Many users of Internet interventions do not persist with the full treatment program. As persistence may influence outcomes of such interventions, being able to maximize persistence is vital. However, while studies have begun to explore the predictors of dropout in Internet interventions, few have explored reasons why users persist with the programs, which may not just be the converse of the reasons for dropout.To answer the question of what influences persistence with online interventions.We interviewed participants in the Cardiovascular Risk E-couch Depression Outcome (CREDO), a trial evaluating the efficacy of an eHealth intervention (e-couch) in treating depressive symptoms in those with comorbid depression and cardiovascular risk factors. Interviews were semistructured in nature and were analyzed using a grounded theory approach. Interview numbers were curtailed (n = 12) after theoretical saturation.All participants reported substantial barriers to completing the program including time constraints, competing priorities, anxiety about spending time on the computer, and perception of limited worth of the intervention. Participants who persisted with the trial reported intrinsic motivations such as personal values about task completion and sense of control, and recognized external motivators that aided the development of habits and identified personal benefits attributable to the program.Online interventions may benefit from content that enhances the intrinsic motivations such as a having sense of control and being able to identify with the program, and by increasing the relative value of the program in order to enhance persistence. Persistence within a trial setting appears modifiable through explicit messages regarding supporting others. In terms of motivators, the use of a hook to engage participants who are starting the intervention due to curiosity and the use of reminder systems to prompt participants may also improve persistence. The worth of such additions should be evaluated using adherence and outcomes metrics.Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12610000085077; http://www.anzctr.org.au/ACTRN12610000085077.aspx (Archived by WebCite at http://www.webcitation.org/68MtyPO3w) As acceptability of the Internet and Web-based programs increases -5, so toIncreasing interest in online interventions or e-therapy has largely been driven by the belief that delivering interventions over the Internet overcomes some of the barriers of traditional face-to-face therapy. Such issues include geographical access, time restrictions, cost of accessing services, and reluctance to seek help -18. The While online interventions have been shown to be effective -27, littResearch into program engagement, particularly the reasons for nonadherence and poor persistence, is sparse despite the thinking that the degree of engagement in self-directed or self-help online therapy is as important as engagement in face-to-face therapies ,38,39 foMaximizing a participant’s motivations to persist seems a self-evident goal of an e-therapy program ,42, evenWhile these two studies provide an understanding of some of the factors that influence adherence and the translation of online to offline behavior in their perspective populations, no clear model exists to explain the role of motivation in influencing persistence in online interventions. Grounded theory is a form of systematic qualitative inquiry that allows the generation of theory from data. This methodology is useful for the development of new theories and ideas in areas where theory does not readily exist, such as the area of motivation in online interventions and e-therapy. This study took a grounded theory approach to address the question of what influences persistence with online interventions in order to propose a model to aid program development.We conducted face-to-face and telephone interviews with participants in the Cardiovascular Risk E-couch Depression Outcome (CREDO) study, which evaluated the efficacy of an eHealth intervention (e-couch) in treating depressive symptoms in those with comorbid depression and cardiovascular risk factors .Participants, recruited from those engaged in the study between the 6- and 12-month follow-up stages, were invited by email to participate in the qualitative study. Participants who were less than 2 weeks away from an assessment were excluded, so as not to add burden to their participation for the final survey. We sent 80 email invitations to participants with the aim of recruiting approximately 10% of these. Participants were invited only once. Participants were selected at random from those eligible using a random number generator. Recruitment occurred in three waves and continued until theoretical saturation (where no new insights emerged from the data)  was reacE-couch is a 12-week online cognitive behavioral therapy intervention for depression. The program contains elements pertaining to psychoeducation, activity scheduling, thought challenging, problem solving, and interpersonal therapy. The program is fully automated with a new module opening each week. During the trial, participants received a reminder email 3–4 days after the module had opened. They also received a scripted reminder, but not therapeutically assisting, telephone call from a research assistant if they had still not completed the module 1 week after it had opened.We obtained written informed consent from all participants. Ethics approval for the CREDO trial and the qualitative substudy was obtained from the University of Sydney Human Research Ethics Committee. The University of New South Wales Human Research Ethics Committee provided ethics approval for the 45 and Up Study.Participants in the study were residents of New South Wales, Australia, who had enrolled previously in the 45 and Up Study  and, subWe conducted 10 telephone and two face-to-face interviews between August and December 2011 at a location and time of the interviewee’s convenience. The interviews were conducted and analyzed using a grounded theory approach, where themes and theories were allowed to emerge without using preconceived hypotheses and ideas . An initAfter we received informed consent from participants, we audio recorded interviews using an electronic dictating machine.On completion of interviews, the interviewer transcribed audio files verbatim and then checked the transcriptions against the audio recordings. Interview transcripts were analyzed and coded as soon as possible after each interview. Handwritten field notes and impressions of the interview were also used to inform the analysis and generation of memos. The lead researcher analyzed typed transcripts of the interview using codes developed from written comments on the transcription. These comments were analyzed using a grounded theory approach emphasizing the use of iterative techniques, whereby data and emergent theory are constantly compared . During In total, two central themes of barriers and motivators emerged, each with several subthemes. All participants reported substantial barriers that decreased their motivation to continue with the program. These included time constraints and competing priorities, technology fatigue from spending the day on the computer at work, anxiety about spending time on the computer away from other demands of day-to-day life, and perception of limited worth of the intervention.Participants who persisted with the trial identified intrinsic motivations and extrinsically motivated strategies to overcome the barriers that they faced in persisting with the trial. These included developing habits, recognizing personal values about completion, and identifying the benefits for others if the benefits for themselves were not immediately obvious.The competing demands for time and need to prioritize also appeared to result in many participants “forgetting” to complete their module and associated activities on a weekly basis. Many participants alluded to relying on weekly reminders that a module had opened, rather than initiating seeking out the new module themselves. Forgetfulness appeared to be particularly prevalent when the participants postponed completing a module until a later time. Many stated that they would put the module off, intending to complete it when they would be able to focus on the intervention without competing demands, but often forgot to return to it. They often forgot when other demands on their time arose or when they lacked regular computer time, resulting in a lack of visible reminders that the program was open.Interviewee #11It was good, but I did especially appreciate the reminder because sometimes it came through at a busy time, um, I didn’t mean to forget about it but it happens. And I was thankful for the reminder.Mood and anxiety were also often cited as barriers to engagement. When anxiety was high, participants found it difficult to find the time or concentrate on the program. When mood was low, participants lacked motivation to complete the program, often feeling overwhelmed by its demands. This resulted in participants focusing on other more manageable tasks, which allowed them to feel that they had completed at least some tasks throughout the day. Tasks of daily living were often prioritized ahead of the program, even when participants believed that they would benefit from taking the time to complete the program and objectively had the time.Interviewee #1...I get anxious, and then I begin to think to myself that “I’ve got to do this” and “I’ve got to do that,” and “I can’t do my [program] now, I’ll do it later,” and really there is nothing that can’t wait. Nothing at all. I have a set routine. I’m retired. But I get myself into such a state of anxiety that I can’t relax and do my [program]. So I leave it and go and do my silly little things such as taking my dog for a walk and doing my shopping. All sorts of, you know, mundane things that are not important, well they are important, but I could give myself the time to relax and do it...Several participants reported frustration with the program’s lack of personalization. This was particularly reinforced by frustration with the standardized questionnaires embedded in the program and trial. Participants frequently