in anticipation and response strategies, it is advisable for no-suppression and invisible trials to be randomly intermixed and, when possible, for response accuracy to be measured rather than RT . In factPutting aside, for now, the methodological challenges associated with designing a no-suppression condition for b-CFS, we would like to conclude with a conceptual point. Many studies using b-CFS have now reported stimulus factors that influence the time it takes to report an initially suppressed stimulus. Indeed, such effects have been reported for a broad range of stimulus categories, including words, scenes and faces. True to the logic of no-suppression conditions outlined above, these same studies did not find the same effects on RT when the stimuli were not suppressed, indicating the specific involvement of processes outside of awareness. This remarkable situation raises the question why preferential processing should occur only in the absence of awareness, and why the same mechanisms that affect processing outside of awareness would not influence conscious processing as well. One possibility is that similar influences do affect conscious processing as well, but that these influences can remain undetected for methodological reasons such as the ones outlined above. For instance, early studies have reported shorter RTs for upright faces than for inverted faces during b-CFS but not during visible control conditions, yet more recent work has shown similar effects for visible conditions as well . The morAs a final methodological suggestion for all CFS paradigms we would like to point to an approach in the literature that has not yet been used in combination with CFS, but whose properties may enable this approach to circumvent some concerns associated with no-suppression conditions. This type of approach, traditionally known as the process-dissociation method, capitalizes on experimental measures that differ qualitatively in awareness . This reWhile the relationship between attention and awareness remains controversial, there is growing consensus that that attention and awareness can to some extent be dissociated  but to an extent that depends on the similarity between that stimulus and the stimulus doing the suppression [feature-selective suppression, similar to that described by We recommend that observers’ attentional state be carefully controlled during tasks involving CFS. For example, attention can be cued to the location of the suppressed stimulus to maximize visual processing under suppression. Importantly, when select visual processes are hypothesized as being engaged automatically or in the absence of awareness, it should be made explicit whether these processes are also independent of observers’ attentional engagement. One common approach to examining the role of attention in visual processing outside of awareness is to compare the strength of stimulus-driven effects under conditions where attention is directed toward versus away from suppressed target stimuli.To avoid the potential effects of feature-selective suppression described above, we recommend that experimenters select target images that are similar in spatial composition  within and across stimulus categories or, better yet, create images comprised of different phase spectra but identical amplitude spectra using image processing techniques. Secondly, certain spatial properties can be normalized across stimuli such as spatial frequency amplitude, contrast, mean luminance, orientation content, shape, and size. Finally, we recommend that experimenters use CFS displays that are similar in spatial profile as the target stimuli to be suppressed, in order to maximize suppression of all components of the target stimulus. Alternatively, one can manipulate stimuli to have similar spatial profiles as the CFS display without necessarily altering stimulus recognition. Based on previous studies , achromaThose of us interested in visual processing outside of awareness have at our disposal an impressive array of tools for manipulating visual awareness. Among those tools, CFS is particularly appealing, since it offers several advantages compared to other techniques at rendering stimuli invisible. Not surprisingly, the technique has caught on within the field, and the volume of results for processing outside of awareness obtained with this technique is already quite substantial despite the technique’s appearance on the scene <10 years ago. Surveying studies that have employed CFS, one sees several different ways in which CFS is being exploited, each with its own subtleties that may influence the likelihood of finding evidence for visual processing outside of awareness. Researchers intending to use CFS will learn that multiple factors must be taken into consideration when using this technique; we believe the majority of those considerations can be grouped into four primary questions. In this paper, we have provided recommendations for addressing those questions, and those recommendations are briefly reiterated below.(1) What are suitable paradigms to use with CFS to study processing outside of awareness?• Measures of adaptation aftereffects are useful in examining neural processes involved in the encoding of visual attributes of a stimulus.• Priming paradigms allow researchers to examine processing of physical and conceptual (semantic) characteristics shared by two stimuli.• The b-CFS paradigm relies on the speed at which stimuli emerge from suppression to infer the relative strength of stimulus processing outside of awareness. This technique has been widely used in examining semantic processes outside of awareness.(2) What are the optimal ways to determine whether a stimulus is genuinely invisible?• To obtain a more complete evaluation of observers’ perceptual state under CFS with adaptation or priming paradigms, we advise the employment of multiple measures of awareness, which include measures that assess different states of awareness  and measures that gage different stages of stimulus analysis.• We recommend the use of statistical analyses that reduce the likelihood of falsely accepting the null hypothesis that observers’ performance on an awareness measure is not significantly different from chance, with the implication that stimuli were sufficiently rendered invisible.•within the main experiment, and that those measures be administered in ways that recreate, as nearly as possible, the attentional state and response strategies engendered during the main task when the target stimulus is putatively suppressed from awareness by CFS. We urge that awareness measures be implemented (3) What are effective methods for gaging the specificity and strength of stimulus processing outside of awareness?• Studies should strive to compare stimulus-driven effects found with CFS to those measured without suppression.• The no-suppression condition(s) should be matched as closely as possible to the invisible condition.• We recommend that no-suppression and invisible trials be randomly intermixed to minimize potential differences between conditions .• For the b-CFS procedure, the no-suppression condition should be individually tailored for each participant to produce similar RT distributions in the behavioral task and similar perceptual experiences for the no-suppression and invisible conditions.(4) What are factors that influence the robustness of stimulus-driven effects under CFS?• The spatial location of an observer’s attentional engagement can modulate visual processing under CFS. Thus:◦ It is advisable to hold an observer’s spatial attention constant across trials, particularly directing attention to the location of the stimulus being suppressed so as to maximize the likelihood of stimulus processing under CFS.◦and without attention. Researchers should also consider manipulating an observers’ spatial attention to test whether stimulus processing is engaged without awareness • Low-level features of target stimulus and CFS display can modulate the strength and selectivity of suppression.◦ Target images should be closely matched in spatial composition with one another and with the CFS mask.The Unconscious (In closing, we are excited about the future opportunities for learning more about stimulus processing outside of awareness, and we are confident that CFS can provide one effective means for pursuing that question. Coincidentally, the recent resurgence of interest in processing outside of awareness coincides with the forthcoming one-hundredth anniversary of the publication of one of Sigmund Freud’s most famous essays, onscious . For deconscious  to arrivThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In modern day radiotherapy, the emphasis on reduction on volume exposed to high radiotherapy doses, improving treatment precision as well as reducing radiation-related normal tissue toxicity has increased, and thus there is greater importance given to accurate position verification and correction before delivering radiotherapy. At present, several techniques that accomplish these goals impeccably have been developed, though all of them have their limitations. There is no single method available that eliminates treatment-related uncertainties without considerably adding to the cost. However, delivering “high precision radiotherapy” without periodic image guidance would do more harm than treating large volumes to compensate for setup errors. In the present review, we discuss the concept of image guidance in radiotherapy, the current techniques available, and their expected benefits and pitfalls. Radiotherapy has always required inputs from imaging for treatment planning as well as execution, when the treatment target is not located on the surface and inspection and visual confirmation are not feasible. Traditional radiotherapy practices incorporate use of anatomic surface landmarks as well as radiologic correlation with two-dimensional imaging in the form of port films or fluoroscopic imaging.Sophisticated imaging techniques such as contrast enhanced computed tomography (CECT) scans, magnetic resonance imaging (MRI), positron emission tomography (PET) scans, and angiography obtain three-dimensional (3D) structural and biologic information which is used to precisely define the target and thus enable precise and accurate treatment planning with shaped beams in isocentric or non-isocentric geometry.