cited that the answers were unlikely to reflect their true state, as they did not believe that they fit within the categories provided. This resulted in them feeling frustrated and wanting to stop completing the questionnaires. Many indicated that the capacity to provide additional information in terms of a free-text box would be beneficial and would enhance their desire to persist with the program.Interviewee #4I found the questions strange. The question that said “Do you do what your doctor tells you?,” I’m going “I’m not 4,...how am I going to respond to this?” And in the end I throw my arms in despair and go “Urgh! I don’t know, I don’t know the answer!” and put it in the appropriate box...I found that when I clicked on the “Do I take x, y, z medicine” and put “Yes” and the window came up and gave me space to write in it. I want more of those windows.Other participants were concerned that their answers were being misinterpreted, which may have influenced their answers and therefore the time taken to answer the questionnaires or complete program activities. This was reflected in the number of emails participants sent, unsolicited, to the project team to contextualize their answers to the questionnaires.The perceived unidirectional relationship between the user and the program also led participants to consider that how they engaged in the program was irrelevant and decreased their motivation to continue. They felt that the computer offered little in terms of support, interaction, and feedback. Participants frequently reported thoughts of giving up on the questionnaires and the trial due to feeling that their information would be misrepresented and therefore not useful to the trial team.Interviewee #1Because you need a bit of feedback...It’s a bit of an empty system, because...you know...you put out a few stories about your thoughts and experiences, but you know...nothing comes back.Although some participants reported that this unidirectional nature of the relationship was beneficial, for others, it reduced any obligation to log on to the system and use it. The sense of accountability was decreased, as there was no feedback or cost of engaging with the program. A lack of perception of the program being a therapeutic relationship and of someone relying on their attendance meant that several participants felt that they were able to put off completing the program. The freely available nature of the program, without cost, waiting lists, or time restrictions, meant that participants placed less value on completing the program when it was opened to them.Interviewee #1...it’s a lot easier to ignore. Um...and truly and if you’ve made an appointment and you know you have to pay for it, you’re going to turn up.Participants who felt they were not learning anything reported lower levels of motivation to persist with the program. Some had had previous psychological treatment for depression and felt that the program did not offer them anything substantially different.Interviewee #1...think I was a bit of a difficult ah...participant, because I know a lot about uh...um...emotional therapies. And...and...a lot about the theories, so...um... it was uh...it was a bit tedious to go through each of the programs and the theories and then the examples for me...Interviewee #2...a lot of the stuff we did online was not new...and that frustrated me.Interviewee #10Yeah, and the other thing you know, it’s long-winded. I can, sure there are some people, but for people who have, maybe, who have been to university, who have a certain level of understanding of these problems or a certain level of education um, it was too simplistic for me.Previous therapy experiences also meant that some had different expectations of the therapeutic relationship. The program therefore offered reminders and information, but not therapy as such. This meant that participants struggled to engage in the computer intervention and to see it as therapy.Interviewee #1Um...ah...because you have no feedback, they’re like chalk and cheese really. Well, yes...it was educative, but I didn’t feel like it was a therapy session...Those participants who perceived little personal benefit from the program reported persisting due to the perception of obligation to the researcher or the belief that their input would help the wider community. They appeared to hold values around completion and contributing to society that allowed them to continue to persist with the program.Participants reported several strategies and motivations that allowed them to complete the program on a (semi)regular basis. These strategies were rarely linked to a specific intervention barrier but were rather seen as intrinsic traits or behaviors that allowed them to continue engaging with the program.The perception of receiving a benefit from the program was considered to be the primary reason many participants persisted with the intervention. Participants reported experiencing benefits of the program beyond a sense of accomplishment and looking forward to completing the next part of the program. These participants frequently spoke about the benefits of the program and how they were implementing changes from the program into their daily lives.Interviewee #3...if I thought to myself this is useless I’m not getting anywhere. I possibly wouldn’t finish it...I think that it was very, very interesting. There was a heck of a lot to learn from it, there was a lot I found, the reading, everything, I found I quite...I enjoyed it. There you go.Participants who saw personal value often used the language of the program. They were able to recall tasks they completed, appeared to have a better recall of the program, and continued to refer back to the program after they had completed it. They reported an overall sense of satisfaction that carried through each module and they appeared engaged in the program as a whole.Interviewee #5I felt satisfied at the end of it. That I’d done it, it was interesting and it was...I suppose it reinforced what I probably should be doing myself. It was easy to do and pleasant, and yeah I felt good about doing it...Several participants reported that they had dropped out of previous face-to-face therapeutic relationships due to the nature of the interactions. They reported feeling as though the therapist had been restrained by the amount of time available and that they were rushed by the time allocated to individual sessions. This resulted in their feeling unimportant and merely a patient rather than a person, and that their agenda was not as important as the agenda of the therapist.Interviewee #3...you could sit there and just actually take your time to do it. You know you could really think. Whereas when you’re talking to somebody and you’ve got an hour or three-quarters of an hour or something, you really kind of you know...so time to me is the important thing.Being able to dictate the pace in the online intervention meant that they felt they got more benefit from it. The ability to choose which activities to complete and what areas to focus on for the activities resulted in many feeling as though their agendas were catered for in the program rather than that of the therapist. Due to this, they felt that they could be more honest with the program, and this led to greater reflection.Interviewee #11I think when I reflect on therapy you’ve got the lead up, going to [laughs] therapy, the anxiety beforehand, you know. You’re going into the therapist’s room, you’re in someone else’s domain, but it’s like being in front of a stranger, you know, you’re in front of a stranger and you feel you have to perform a little bit. [Laughs] whereas if you’re by yourself, controlling it yourself, I think you can be more reflective about yourself.Many participants reported returning to the intervention several times. This allowed them to reflect on what they had learned. This appeared to facilitate the program being incorporated into everyday life. Those who returned to the program several times were more likely to find the program material novel and interesting. Several of these users often frequently returned to other trusted websites when they needed information. These participants appeared to view the program as a useful tool for themselves, rather than a research project where their contribution would be more valuable to others instead of themselves.The ability to be flexible in the completion of the program also meant that participants were able to overcome time-related barriers, allowing them to complete modules in sections, when time allowed. Sometimes this appeared to be prompted by reminders that were part of the program.Interviewee #4I remember doing a long one and thinking, “Oh yeah! I missed one! I can go in and do that one again”. It always good because you know you can go in and do it again. That’s the first time I actually left the memory one and come back and do it again and I’m going “I’ll do that later”The perception of flexibility and control of the program appeared to enhance engagement in the program and therefore improved motivation to persist with the trial.Many participants cited value in completing what they had started as a reason to continue the program despite being frustrated with it. The participants often contextualized these values as being a learning of their generation, with many indicating that these values had developed as they had grown up and had persisted into adulthood.Interviewee #2...because I had committed to doing something and so I did it.Due to having these values, they were going to complete the program regardless of how frustrating or tedious it was. They completed it because their sense of what they should and shouldn’t do directed them. Holding such values meant that they did not consider stopping the program or dropping out from the study.Interviewee #3Sheer determination to see what’s this one going to be about, curiosity perhaps and it was determination. I feel you know if a job’s worth doing you do it as good as you can. There’s no point in giving up in the middle of [a] thing you just keep on plodding on.On completing the entire program they described a sense of accomplishment coupled with relief, a sense of having done something that was difficult, but