“In-room” imaging methods  obtain periodic information on target position and movement (within the same session or between consecutive sessions), compare it with reference imaging, and give feedback to correct the patient setup and optimize target localization. They also have the potential to provide feedback that may help to adapt subsequent treatment sessions according to tumor response.Broadly, imaging has two major roles in radiotherapy:More specifically, modern day radiotherapy regards the latter application with “in-room” imaging as “image guided radiation therapy” (IGRT).Modern external beam radiotherapy techniques such as intensity modulated radiation therapy (IMRT), volumetric modulated arc therapy (VMAT), stereotactic radiosurgery (SRS), or stereotactic radiotherapy (SRT) have helped reduce the safety margin around the target volumes thus allowing for lower normal tissue doses without compromising delivery of tumoricidal doses. However, there is a great deal of uncertainty in accurately defining of the position of targets during the delivery of fractionated radiotherapy, both during a given fraction and between successive fractions. Targets that may move during treatment due to respiratory or peristaltic movements or with cardiac pulsations create an even bigger challenge. Hence, there is need to develop and implement strategies to measure, monitor, and correct these uncertainties. This has led to evolution of various in-room imaging technologies which enable evaluation and correction of setup errors, anatomic changes related to weight loss or deformation, or internal organ motion related to respiration, peristalsis, or rectal/bladder filling.Brachytherapy treatment planning also incorporates orthogonal X-ray imaging and fluoroscopy for guiding brachytherapy catheter/applicator placement, volumetric imaging with CT or MRI for applicator identification and reconstruction, and plan optimization in three dimensions based on imaging. Isodose distribution is reviewed and optimized on visualizing dose distribution to the target as well as critical structures. This adds to treatment efficacy and safety.Though technically complex, surgical procedures enable the operating surgeons to directly visualize and handle their targets, thus eliminating the ambiguity in identification and appropriate management. Radiotherapy, despite being a local therapy that aims to achieve similar goals, inherently carries the disadvantage of making a significant number of assumptions when using traditional treatment techniques. The 3D image dataset acquired at simulation is a snapshot of the tumor, its relation to normal structures, and the patient's shape and position at a single time point, and it is this model that is used for plan development and dose calculation. During the planning stage, a lot of assumptions based on prior experience and literature are used with regard to clinical target volume (CTV) margins to define the microscopic spread around the tumor and planning target volume (PTV) margins to incorporate the expected range of internal organ motion and setup errors. The treatment is then carried out with the belief that all those assumptions would hold true for any given patient during daily treatment sessions, the foremost being that the patient and tumor anatomy and their positions with respect to the positioning devices have remained unchanged since the time of simulation. However, the assumption that the dose calculated on the CT dataset on the planning system matches the dose delivered through each fraction or through the entire radiation therapy course is grossly in error. Additionally, the internal organs and targets move with respiration and peristalsis and planning radiotherapy on a static image dataset is unable to account for errors due to this motion. To ensure that all of these assumptions do not compromise the dose delivery to the CTV, wider PTV margins are taken. This causes a large volume of normal tissues to be included in the irradiated volume. IGRT gives a method to capture this information regularly during the treatment course in the form of serial “snapshots” and is a means of verifying accurate and precise radiation delivery. In simple terms, the IGRT process ensures that the delivered treatment matches the intended treatment in accurately targeting the tumor while minimizing “collateral damage.” Changes to the composite delivered dose and their impact on disease control as well as toxicity may be minimized by use of appropriate localization devices and PTV margins. Occasionally, replanning may be required if gross deviations beyond predetermined tolerances are observed –3.IGRT allows assessment of geometric accuracy of the “patient model” during treatment delivery. It provides a method whereby deviations of anatomy from initial plan are determined and this information is used to update dosimetric assumptions. Correction strategies may include daily repositioning to register patient position in accordance with the base plan or recalculation of treatment delivery in real time to reflect the patient's presentation during a given fraction. This philosophy of reevaluating treatment and accounting for the differences between actual patient anatomy on a given day and the snapshot of planned treatment is known as adaptive radiotherapy . The eveAn error in radiotherapy delivery is defined as any deviation from intended or planned treatment. A great degree of uncertainty is inherent to radiotherapy practices and may be in the form of mechanical uncertainties related to treatment unit parameters such as couch and gantry motion, patient uncertainties related to ability to lie comfortably in a certain position and cooperate during the treatment time, geometric uncertainties related to position and motion of target, and dosimetric uncertainties. IGRT deals with the geometric uncertainties, which may be either intrafractional or interfractional , 6.Both inter- and intrafractional uncertainties may be a result of a combination of systematic and random errors.A systematic error is essentially a treatment preparation error and is introduced into the chain during the process of positioning, simulation, or target delineation. This error, if uncorrected, would affect all treatment fractions uniformly. A random error, on the other hand, is a treatment execution error, is unpredictable, and varies with each fraction. Systematic errors shift the entire dose distribution away from the CTV, while random errors blur this distribution around the CTV. Of the two, systematic error is more ominous since it would have a much larger impact on treatment accuracy and hence the therapeutic ratio.Margins are added to the CTV to take these errors into account. These margins are geometric expansions around the CTV and may be non-uniform in all dimensions depending on the expected errors. These margins ensure that dosimetric planning goals are met despite the variations during and between fractions. ICRU 62 defines the expansion of PTV around the CTV as a composite of two factors—internal target motion  and setup variations (setup margin) . DependiOffline and online IGRT correction strategies refer to whether the patient is on the treatment couch while the verification is being done and whether the correction would be applied to the same or subsequent sessions.In the offline strategy, images are acquired before treatment and matched to the reference image at a later time point. This strategy aims to determine the individual systematic setup error and thus reduce it. When combined with setup data of other patients treated under the same protocol, it helps define the population standard error for that treatment in that institution. Widely used offline correction protocols include Shrinking action level and No action level protocols , 11. PTVAn online strategy, on the other hand, employs acquisition of images and their verification and correction prior to the day's treatment. It aims to reduce both random and systematic errors. The treatment site and the expected magnitude of error may determine the frequency of online imaging. Sites where large daily shifts are anticipated  or where even slight shifts will alter the dose distribution within adjacent critical structures  are best managed with daily imaging. Our experience with online corrections showed the maximum errors in thorax followed closely by abdomen and pelvis. The minimum errors were observed in head and neck region . AdditioDepending on the imaging methods used, the IGRT systems may broadly be divided into radiation based and nonradiation based systems , 15.These may employ ultrasound, camera-based systems, electromagnetic tracking, and MRI systems integrated into the treatment room.These systems  acquire 3D images that help align targets to correct for interfractional errors. Geometric accuracy is 3–5 mm and the greatest advantage is lack of any ionizing radiation. Sites of common application include prostate, lung, and breast radiotherapy.These systems identify the patient reference setup point positions in comparison to their location in the planning CT coordinate system, which aids in computing the treatment couch translation to align the treatment isocenter with plan isocenter. Optical tracking may also be used for intrafraction position monitoring for either gating (treatment delivery only at a certain position of target) or repositioning for correction. Tools such as AlignRT image the patient directly and track the skin surface to give real time feedback for necessary corrections. These systems have found application in treatment of prostate and breast cancer and for respiratory gating using external surrogates. Geometric accuracy is 1-2 mm, but application is limited only to situations where external surface may act as a reliable surrogate for internal position or motion.These systems  make use of electromagnetic transponders (beacons) embedded within the tumor, and motion of these beacons may be tracked in real time using a detector array system. Beacons need to be placed through a minimally invasive procedure, their presence may introduce artifacts in MR images, and there are limitations to the patient size. Calypso