that they had overcome their difficulties. These participants placed value in hard work and were often scornful of those who dropped out, implying this was a weakness of character. They placed pride in completion where they saw the task as a chore rather than a personally useful tool or activity.Participants who integrated the program into their day-to-day routine appeared to be less likely to struggle to be adherent. They did not see the program as an activity to be reflected on, but rather as a task to be completed when it was available. These people did not report the frustrations with the program that those who saw completing the program due to a sense of duty did; rather, they saw it as a task to be done with little emotional connection to it.Interviewee #2I log in to my Internet every morning. I spend an hour or two hours, depending on what I have to do. And...um...and I’ll always complete task as they come up. So with my emails, it’s the same. I’ll answer them on the same day or possibly the next day.This was more common in participants who logged on to the computer daily and who often referred to themselves in terms that implied a perception of being organized or task driven. Routine was often highly valued and the ability of the program to fit into this allowed it to become routine. These participants saw the program as something that needed to be done, rather than something that they wanted to do or enjoyed doing. They often described it as part of the daily chores or to-do list for that day. The development of habit and the perception of the modules as needing to be done is particularly interesting given that participants were able to opt out of the trial at any stage. Instead, they conceptualized the trial as medicine that needs to be taken.Many participants used words such as commitment and obligation to describe the things that made them remain engaged in the program. They described feeling a sense of commitment to the researcher or to the research. Many of these participants had been part of research teams in the past and therefore placed heavy emphasis on participating in research projects. Some were also aware of the numbers required to successfully complete a research project and aware of the impact of dropout on research outcomes.Interviewee #9Well, I mean, you really, you have to keep going. Um, when you start something you got to finish it, unless it drives you crazy. And it didn’t drive me crazy. There were some days when I did it and I thought “God how much more of this?,” but you made a commitment, so you followed through and you did it. And I mean if you don’t finish it how can you help if you’re only giving half information how can you help? And as I say the main criteria was perhaps I could help.Interviewee #2I am thinking of the fact that I have committed. Commitment is all I can think of. And I am thinking that the person I have committed to is relying on my support. And um...it would be very unfair to let them down. I know that I am only one of many people, but if everybody would drop out...where would we be?When participants had engaged previously in programs that were not obviously linked to research, they were less likely to adhere to the program outline. This research-related engagement appeared to be driven by an obligation to others, rather than an obligation and perception of the benefits that they might have received themselves. Participants reported that when they were completing a program for only themselves, program use was more ad hoc, as they believed that they could complete the program at their own pace without affecting others. However, when engaged in research, there was more pressure to complete the program in a timely manner and in its entirety to ensure their contribution was able to benefit others.Interviewee #2For instance, with my [other program]. I drop in and I drop out depending on my other commitments because I know that nobody else is involved but myself.This motivating factor appears to be linked to the completion of research and may not enhance motivation in open access users of freely available online programs. The removal of an obligation to others when there is no research project attached to the program may lead to open access users not completing programs, not obtaining the full benefits of programs, or dismissing online programs as ineffective. Such experiences may also lead to participants dismissing psychological therapies as ineffective for their problem and may actually decrease help seeking.Overall, participants who had persisted with the e-therapy intervention reported multiple barriers and motivating factors in completing the program. It appeared that all participants initially engaged in the program with the hope that the program would be beneficial for themselves or for others. However, only half of the interviewed participants reported being able to complete their modules on a weekly basis. These participants appeared to have different barriers from those who completed the program less frequently, who often mentioned a lack of time and competing demands such as work and family. Motivating factors for less-adherent participants included believing that the research was important and feeling obligated to others. The more regularly adherent participants appeared to be older and reported more confusion around technology use but fewer issues with time demands and program frustrations. More-adherent participants were more likely to perceive the program as beneficial. These benefits consisted of mood improvements and a sense of getting things done, satisfaction at completing the program, and a sense of contributing to the wider community through research. However, over time the motivation to persist with the program decreased in many participants due to ongoing program frustration and the influence of personal factors such as mood issues and forgetfulness. To overcome these barriers, participants needed external motivators in the forms of reminders of their reasons for engagement and demonstrations of the benefits of completing the program . Consistent with previous research, these factors were often varied and multifaceted ,44,45 buSeveral factors have emerged as themes about why people choose to engage and persist in an online intervention see .. ConsisHowever, this study suggests two additional areas that influenced persistence other than personal benefits. The first of these, which is trial specific and may not be replicated in a nontrial or real-world setting, is that participants persisted with the intervention and study because of beliefs that research is important, even when they felt little personal benefit from the program, or feelings of commitment to researchers. This is consistent with medical literature that has indicated that people who participate in research trials are more likely to view research more favorably  and are The second factor was the perception that through use of the program they may benefit the wider community. Consumers rarely appear to use interventions with the view that their behavior would affect the wider community, with the exception of vaccinations, which reduce the chance of disease for everyone through herd immunity, by decreasing exposure to pathogens. In terms of Internet-based interventions, users may be encouraged to reflect on the benefits of program completion. This might include consideration of the impact of their mood on others, the ability to provide feedback on the program so that it can be improved for others, and the importance of completing the program to determine for whom the program may be most useful, even if the participant is not finding it beneficial. While these are not necessarily trial factors per se, they may be more salient in a trial situation where participant information sheets and messages throughout the trial reinforce the experimental nature of the trial and the future benefits for others.The influence of the research process on outcome has also been noted previously in online interventions ,44. GerhCertain predicted barriers and motivational factors were absent from the themes that emerged. We expected physical discomfort and health problems to be a barrier given the older comorbid nature of the population. Many participants reported physical discomfort to the trial team through email communication, and they also often mentioned physical health problems in the interviews, but participants did not talk about these as barriers to persistence. Participants considered psychological barriers and motivators to be of greater importance than physical ones. This may reflect either that the intervention had a primary psychological target or that the Internet-based method of delivery may reduce travel-associated physical barriers. It may also represent a bias in the questions we used or in participants’ placing greater emphasis on psychological burdens and motivators. Regardless, this is consistent with other literature on uptake and motivation, which has only found psychological barriers and facilitators of persistence with interventions ,44,45.First and most important, these findings, like all qualitative research, are unique to this study population and the analysis of this research team, and the analysis of these data was influenced by the perspective of the researchers. If another team, with a different theoretical position, were to analyze the transcripts, they would likely find different results. The primary author has worked in the area of medication adherence and also works as a clinical and health psychologist, while the secondary author is a psychiatrist with a background in epidemiology. These positions have resulted in the development of views of what influences adherence behavior in a clinical setting. This, however, may be different from the influences of adherence in a research or online forum. Given this, we attempted to be as objective as possible, but the analysis is likely to have been couched within these experiences.Second, the participants interviewed were people who volunteered to participate in a qualitative interview, from a group that volunteered to participate in an online study, from a group that volunteered to participate in a longitudinal study of health. Therefore, this group was likely to be highly motivated to participate in research and may have been different from those who did not wish to share their views. This played out in the reported emphasis of research as an important motivator by all interview participants.Due to trial logistic constraints, participants were not contacted until approximately 9 months after they commenced the intervention, or 6 months after they had completed it. This meant that several of the participants may have struggled to recall their intrinsic motivations for continuing to engage in the trial, and their recall was likely influenced by their present situation in order to compensate for the information that they had forgotten . While sFinally, we were not able to recruit anyone who had not completed the trial or withdrawn, preventing us from exploring the barriers and motivators in people who do not persist with the intervention. Of those who did not persist and who did provide additional contact with the researchers, their reasons for dropping out of the trial tended to comprise a lack of time to complete the program at present, and they were therefore unwilling to participate in a further interview. Future research would benefit from interviewing participants who dropped out of online interventions to determine their reasons for doing so.In terms of maintaining participants in online programs, it may be useful to consider ways to maximize intrinsic motivations and overcome the barriers (through extrinsic motivators) that participants mentioned. This needs to occur in order to meet the dynamic state of motivation. Given this, we propose four steps.First, briefly, initial hope could be enhanced through educating users about the benefits of Internet interventions and testimonies from previous users about the program. The use of a hook (a message or program design to build curiosity) to engage participants who are starting the intervention due to interest rather than hope for improvement may form a prelude to this and engage users early on.Second, ongoing engagement could be enhanced through using free-text boxes and providing feedback to activities. While researchers have traditionally been cautious about free text due to the need to manage risk, participants appear to want this to enhance their experience of using the program. These aspects need to be considered if online interventions are to be used to treat people in the community without the intensive follow-up and potential obligation of a research trial.Third, time barriers could be overcome through the use of habit-forming strategies such as having the program scheduled to be completed each week. Such scheduling would need to be balanced to ensure that program flexibility is not lost, but rather that participants could nominate a time to complete the program next week and be assigned a reminder service for this. Users could be educated about the importance of completing the program regularly and prioritizing this, in a manner similar to how they are educated about taking medication correctly. The findings from this study indicate that online interventions benefit significantly from the use of reminder systems to prompt participants who have forgotten to continue with the interventions.Fourth, motivation could be enhanced through messaging throughout the program about the benefits of completing the program on a regular basis. Participants may benefit from personal reinforcement through having their outcome scores tracked over time and receiving tailored feedback on this. This may be particularly useful for patients who may struggle to perceive their personal improvements over the course of the program. This type of feedback needs to be provided repeatedly to keep participants motivated and to highlight their achievements and progress through the course of the program. Such an approach is similar to building mastery, which has been shown to enhance persistence with educational study  and withHowever, such communication would benefit from being delivered on a variable-ratio schedule to decrease the predictability of messaging and increase interest in the communications. Further knowledge about building engagement and persistence could be drawn from the self-help book literature to further explore this area of research . FinallyThis study is unique in that participants were older adults with physical health comorbidities, and little is known about the use of online interventions in such groups. In addition, the intervention consisted of 12 weekly modules, longer than the 9-module intervention studied by Gerhards et al  and the


Although researchers develop evidence-based programs for public health practice, rates of adoption and implementation are often low. This qualitative study aimed to better understand implementation of the Program to Encourage Active, Rewarding Lives for Seniors (PEARLS), a depression care management program at a Seattle-King County area agency on aging.We used stratified, purposive sampling in 2008 to identify 38 PEARLS clients and agency staff for participation. In 9 focus groups and 1 one-on-one interview, we asked participants to identify benefits and negative consequences of PEARLS, facilitators of and barriers to program implementation, and strategies for overcoming the barriers. Two independent researchers used thematic analysis to categorize data into key themes and subthemes.PEARLS benefits clients by decreasing depression symptoms and addressing other concerns, such as health problems. For staff, PEARLS provides "another set of eyes" and is a comprehensive program to help them meet clients' mental health needs. Barriers included issues with implementation process  and the perception that eligibility criteria were more rigid than those of other agency programs. Recommended solutions included changing eligibility criteria, providing additional staff training, increasing communication, and clarifying referral procedures, roles, and responsibilities.Barriers to PEARLS delivery discourage referrals to what is generally viewed as a beneficial program. Implementing participants' strategies for overcoming these barriers can enhance delivery of PEARLS to a greater number of older adults and help them improve their depression symptoms. Although researchers develop evidence-based programs for public health practice, rates of adoption and implementation are often low , and incwww.pearlsprogram.org). PEARLS was developed in partnership with community-based aging organizations and designed for delivery outside of traditional mental health settings. Two randomized controlled trials  is a home-based, care management program to detect and manage depression among community-based older adults ; facilitators of and barriers to program implementation; and strategies for improving PEARLS implementation.A qualitative research design is well-suited for understanding context . We usedSince the original PEARLS effectiveness study was completed, the local area agency on aging (Aging and Disability Services [ADS]) has continued to implement PEARLS. Most ADS clients are low-income, frail elders who are homebound and socially isolated. Although ADS has some components for successfully implementing evidence-based programs ,7, its aWe used stratified, purposive sampling  in 2008 The research team developed a focus group guide. Each topic was introduced by a question, followed by several probes. A research interviewer trained in semistructured interviewing and focus group techniques conducted 10 interviews (9 focus groups and 1 one-on-one interview) . Focus gWe used thematic analysis, a research method to analyze text data with attention to the content or contextual meaning . Using tTwo members of the research team (M.C. and L.S.) analyzed and coded the data. They used transcripts to independently identify subthemes in each major theme , then met to compare their coding. Several passages used to identify each subtheme were compared to provide evidence for thematic selections. When disagreements arose, coders presented supporting textual passages and reasoning until a final subtheme list was developed. Coders met with study participants and other ADS staff to confirm whether interpretations and findings were appropriate; after this comparison, no results needed to be changed. No statistical software was used to analyze the data.For clientsIn addition to treating depression, PEARLS was credited with other benefits, such as increasing clients' physical and pleasurable activities, social support, quality of life, function, physical health, and ability to live independently, all of which improve clients' depression symptoms and are benefits unto themselves. As a PEARLS staff member (focus group no. 5) shared,I think the treatment really does improve their depression. Once that happens, or at least along with that happening, I think people become more activated; they tend to take better charge of their medical care and their physical health, in addition to being less depressed. They tend to manage things in their lives a bit better.Participants cited beneficial program components. Its home-based setting was discussed as being essential in reaching frail, homebound older adults who are hindered by transportation barriers and stigmas, particularly about accessing traditional mental health care. Certain program processes were also viewed favorably. As a former client (focus group no. 9) shared,That form  was really beneficial too, [be]cause it helped me focus on certain issues instead of just being so scatter-brain[ed] and not knowing what to do with it all. And having something on paper helped, as I had something to refer to.One case manager (focus group no. 3) articulated that these components became tools that "can be applied to the other stresses that come up in clients' lives."For staffPEARLS counselors provide support to the case manager and the goals of case management, and case managers and other agency staff benefited from having someone else in the client's home to provide feedback on the client's status and to assist with other concerns. As a case manager (focus group no. 7) described,When the [PEARLS counselor] goes out to see people, that is another set of