has a geometric accuracy of <2 mm, but its use at present is limited to prostate radiotherapy.These systems  help real time assessment of internal soft tissue anatomy and motion using continual soft tissue imaging and allow for intrafractional corrections. Geometric accuracy of the system is 1-2 mm. However, MRI has certain drawbacks such as motion artifacts, distortion with non-uniform magnetic fields, and cannot be performed for patients with pacemakers or metallic implants. All these limitations of diagnostic MRI apply to this IGRT system as well. A wide application potential exists in treatment of prostate, liver, and brain, as well as for brachytherapy.These include static as well as real time tracking, using either kilovoltage (KV), megavoltage (MV), or hybrid methods.EPID was developed as a replacement of film dosimetry for treatment field verification and is based on indirect detection active matrix flat panel imagers (AMFPIs). They are offered as standard equipment by nearly all linear accelerator (LINAC) vendors as both field verification and quality assurance (QA) tools. Image acquisition is 2D, with a geometric accuracy of 2 mm. Bony landmarks on planar images are used as surrogates for defining positional variations respective to the digital reconstructed radiographs (DRRs) developed from the planning CT dataset . DiffereThese systems consist of retractable X-ray tube and amorphous silicon detectors mounted either orthogonal to  or along the treatment beam axis (Siemens Artiste). These have capability of 2D, fluoroscopic and CBCT imaging. Another system  consists of a gimbaled X-ray treatment head mounted on an O-ring with two KV X-ray tubes, two flat panel detectors, and an EPID. The O-ring can be rotated 360 degrees around the isocenter and can be skewed 60 degrees around its vertical axis. Geometric accuracy is 1 mm or lesser with possibility of 2D and 3D matching with DRRs or X-ray volumetric images generated from planning CT data sets. Scanning is done through a continuous partial or complete gantry rotation around the couch, acquiring the “average” position of organs with respiratory motion. Both interfraction setup changes and anatomical changes related to weight changes or organ filling  may be monitored . Repeat This system has an in-room CT scanner and gantry that moves across the treatment couch/patient, which can be rotated towards either the scanner or the gantry for imaging and treatment, respectively. 3D images are taken with the patient immobilized on the couch, the difference from a diagnostic CT being a larger bore size (>80 cm diameter) to accommodate bulky immobilization devices, and a multislice detector. Accuracy and applications are similar to CBCT with average dose of 10–50 mGy per image .This includes an on-board imaging system to obtain MV CT images of the patient in treatment position. The same LINAC is used to generate both the treatment (6 MV) and imaging beam (3.5 MV). A xenon detector located on the gantry opposite the LINAC collects exit data for generation of MV CT images. Patient dose from imaging varies with pitch setting and is typically 10–30 mGy per scan .2D KV Stereoscopic Imaging (CyberKnife). The Accuray CyberKnife robotic radiosurgery system consists of a compact LINAC mounted on an industrial robotic manipulator arm which directs the radiation beams to the desired target based on inputs from two orthogonal X-ray imaging systems mounted on the room ceiling with flat panel floor detectors on either side of couch, integrated to provide image guidance for the treatment process. Images are acquired throughout the treatment duration at periodic intervals ranging from 5 to 90 seconds, and the couch and robotic head movements are guided through an automatic process. Several tracking methods may be used depending upon the treatment site  System. This system is designed for real time tracking of tumors by imaging implanted fiducials and using this information for gating. It consists of four X-ray camera systems mounted on the floor, a ceiling-mounted image intensifier, and a high-voltage X-ray generator. The LINAC is gated to irradiate the tumor only when the marker is within a given tolerance from its planned coordinates relative to the isocenter [socenter , 35.VERO. This system has two X-ray tubes and corresponding flat panel detectors and uses a combination of initial couch motion and a pair of radiographs for patient alignment. The couch is capable of 3D alignment for initial coarse setup and then the on-board imaging subsystem helps fine-tuning. A pair of radiographs is acquired and registered with prior DRRs using bony landmarks to evaluate the translational and rotational shifts. The system can also compensate for organ motion [n motion .ExacTrac X-Ray 6-D Stereotactic IGRT System. It uses a combination of optical positioning and KV radiographic imaging for online positioning corrections. There are two main subsystems: an infrared-based system for initial patient setup and precise control of couch movement using a robotic couch and a radiographic KV X-ray imaging system for position verification and readjustment based on