eyes and ears with the client that can see things that aren't quite right. . . . He can convey to us the client's needs and concerns.PEARLS also offers a more in-depth service than the triage and referral that typically defines case management. As a case manager stated (focus group no. 4), PEARLS "touches the client directly," providing a more "comprehensive" and "proactive" tool that case managers can offer.For agencyParticipants indicated that implementing PEARLS has been innovative for ADS because area agencies on aging typically do not provide mental health services and that delivering PEARLS helps the agency feel they are doing more about depression for their clients. ADS has also "garnered a lot of respect from the national case management community" , serving as a model for policy making and as a source of pride. Through PEARLS, ADS has gained experience with evidence-based programming by partnering with the University of Washington and now serves new populations with novel programming. Finally, PEARLS provided ADS new funding opportunities, such as a county health and human services levy to offer PEARLS to veterans and their spouses.Others outside the agencyParticipants indicated that PEARLS clients' family, friends, and neighbors, as well as providers of other health, mental health, and social service systems, benefit from clients being less depressed and having enhanced skills to manage their lives. According to a PEARLS counselor (focus group no. 8), the program has the potential to save or offset care costs because of decreased use of other services:If people are caring for their depression as well as their health, it would certainly help the cost of the health care. They will be using services more appropriately and maybe they will also not let conditions get so severe that they are an emergency.Few negative consequences of the PEARLS program were identified by clients; some focus group participants reported no negative consequences. Negative consequences included clients being disappointed if determined to be ineligible for the program and stress or embarrassment because of the stigma of mental illness. As a PEARLS team member (focus group no. 2) described, "Sooner or later people have to accept that they're depressed, and that does put them face-to-face with stigma about being mentally ill." Participants also indicated that challenges may arise when the client drops out  or finishes the program .Negative consequences for staff and the agency were framed as barriers to program implementation.Participants spoke of processes that facilitated program delivery. Counselors aid implementation through advertising PEARLS in the agency by using personalized flyers, presentations at staff meetings, and individual contacts with case managers. Counselors also provide tools for case managers to connect clients to PEARLS. As a case manager (focus group no. 6) stated,What I like is the picture [of the PEARLS counselor] . . . and the little advertisement flyer about the program because I take those with me just like I do the forms. And when I'm doing the assessment and the client already scored that mark of 5 or better, I already have that something to hand to the client. So it's a flyer that's used as an advertising tool as well as a door-opening.Program exclusion criteria were the most commonly identified barriers to program implementation. The following exclusion criteria, in order of importance, were mentioned: speaking a non-English language, having too few depressive symptoms or too many , having comorbid psychiatric conditions , having substance abuse disorders, being younger, and having cognitive impairment. As a case manager (focus group no. 6) said, "It seemed to me that there was more screening out than screening in." These program eligibility criteria were viewed as too stringent in the context of an increasingly younger and sicker client population, coupled with frequent pressure to refer to PEARLS. The criteria also present a discrimination issue, conflicting with the agency's goal to serve all clients, particularly those who are non-English–speaking, who comprise one-third of the agency's client population and are served by other programs through contracts with interpreters.These eligibility criteria dissuaded case managers and supervisors from referring clients to PEARLS, despite their beliefs in the many benefits of the program. As a case manager (focus group no. 7) described,Sometimes it is kind of a hopeless feeling, like, gosh, they are going to be screened out right away . . . we feel like we've got this option, and we would love to connect you up with the program, but we can't because you're not going to be eligible.Another widespread barrier for delivering PEARLS was the depression screening instrument. PEARLS actively screens for depression, which is often unrecognized in older people. Potential clients are first screened using the 11-item Center for Epidemiologic Studies Depression Scale (CES-D-11) ,21. ClieWorkload was another barrier to program implementation. Case managers who have high caseloads indicated that referring clients to PEARLS can become burdensome. One case manager (focus group no. 7) said,There is more expected of the case managers for each client, so sometimes when you see someone who is depressed and you are required to offer the person a referral, you think, "I've been with this client almost 2 hours already . . . I'm not even halfway through the assessment, and I have got to be back in the office in an hour." And I don't feel you have the opportunity to present the program to the client.Focus group participants identified possible strategies for addressing barriers to program delivery and enhancing program benefits and facilitators. Changing the eligibility criteria was the most common approach mentioned. As a case manager (focus group no. 4) stated, "Boy, if you opened up English-as-a-second-language clients, I probably have 3 clients I could refer right now." Another strategy was marketing PEARLS outside of the agency, as many other providers  see agency clients more regularly, presenting more opportunities to refer.Improving communication was mentioned by participants in every interview. Better communication and feedback  is needed between case managers and counselors. More communication is also needed about roles and responsibilities for implementation, such as who is responsible for following up on the outcome of the PEARLS referral. Participants also asked for more frequent program updates from administrators to staff members to better institutionalize PEARLS into regular agency practice.Our findings are similar to recent research on implementing evidence-based mental health programs in community settings. Allocating adequate staff resources, training, and communication is essential when integrating a new program into settings that have a high staff turnover and among staff who have a heavy workload and limited time ,24. Our These findings have informed the development and execution of a strategic implementation plan at ADS to improve PEARLS program delivery. During the past 3 years, ADS has expanded program eligibility criteria, standardized implementation processes, and conducted new education and training activities to address barriers to program implementation and provide education and support that was lost after the efficacy trial ended. ADS has experienced modest improvements in rates of referral (from 9% to 15%) and enrollment (from 4% to 8%) during this time. Findings from this research have also benefited other PEARLS programs around the country, many of which experience similar issues. Study findings are shared during monthly technical assistance calls with PEARLS providers, who are able to adapt and use them to augment program delivery in their agencies.This study provides a detailed picture of implementation from the perspectives of those involved; contrary to an efficacy study, in which context is capable of being controlled, "implementation research occurs in real world settings distinguished by their complexity and variation in context" . InterviThis study had several limitations. Bias is inherent if participants have preexisting, ongoing relationships or if they feel more or less likely to participate on the basis of perceived incentives or liabilities of participation. In groups, participants may respond according to what other people say  or not offer new ideas when the group is in agreement ("groupthink" bias) . The intFindings suggest that purposive sampling and thematic analyses can identify a range of key factors for implementation of evidence-based programs. These factors have helped target improvements in PEARLS delivery at ADS and are also relevant for researchers and practitioners interested in improving translation of evidence-based practices.