internal anatomy or implanted markers. Infrared system may also be used for respiratory monitoring and signaling to LINAC for beam tracking and gating. Novalis Tx combines this system with an additional on-board imaging system  on a multiphoton/electron beam LINAC [am LINAC , 38.American College of Radiology (ACR) and the American Society for Radiation Oncology (ASTRO) jointly developed guidelines for IGRT that define the qualifications and responsibilities of personnel including radiation oncologists, medical physicists, dosimetrists and radiation therapists, QA standards, clinical implementation, and suggested documentation. Similar guidelines have also been proposed by European agencies –41. A suQualifications. Respective personnel should obtain appropriate certification with specific training in IGRT before performing any stereotactic procedures.ResponsibilitiesRadiation Oncologist. (i) Conduct of disease-specific treatment, staging, evaluation of comorbid conditions and prior treatments, exploration of all available treatments including discussion of pros and cons of IGRT, treatment, and subsequent follow-up.(ii) Determination of the most appropriate patient positioning method, recommendation of the appropriate approach to manage organ motion, supervision of simulation paying particular attention to positioning, immobilization and appropriate motion management, determination and delineation of target volumes and relevant normal critical structures using available imaging techniques, communication of expected goals and constraints and collaboration with the physicist in the iterative process of plan development to achieve the desired goals, supervision of treatment delivery and determination of acceptable day-to-day setup variations, and participation in the QA process and subsequent approval.Medical Physicist. (i) Acceptance testing and commissioning, assuring mechanical, software, and geometric precision and accuracy, as well as image quality verification and documentation in a given IGRT system.(ii) Implementation and management of a QA program.(iii) Development and implementation of standard operating procedures (SOPs) for IGRT use, in collaboration with the radiation oncologist.Dosimetrist. (i) Normal structure delineation under the guidance of radiation oncologist.(ii) Management of volumetric patient image data (CT and other fused data sets) on radiation treatment planning (RTP) system.(iii) Generation of a treatment plan under oncologist's and physicist's guidance.(iv) Generation of all technical documentation for IGRT plan implementation.(v) Assisting with treatment verification.Radiation Therapist. (i) Understanding and appropriate use of immobilization/repositioning systems.(ii) Performance of simulation and generation of imaging data for planning, implementation of treatment plan, acquisition of periodic verification images under supervision and periodic evaluation of stability and reproducibility of the immobilization/repositioning system, and reporting inconsistencies immediately.Fiducial Markers. These serve as surrogates to soft tissue targets when they are difficult to visualize and their alignment cannot be related to bony anatomy. These may be tracked in real time to obtain 3D coordinates of the target for subsequent corrections.Moving Targets and Delineation. Intrafraction target motion or interfraction displacement, deformation, or alteration of targets and other tissues should be accounted for during determination of PTVs. Appropriate motion management methods should be chosen depending on available expertise and degree and type of motion. This process starts at the time of simulation and continues throughout till the end of therapy.Patient Positioning. It is imperative to ensure the accuracy of patient position and its reproducibility for fractionated treatments relative to the chosen IGRT device as well as treatment unit.Image Acquisition. The IGRT system should be calibrated to ensure high imaging quality with attention to slice thickness uniformity, image contrast, spatial resolution, isocenter alignment between imaging and treatment planning and delivery systems, accuracy of software used for identification, and correction of couch misalignments. Relevant QA procedures should ensure reliability and reproducibility of the entire process.Treatment Verification. Image review by radiation oncologist at the first fraction and then periodically is necessary to ensure treatment accuracy and reproducibility. Each department should determine its own threshold of couch positioning changes that would necessitate setup review or change before treatment delivery.Quality Assurance and Documentation. A documentation of all the necessary QA procedures throughout the course of simulation, treatment, and periodic verification should be maintained. These would help determine departmental thresholds for action as well as serve as guides for modification of the processes involved following review of findings.Use of the IGRT process has improved our awareness and understanding of daily inter- and intrafractional setup variations and motion. Real time tracking has helped