ARID1A gene encodes adenine-thymine (AT)-rich interactive domain-containing protein 1A, which participates in chromatin remodeling. ARID1A has been showed to function as a tumor suppressor in various cancer types. In the current study, we investigated the expression and prognosis value of ARID1A in primary gastric cancer. Meanwhile, the biological role of ARID1A was further investigated using cell model in vitro.The ARID1A gene in primary gastric cancer pathogenesis, real-time quantitative PCR and western blotting were used to examine the ARID1A expression in paired cancerous and noncancerous tissues. Results revealed decreased ARID1A mRNA (P = 0.0029) and protein (P = 0.0015) expression in most tumor-bearing tissues compared with the matched adjacent non-tumor tissues, and in gastric cancer cell lines. To further investigate the clinicopathological and prognostic roles of ARID1A expression, we performed immunohistochemical analyses of the 224 paraffin-embedded gastric cancer tissue blocks. Data revealed that the loss of ARID1A expression was significantly correlated with T stage (P = 0.001) and grade (P = 0.006). Consistent with these results, we found that loss of ARID1A expression was significantly correlated with poor survival in gastric cancer patients (P = 0.003). Cox regression analyses showed that ARID1A expression was an independent predictor of overall survival (P = 0.029). Furthermore, the functions of ARID1A in the proliferation and colony formation of gastric cell lines were analyzed by transfecting cells with full-length ARID1A expression vector or siRNA targeting ARID1A. Restoring ARID1A expression in gastric cancer cells significantly inhibited cell proliferation and colony formation. Silencing ARID1A expression in gastric epithelial cell line significantly enhanced cell growth rate.To investigate the role of ARID1A may play an important role in gastric cancer and may serve as a valuable prognostic marker and potential target for gene therapy in the treatment of gastric cancer.Our data suggest that  Gastric cancer is the fourth most common malignancy worldwide and the second most common cause of cancer-related deaths each year (10.4% of cancer deaths) ARID1A in a leukemia cell line confers resistance to Fas-mediated apoptosis The eukaryotically conserved SWI/SNF chromatin-remodeling complex plays essential roles in a variety of cellular processes, including differentiation, proliferation and DNA repair ARID1A mutations and loss of BAF250a protein have been found to correlate strongly with the ovarian clear-cell carcinoma and uterine low-grade endometrioid carcinoma ARID1A is a potential candidate tumor suppressor gene. However, the clinical significance of such differential expression and the function of the ARID1A protein remain undefined due to the lack of studies using fresh human tumor samples. In the present study, we analyzed the ARID1A expression level in gastric cancer using real-time quantitative RT-PCR, western blotting and immunohistochemistry. Meanwhile, we identified the relationship between ARID1A expression and clinicopathological features and evaluated its prognostic value in post-resection survival of gastric cancer patients. Furthermore, we evaluated the functional role of ARID1A in the tumorigenesis of primary gastric cancer by examining the in vitro proliferation and colony formation in gastric cell lines.Recently, ARID1A was determined by real-time quantitative RT-PCR assays in 66 paired cancerous and the matched adjacent normal gastric mucosa tissues. The ARID1A expression level was significantly lower in 43 (65.15%) tumor-bearing tissues compared with the adjacent non-tumor tissues  of the gastric tumor tissues compared with matched adjacent non-tumor tissues (P = 0.001) and tumor grade (P = 0.006), but not with age, gender, tumor size, distant metastasis (M stage), and tumor locus or local lymph node metastasis (N stage).To further investigate the clinicopathological and prognostic roles of ARID1A expression, we performed immunohistochemical analyses of the 224 paraffin-embedded gastric cancer tissue blocks. Overall, 115 of 224 (51.3%) cases showed negative ARID1A expression in cancerous tissues , whereasP = 0.003, log-rank test, The 5-year overall survival rates in patients with positive and negative ARID1A expression were 68.8% and 52.2%, respectively. The overall survival of patients with negative ARID1A expression was significantly worse than that of ARID1A-positive patients  inhibited in ARID1A-transfected gastric cancer cells compared with control vector-transfected gastric cancer cells  gastric cancer samples, with 39 (85%) truncated mutations ARID1A mutations show either loss of or substantially reduced protein expression compared to those without ARID1A mutation. In contrast, there are only 6 gastric cancer samples showing absent or weak protein expression in the absence of detectable ARID1A mutation, which suggests that other mechanisms may contribute to ARID1A inactivation. Recently, another exome sequencing research by Zang et al. also showed ARID1A mutations in 8% of gastric samples, of which 75% lost or reduced the protein expression ARID1A alterations in gastric cancer samples with microsatellite instability (MSI) than those with microsatellite stability (MSS). Moreover, the mutation spectrum of ARID1A is distinct between the two genetic types of gastric cancer, with most indels in the MSI type and more single-neucliotide variations in the MSS type. MSI is defined as indel mutations within nucleotide repeats (known as microsatellite regions) resulted from DNA mismatch repair gene inactivation-induced replication errors et al., the indel mutation rate (78%) of ARID1A in MSI gastric cancer is comparable to that of TGFBR2 in MSI colon cancer, a well-established and functionally validated driver gene inactivated by MSI ARID1A together with MSI may play an important role in gastric carcinogenesis. Therefore, the relationship between ARID1A alterations and MSI status in gastric cancer, as well as its clinicopathological significance, needs further investigation in the future research.To date, the causes of et al., the expression of ARID1A was only detected in a small-size sample (32 cases), and there was no further exploration of its clinical significance ARID1A expression was significantly correlated with a higher T stage of gastric cancer, implying that absence of ARID1A expression may promote tumor growth and invasion. In addition, we detected lower ARID1A immunoreactivity in poorly differentiated gastric cancer tissues than in well-differentiated ones, suggesting that decreased ARID1A expression might play a role in tumor de-differentiation. Consistent with our findings, other investigators also found that decreased ARID1A expression is significantly associated with a higher grade of breast cancer In the study by Wang ARID1A expression and poorer clinical outcome of gastric cancer patients after radical operation. Cox hazard ratio regression analyses further demonstrated that the ARID1A expression level was an independent risk factor for survival, suggesting that it may serve as a valuable prognostic biomarker for gastric cancer patients after surgery and a potential target for gene therapy in the treatment of gastric cancer. In ovarian clear cell carcinoma, it was also reported that patients with positive ARID1A expression had a longer progression-free survival than those with negative ARID1A expression ARID1A expression is significantly correlated with chemoresistance in ovarian clear cell carcinoma, which is also associated with a poor prognosis of cancer. These data suggest that ARID1A expression and mutation examination might be helpful to guiding clinical management. Taken together, our observations that the loss of ARID1A expression in gastric cancer is associated with more malignant phenotypes and a worse prognosis imply that it may play a tumor suppressor role in gastric carcinogenesis.A Kaplan-Meier survival analysis showed a significant correlation between the loss of ARID1A in gastric cell lines. Restoring ARID1A expression in gastric cancer cells significantly inhibited cell proliferation and colony formation. Silencing the expression of ARID1A in gastric epithelial cells significantly enhanced the cell growth rate. These results indicated that ARID1A may play an import role in inhibiting tumor cell growth. Recently, functional assays of ARID1A in gastric cancer cell lines by Zang et al. suggested that ARID1A exert tumor-suppressor activity et al. demonstrated that restoring the expression of wild-type ARID1A is sufficient to suppress the proliferation and tumorigenecity of xenografts with human ovarian cancer cell lines harboring ARID1A mutations, while RNA interference-mediated ARID1A silencing enhances cellular proliferation and tumorigenicity in two non-transformed human ovarian epithelial cell lines, IOSE-80PC and OSE4 ARID1A may play an important role in the process of carcinogenesis.We further investigated the functional role of ARID1A expression in gastric cancer and its correlation with a more malignant phenotype and poorer prognosis in a large number of clinical samples. In addition, we proved that ARID1A can inhibit tumor cell growth and colony formation in vitro. To the best of our knowledge, the data generated in the current study represent the first report correlating the presence of ARID1A with clinicopathological characteristics and the overall survival of gastric cancer patients. Taken together with the results of Wang et al. and Zang et al. ARID1A might serve as a candidate tumor suppressor and prognostic biomarker in gastric carcinogenesis.In conclusion, we have demonstrated the loss of The research was approved by the Ethics Committee of Sun Yat-sen University Cancer Center, and written informed consent was obtained from each patient involved in the study.2.The gastric cancer cell lines, SGC7901, AGS, MGC803, and the gastric epithelial mucosa cell line GES1 were obtained from the Committee of Type Culture Collection of Chinese Academy of Sciences . The cell lines were cultured in RPMI 1640 media supplied with 10% heat-inactive fetal bovine serum (FBS). The cells were incubated at 37°C in a humidified chamber containing 5% COA total of 66 paired cancerous and matched adjacent noncancerous gastric mucosa tissues were collected from gastric cancer patients undergoing gastrectomy at Sun Yat-sen University Cancer Center between 2009 and 2011, and the diagnosis was confirmed by pathological examination. The 25 paired cancerous and corresponding adjacent noncancerous gastric mucosa tissues used to detect the ARID1A protein expression in western blotting were selected from the 66 paired samples. After surgical resection, fresh tissues were immediately immerged in RNAlater  to avoid RNA degradation, stored at 4°C overnight to allow thorough penetration of RNAlater into the tissue and then frozen at −80°C until RNA and protein extraction was performed. Another 224 paraffin-embedded primary gastric carcinoma samples which had been collected between 2003 and 2005, were obtained from the Sun Yat-sen University Cancer Center. None of these patients had received radiotherapy or chemotherapy prior to surgery. The follow-up data of the gastric cancer patients in this study are available and complete. Postoperative follow-up occurred at our outpatient department and included clinical and laboratory examinations every 3 months for the first 2 years, every 6 months during the third to fifth years, annually for an additional 5 years or until patient death, whichever occurred first. The histopathological type and stage of gastric cancer were determined according to the criteria of the World Health Organization classification and the TNM stage set out by the Union for International Cancer Control.ARID1A and GAPDH  with the following primers: ARID1A forward: 5′-CTTCAACCTCAGTCAGCTCCCA-3′, and reverse: 5′-GGTCACCCACCTCATACTCCTTT-3′; GAPDH forward: 5′-CTCCTCCTGTTCGACAGTCAGC-3′, and reverse: 5′-CCCAATACGACCAAATCCGTT-3′. Gene-specific amplification was performed using an ABI 7900HT real-time PCR system  with a 15 µl PCR mix containing 0.5 µl of cDNA, 7.5 µl of 2 x SYBR Green master mix , and 200 nM of the appropriate oligonucleotide primers. The mix was preheated at 95°C (10 min) and then amplified at 95°C (30 sec) and 60°C (1 min) for 45 cycles. The resolution curve was measured at 95°C for 15 sec, 60°C for 15 sec and 95°C for 15 sec. The Ct (threshold cycle) value of each sample was calculated from the threshold cycles with the instrument’s software (SDS 2.3), and the relative expression of ARID1A mRNA was normalized to the GAPDH value. Data were analyzed using the comparative threshold cycle (2-ΔCT) method.Total RNA was extracted using TRIzol  according to the manufacturer’s protocol. Total RNA concentration was assessed by measuring absorbance at 260 nm using a NANO DROP spectrophotometer . Reverse transcription (RT) to synthesize the first-strand of cDNA was performed with 2 µg of total RNA treated with M-MLV reverse transcriptase  according to the manufacturer’s recommendations. The resulting cDNA was then subjected to real-time quantitative PCR for evaluation of the relative mRNA levels of The homogenized gastric cancer samples, including tumor and nontumor tissues, as well as cell lines, were lysed in RIPA lysis buffer, and the lysates were harvested by centrifugation  at 4°C for 30 min. Approximately 50 µg protein samples were then separated by electrophoresis in a 12% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane. After blocking the non-specific binding sites for 60 min with 5% non-fat milk, the membranes were incubated overnight at 4°C with a mouse monoclonal antibody against ARID1A . The membranes were then washed three times with TBST  for 10 min and probed with the horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibody  at 37°C for 1 hour. After three washes, the membranes were developed by an enhanced chemiluminescence system . The band intensity was measured by densitometry using the Quantity One software . The protein levels were normalized to that of GAPDH detected using mouse anti-human GAPDH monoclonal antibody .The tissue sections were deparaffinized with dimethylbenzene and rehydrated through 100%, 95%, 90%, 80% and 70% ethanol. After three washes in PBS , the slides were boiled in antigen retrieval buffer containing 0.01 M sodium citrate-hydrochloric acid (pH = 6.0) for 15 min in a microwave oven. After rinsing with PBS, the tissue sections were incubated with primary antibody and the slides were then rinsed in 3% peroxidase quenching solution (Invitrogen) to block endogenous peroxidase. The sections were then incubated with a mouse monoclonal antibody against ARID1A  at 4°C overnight and then incubated with horseradish peroxidase (HRP) (ChemMateTM DAKO EnVisionTM Detection Kit) at room temperature for 30 min. After washing in PBS, the visualization signal was developed with 3, 3′-diaminobenzidine (DAB) solution, and all of the slides were counterstained with hematoxylin. As negative controls, adjacent sections were processed as described above except that they were incubated overnight at 4°C in blocking solution without the primary antibody.The total ARID1A immunostaining score was calculated as the sum of the percent of positively stained tumor cells and the staining intensity. Briefly, the percentage of positive staining was scored as 0 , 1 , 2  or 3 , and the intensity as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (dark staining). The total immunostaining score was calculated with the value of percent positivity score × staining intensity score, which ranged from 0 to 9. The expression level of ARID1A was defined as following: “−” , “+” , “++” , “+++” . Based on the ARID1A expression levels, the gastric cancer patients were divided into two groups: negative ARID1A expression group (ARID1A-) and positive ARID1A expression group .ARID1A cDNA was obtained from the Asbio Technology Company . Empty vector was used as negative control. MGC803 cells were cultured in 6-well plates until they reached 85–90% confluence, and then transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, gene expression was examined by western blotting analysis. And then, cell proliferation and colony formation were performed.A eukaryotic expression plasmid pCMV6-Entry containing the full-length of human ARID1A expression, the siRNAs were synthesized by GenePharma Company . The siRNA sequences were as follows: siRNA-ARID1A, sense: 5′GCCCUAACAUGGCCAAUAUTT3′, antisense: 5′AUAUUGGCCAUGUUAGGGCTT3′. The negative control (NC), sense: 5′UUCUCCGAACGUGUCACGUTT3′, antisense: 5′ACGUGACACGUUCGGAGAATT3′. 400 pmol siRNA-ARID1A or NC were transfected into 2×105 GES1 cells using Lipofectamine RNAi MAX reagent  according to the manufacturer’s protocol. After that, cell proliferation was then performed.For knockdown of 2 cells per well. The cell growth rate was detected using cell proliferation MTS kit according to the manufacturer's instruction . Each experiment was performed in triplicate.Cell growth rate of MGC803 or GES1 cells was detected by MTS cell proliferation assay. Cells were seeded in a 96-well plate at a density of 5×10ARID1A-expressing MGC803 cells or control MGC803 cells were plated in a 6-well plate at a density of 5×102 cells per well. After 10 days of culture, surviving colonies (>50 cells per colony) were counted with crystal violet (0.5%) staining. Colony-forming efficiency (CFE %) was defined as the ratio of the number of colonies formed in culture to the number of cells inoculated. The experiment was done in triplicate.For the colony formation assay, 2 test was used to analyze the relationships between ARID1A expression and various clinicopathological parameters. Survival curves were calculated using the Kaplan–Meier method and compared by the log-rank test. The Cox proportional hazard regression model was used for univariate and multivariate analyses to study the effects of the clinicopathological variables and ARID1A expression on survival. The two-tailed unpaired Student’s t test was used to assess differences in cell growth rate and colony formation. Statistical analyses were performed with the Statistical Package for the Social Sciences, version 17.0 , and a two-sided P value less than 0.05 was considered to be statistically significant.Differences in mRNA and protein expression between tumor samples and the paired adjacent non-tumor tissue samples were evaluated with the paired-samples t-test. The χ


To investigate the expression of the preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia and its clinical significance.The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia (AL) and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR. Correlation analyses between PRAME gene expression and the clinical characteristics  of the patients were performed.n=27), and in 28.6% of the patients with acute lymphoblastic leukemia , but was not expressed in the healthy volunteers. The difference in the expression levels between AML and ALL patients was statistically significant. The rate of gene expression was 80% in M3, 33.3% in M2, and 28.6% in M5. Gene expression was also found to be correlated with CD15 and CD33 expression and abnormal karyotype, but not with age, gender, white blood count or percentage of blast cells.The PRAME gene was expressed in 38.2% of all 34 patients, in 40.7% of the patients with acute myelogenous leukemia (AML, The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute leukemia. This gene, which is located in 22q11 of the human chromosome and codes for a protein with 509 amino acids, is a tumor antigen recognized by HLA-24 and afterward presented to cytotoxic lymphocytes (CTL). In normal tissues, PRAME is expressed in the testis, adrenal gland, ovary, and endometrium, and its expression levels are lower by more than 3 logarithmic series compared with that in tumor tissues . Studies abroad suggest that the PRAME gene is highly expressed in leukemic cells , and its expression levels are correlated with the relapse and the remission of leukemia .The PRAME gene was discovered by Ikeda and his colleagues in a melanoma patient in 1997 . Until now, however, local investigations and data on PRAME gene are rare. Hence, this study investigated PRAME mRNA expression in leukemic patients and its relationship with clinical data such as white blood cell count, bone marrow blast count, immunophenotype of leukemia, and karyotype. This study also presents theoretical values to monitor minimal residual disease (MRD) and the immunotherapy of leukemia.Continuous gene expression monitoring during treatment could determine the effects of chemotherapy and whether leukemic cells are drug resistant 2 cases , 5 M3 cases , 7 M5 cases , and 1 M4EO case. Seven had acute lymphoid leukemia (ALL) (3 children and 4 adults). Twelve healthy volunteers were enrolled as control.Written informed consent was obtained from all patients before the trial. A total of 34 leukemia patients (according to FAB diagnostic criteria) were enrolled in the study, including 24 newly diagnosed and 10 remission patients. Fifteen patients were male, and 19 were female. Their median age was 42.5 years (ranged from 9 to 72 years). Twenty-seven patients had acute myelogenous leukemia) (AML): 12 MBone marrow samples, 1 mL each, were obtained and anti-coagulated with 2% EDTA, dealed with 30 mL hemolysin (1:10 diluted), and then stored in a dark place for 10 min. The samples were centrifugated at 3,000 r/min for 10 min, and the sediments obtained were washed with PBS. The sediments were bone marrow mononuclear cells.6 cells, 10 µL lysis buffer and 0.72 µL β mercaptoethanol were added, and the resulting mixtures were mixed for 5 min.