quantify interpatient and intrapatient variations in lung and liver tumor motion related to breathing and complexities of such motion have become clearer. We now understand that even when breath-holds are repeated, the relative position of soft tissue and skeletal structures may vary, rendering use of bony landmarks useless for such endeavors. Changes in prostate position  have been quantified and we can better correct for these errors as well as tailor PTV margins to these findings, thus allowing more accurate targeting. Understanding of the various IGRT techniques, their applicability, limitations, and additional radiation hazards helps the radiation oncologist take an educated decision on the method best suited to a particular clinical situation for maximizing benefit from radiation therapy. Changes in parotid position relative to the tumor in head and neck cases, change in body contour due to weight loss, seroma, or body fluid collections, change in prostate position relative to bladder or rectal filling and effect of bowel gas, reduction of tumor size during treatment, and changes in spinal position during spinal or head and neck radiotherapy are situations which were never even considered of significance in the pre-IGRT era and their respective roles and solutions are being developed as we are understanding their role during treatment. With better geometric precision, volume of irradiated healthy normal tissue can be significantly reduced with reduction in toxicity risks. Adaptation to reduction in tumor volume may lead to additional gains in normal tissue toxicity reduction.Results from ongoing and future trials will hopefully demonstrate the net gain in therapeutic ratio from application of IGRT technologies and the onus lies on the radiation oncology community to take up the challenge of demonstrating the benefit of these potentially expensive approaches.IGRT is most likely to benefit clinical situations where the tumor is in close proximity to sensitive healthy tissues, when doses required for disease control exceed the tolerance levels of adjacent normal tissues or when large organ motion and setup errors may result in severe consequences of positional errors. All patients treated with conformal radiotherapy, IMRT, and SBRT should, in theory, benefit from IGRT. Thoracic and upper abdominal targets with significant respiratory motion, obese patients, head and neck cancers, paraspinal and retroperitoneal sarcomas, and prostate cancer are situations that are expected to derive maximum benefit with some clinical experience forthcoming. Clinical situations where even low dose irradiation produces excellent local control, palliative radiotherapy delivered using large fields, and superficial tumors that are amenable to direct visual inspection are likely to derive least benefit from IGRT.Limited availability of experienced trained staff is a major hurdle in wide application of the technique despite its demonstrable benefits, even with the simplest approaches. Other factors that need consideration include quality control, algorithms that define the decisions whether to change a plan or continue with original plan, and need for commercial development of software as well as hardware to match clinical needs and demands. Another major concern regarding frequent on-treatment imaging is the radiation dose to normal tissues. Although the doses from IGRT appear insignificant, only long term follow-up will define any potential risk of second malignancies from low dose exposure. Thus, there is an ongoing debate on the necessary frequency of verification imaging especially when using ionizing radiation. Recent developments in MR-LINACs have tried to address these concerns while allowing daily imaging for treatment verification. Another concern is that of treatment safety since the technologies available in the clinic require integration of hardware and software from different vendors. Clinical use of any system should be preceded by proper acceptance testing, commissioning, and routine QA used to assure accurate regular functionality. Education of all users  on safe use and clinical utility is mandatory, along with knowledge of additional dose and possible risks associated with use. No single technology is ideal in every scenario and no single institution can manage to integrate all or most technologies in one place. Only time will tell which of these methods gain wider popularity and acceptance, based on clinical relevance and ease of use.Use of IGRT systems is essential to treatment of any site where setup deviations and organ motion are anticipated. Additional gains are monitoring of treatment response, weight changes, and organ filling on day-to-day basis. With improved precision of planning systems, use of SRS or SRT, and high dose hypofractionated regimens, the chances of small deviations leading to significant errors in treatment delivery are much higher, and the use of IGRT is far more critical in these situations. Integration of LINACs with MR-based soft tissue imaging and PET-based biological imaging may help even further improve targeting accuracy in the future , 43. How