(1) Mononuclear cell lysis. Into each PCR tube with 10(2) Liquid phase separation. Into each of the mixtures from step 1 was added 10 µL sodium acetate (2 mmol/L). The resulting mixtures were mixed well and allowed to stand for 5 min. Then 100 µL phenol water was added, and again, the mixtures were mixed well and allowed to stand for 5 min. Finally, 20 µL chloroform/isoamyl alcohol was added, and the mixtures were mixed well and centrifugated at 12,000 r/min at 4°C for 5 min. Supernatants were collected.(3) RNA precipitation. Into the supernatants was added 100 µL isoamyl alcohol. The resulting mixtures were mixed well, placed on ice for 10 min, and centrifugated at 12,000 r/min at room temperature for 5 min. The sediments obtained were the total RNA.2O). The resulting mixtures were mixed well and then centrifugated at 12,000 r/min at room temperature for 5 min. Sediments were collected.(4) RNA washing (performed twice). Into the sediments was added 75% ethanol .(5) Redissolving RNA. The sediments were dried and then dissolved with DEPC-H(1) Primers. PRAME: upstream primer – 5’-CTG TAC TCA TTT CCA GAG CCA GA- 3’; downstream primer – 5’-TAT TGA GAG GGT TTC CAA GGG GTT-3’; the amplified fragment length had 561 bp. β-actin : upstream primer – 5’-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3’; downstream primer – 5’-CGT CAT ACT CCT GCT GAT CCA CAT CTC-3’; the amplified fragment length was 800 bp.2O, 11 µL; Oligo-p(dT)18, 1 µL; 5×reaction buffer, 4 µL; RNase inhibitor (20 U/µL), 1 µL; dNTP, 2 µL; and M-MuLV reverse transcriptase, 1 µL. The mixtures were incubated at 37°C for 60 min and then at 70°C for 10 min.(2) Reverse reaction. Reaction systems: sample total RNA, 1 µg to 5 µg; DDH2O, 22 µL; and upstream and downstream primers, 1 µL each. PRAME gene amplification conditions: 94°C 5 min,  × 34 cycles. β-actin amplification conditions: 94°C 4 min,  Product analysis. The PCR products (8 µL) were subjected to 2.5% agarose gel electrophoresis at 120 V for 30 min and then analyzed under ultraviolet lamp.(3) PCR amplification. Reaction systems: cDNA, 1 µL 2 × PCR Master 25 µL; DDHt test were performed.SPSS13.0 analysis software was used. Single-factor ANOVA, homogeneity of variance analysis, and two-sample n=34) (n=27), and in the de novo AML patients, the expression rate was 43.75% (n=16). In the ALL patients, the expression rate was 28.6% (n=7). The expression rate of the PRAME gene was higher in the AML patients than in the ALL patients (P>0.05).In the 34 leukemia patients, the expression rate of the PRAME gene was 38.2% (2 and M3 groups than in the M5 and ALL groups (P<0.05).The expression of the PRAME gene did not have an obvious relationship with patients’ gender, age, or white blood cell count. Gene expression was found higher in the M3 (80%) than in M2 (33.3%) (P<0.05). The study also investigated the percentage of blast or blast+immature cells in the bone marrow of the patients; no correlation was found between this factor and PRAME gene expression. The fluorescence intensity of the specific surface markers of the leukemic cells, including CD3, CD5, CD7, CD10, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD34, CD64, and CD117, was measured, and correlation analysis between fluorescence intensity and PRAME gene expression was performed. Results showed obvious differences in the expression of CD15 and CD33 between the PRAME-positive and negative groups  examination. Then after five months, the patient morphologically relapsed.In this study, PRAME gene expression in all patients was continuously monitored before every cycle of chemotherapy. In one patient with ALL, PRAME gene expression disappeared after the first cycle of chemotherapy, and this improvement was accompanied by complete morphological remission . Very interestingly, in an M-4 to 10-6, which is better than that of FCM or fluorescence in situ hybridization. However, the use of PCR in leukemia is limited because only several types of leukemia have specific gene markers such as PML/RARa, AML1/ETO, and bcr/abl. As a result, finding a widely expressed targeting gene is a precondition for the early diagnosis, MRD monitoring, and immunotherapy of leukemia.Until now, the criteria for the clinical remission of leukemia are based on symptoms, physical signs, blood cell count, and percentage of blast and immature cells in the bone marrow. According to these criteria, even patients with CR still have some leukemic cells which cannot be detected by common microscopy. These cells are called MRD. MRD is the primary cause of leukemia relapse. Clearly, in the early stages of leukemia or after CR, the means of monitoring and the eradication of leukemic cells are critical in the treatment of the disease. PCR, a modern biological method, has high sensitivity that can reach up to 10 and Baren , but was lower than that reported by Steinbach . These variations may have been caused by the differences in the subjects examined. This study included both adults and children, Paydas  and Baren focused on adults with acute leukemia, and Steinbach focused on children with acute leukemia. From the subtypes of AML, M3 patients had the highest expression rate (80%), followed by M2 (33.3%), and then M5 (28.6%), similar to the results of Paydas  and Baren . Like the findings of other studies, no PRAME expression was found in the 12 healthy volunteers. To further investigate whether PRAME gene expression is correlated with the clinical characteristics of leukemia, Paydas  examined 74 patients, including 68 patients with de novo acute leukemia, 3 with chronic myeloid leukemia-blastic phase, and 3 with myelodysplastic/myeloproliferative syndrome-blastic transformation. Their results suggested that PRAME gene expression was not correlated with age, gender, size of peripheral lymph nodes, hemoglobin and white blood cell count, platelet count, LDH level, ALP level, albumin level, cell surface antigen, or response to therapy. This study also concluded that PRAME expression was not correlated with age, gender, white blood cell count, or percentage of blast and immature cells in the bone marrow.The expression of PRAME was higher in this study than that reported by Paydas . This study also proved that PRAME was somehow related to chromosome abnormality. However, whether the PRAME gene is caused by chromosome changes needs further investigation. Moreover, this study found that PRAME gene was correlated with CD15 and CD33, similar to the results of Paydas  and other related studies.Several studies have demonstrated that the expression of the PRAME gene is correlated with chromosome translocation , WT1 can only be detected when the titer of leukemic cells is above 10-3 to 10-4. For PRAME, however, the detected titer is lower than 10-4. Furthermore, WT1 is mainly expressed in AML patients. In APL and ALL patients, the WT1 gene is not expressed or is detected at titers lower than 101 to 102 copies/mg RNA. By contrast, PRAME gene is highly expressed in all these types. These findings suggest that the combined use of WT1 and PRAME can improve the detection of MRD . In some diseases, the expression of PRAME gene becomes positive earlier, but disappears later, than that of WT1. Hence, PRAME may be a better candidate than WT1 for MRD monitoring. Through dynamic monitoring of PRAME gene in one of the patients examined, this study found the existence of MRD 3 months earlier than FCM and 5 months earlier than morphological relapse. This particular case proved the value of detecting PRAME gene expression in monitoring MRD. Moreover, the quantitative examination of the PRAME gene can be used to tailor chemotherapy schedules for individual leukemia patients: when the level of the PRAME gene decreases to normal, patients do not need to have chemotherapy; however, when the expression level of PRAME increases rapidly, patients must be treated with appropriate chemotherapy. This practice is different from current chemotherapeutic strategies guided by morphological changes.Compared with Wilm’s tumor (WT1) gene, PRAME gene, as another widely expressed leukemia gene, is more widely expressed in patients with normal karyotypes. According to Matsushima . Assisted by retinoid acid (RA), the PRAME protein combines with RAR, and the resulting complex inhibits ligand-mediated receptor activation. Through chemotaxis for polycomb protein, the RAR element-mediated downstream initiation of transcription is inhibited. As a result, cell differentiation and apoptosis are halted. A recent study also suggests that the PRAME protein is a subunit of ubiquitin ligase, and it participates in the activation of the nuclear factor promoter Y .The function of PRAME gene and its protein is not yet clear. This study found that the expression of PRAME gene was much higher in patients with chromosomal abnormalities than in those with normal chromosomes. Furthermore, chromosomal abnormality always leads to the emergence of fusion genes and proteins, which often participate in intracellular signaling pathways. Thus, speculating that PRAME protein participates in intracellular signaling pathways is reasonable. Some studies have demonstrated that the PRAME protein is an inhibitor of the RARa pathway. Therefore, PRAME is a feasible candidate targeting antigen for tumor immunotherapy or vaccines. A recent study has proven that PRAME protein has antigenicity in vitro, and stimulates the proliferation and activation of specific CD8+ CTL .The PRAME gene is also a promising component in the immunotherapy of leukemia. As a cell membrane protein, the PRAME, presented by HLA-24, can be recognized and lysed by CTL. Hence, PRAME specific CTLs can be extracted and cultivated from melanoma patients. PRAME protein is also not expressed or is expressed only at very low levels in normal tissues, and PRAME mRNA is not expressed in CD34 cells In summary, PRAME gene is a widely expressed leukemia gene that has been the focus of much scientific research in recent years. This gene is not only widely expressed in many hematological malignant diseases but is also intimately related to the progression and the remission of diseases. The hypothesis that the PRAME gene can be used in the monitoring of MRD is gradually being accepted, and the determination of this gene’s function is paving a promising way for the immunotherapy and gene-targeting treatment of leukemia.