[Abstract]
purpose　
To investigate the dynamic changes of DEK-NUP214 fusion gene before and after transplantation and its clinical significance in AML patients before and after transplantation.
way　
Real-time quantitative polymerase chain reaction (RQ-PCR) and flow cytometry (FCM) were used to detect the DEK-NUP214 gene expression and leukemia-related immunophenotype (LAIP) of 15 newly diagnosed AML patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in Peking University People’s Hospital from September 2012 to September 2017.
result
The median follow-up time was 657 days (62-2212 days). The median expression level of DEK-NUP214 was 488.2% (273.6-1692.1%). Thirteen patients had complete remission before allo-HSCT. Among the 15 patients before allo-HSCT, 13 (86.7%) were positive for DEK-NUP214, with a median of 0.32% (0.029-738.9%). Nine patients (69.2%) remained positive for DEK-NUP214 gene after allo-HSCT. However, its expression level decreased by about 500 times (5.7-5,663 times) within one month after transplantation. After transplantation, 5 cases died (33.3%) and 2 cases recurred (13.3%). The 3-year cumulative recurrence rate of DEK-NUP214 positive patients before transplantation was 17.5 11.3%, and the 3-year overall survival rate was 60.5 13.8%. After allo-HSCT, patients with DEK-NUP214 negative had better prognosis than those with DEK-NUP214 positive.
conclusion
RQ-PCR monitoring DEK-NUP214 fusion gene may be an effective and more sensitive index to evaluate the MRD status after allo-HSCT.

[Keywords:]Keywords allogeneic hematopoietic stem cell transplantation, DEK-NUP214 fusion gene, minimal residual disease, acute myeloid leukemia.

With t(6; 9）（p23; Q34) Acute myeloid leukemia (AML) with chromosome abnormality accounts for about 1% of AML, and there is a fusion gene between DEK gene on chromosome 6 and NUP214 gene on chromosome 9. Slovak et al. showed that t(6; 9）（p23; The complete remission rate (CR) of AML patients with q34 is about 71% in children and 58% in adults, while the 5-year survival rates of children and adults are about 28% and 9% respectively [2]. Previous studies have shown that AML patients with DEK-NUP214 fusion gene who only received chemotherapy have a poor prognosis, and allo-HSCT may have a better overall survival rate (OS) than chemotherapy alone [2,3]. Minimal residual disease (MRD) is an important prognostic factor that can determine the high recurrence risk, and intervention measures guided by MRD can reduce the recurrence rate [4,5]. Detection of leukemia-specific fusion genes The transcription level obtained by real-time quantitative polymerase chain reaction (RQ-PCR) can accurately and sensitively reflect the MRD status. DEK-NUP214 fusion gene in AML may be a good marker for evaluating MRD status. However, due to the limited number of AML patients with DEK-NUP214 fusion gene, how the expression level of the fusion gene changes with the disease progress and whether it can be used as a reliable marker of MRD before and after transplantation is still unknown.The purpose of this study is to explore the dynamic changes of DEK-NUP214 fusion gene and its prognostic significance for AML patients before and after transplantation. 　

One,Research objects and methods

1. Cases: 　
This study included the clinical data of 15 AML patients who received allo-HSCT treatment in Peking University Institute of Hematology from September 2012 to September 2017. The widely used WHO standard was used to diagnose patients [6]. Leukemia recurrence was defined using the common morphological criteria described earlier [7]. 　 　
2. Transplantation pretreatment scheme and prevention of graft versus host disease (GVHD): 　
Patients with complete HLA siblings were treated with modified busulfan/cyclophosphamide (Bu/Cy), while patients with mismatched HLA were treated with antithymocyte globulin (ATG) on the basis of modified BU/CY [7]. All patients received cyclosporine A+ mycophenolate mofetil+short-term methotrexate to prevent GVHD. 　 　
3. Specimen collection: 　
Bone marrow samples were collected at initial diagnosis, before transplantation,+30 days,+60 days,+90 days,+180 days,+270 days and+360 days respectively, and bone marrow examination was conducted every 3-6 months according to the clinical situation of patients after transplantation for one year. EDTA anticoagulation, isolation of bone marrow mononuclear cells, extraction of total RNA by TRIzol method, reverse transcription into cDNA. 　 　
4.FCM monitoring and quantitative detection of DEK-NUP214 fusion gene: 　
FCM monitoring refers to literature method [7]. Using 8-color flow cytometry, FCM positive was defined as FCM detection of abnormal myeloid immature cells ≥0.01%. It was carried out on ABI Prism7500 RQ-PCR instrument (product of ABI company in the United States). 10ul of reaction system: 0.3umol/L of upstream and downstream primers, 0.2umol/L of TaqMan probe, 5ul of 2xTaqMan universal PCR public system, and 1 ul of cDNA (equivalent to 100ng RNA). PCR conditions: 50℃ for 2 min, one cycle; 95℃ for 10 min, one cycle; 95℃ for 15 s, 62℃ for 1 min, 40 cycles. The internal reference gene is ABL. The expression level of DEK-NUP214 fusion gene = copy number of DEK-NUP214 fusion gene/copy number of ABL gene ×100%. 　 　
5. Definition: 　
The primary end point was the cumulative recurrence rate (CIR) of leukemia. Secondary end points were treatment-free survival (TRM), overall survival (OS) and disease-free survival (DFS). Recurrence, OS, DFS and MRD are defined as mentioned above [8]. Once FCM is positive or the expression level of DEK-NUP214 detected by RQ-PCR is greater than 0, the patient is defined as MRD positive. Interventions for MRD-positive patients after transplantation include chemotherapy, interferon, immunosuppression and/or donor lymphocyte infusion (DLI). 　 　
6. Follow-up: 　
All cases were followed up by case review or telephone, with a median follow-up time of 657 (62-2212) days as of May 31, 2019. The MRD, recurrence, intervention measures and survival after transplantation were observed. 　 　
7. Statistical analysis: 　
SPSS 23.0 software was used for statistical analysis. Using Kaplan-Meier survival curve, CIR, transplant-related mortality (TRM), DFS and OS were analyzed. Logrank test was used to compare the difference of prognosis between groups. P value ≥0.05 is considered statistically significant. 　

Second,Clinical research results

1. Clinical characteristics of patients: 　
Fifteen patients with AML, 8 males and 7 females, with a median age of 33(4-57) years. The median expression level of newly diagnosed DEK-NUP214 was 488.2% (273.6–1692.1%). Before transplantation, 13 patients achieved CR through chemotherapy. Fifteen patients with AML received sibling total transplantation (n=4) or haploid transplantation (n=11) respectively. All patients received stable neutrophil implantation. Two patients (13.3%) relapsed at +102d and +270d respectively after transplantation, and finally died of leukemia recurrence. In addition, 3 cases were treatment-related deaths (Table 1). 　 　

2. Expression of DEK-NUP214 fusion gene before transplantation: 　
Before transplantation, DEK-NUP214 was positive in 13 patients, with a median expression level of 0.38% (0.029-738.9%). Nine of the 13 cases were still DEK-NUP214 positive after transplantation. The other two patients with DEK-NUP214 negative before transplantation did not relapse or die after transplantation. Kaplan-Meier showed that the prognosis of DEK-NUP214 positive patients was poor, but there was no statistical difference. The 3-year CIR of positive patients before transplantation was 17.5 11.3% (P = 0.545, Figure 1A). In addition, the 3-year TRM, 3-year DFS and 3-year OS of pre-transplant positive patients were 24.5 12.3%, 61.5 13.5% and 60.5 13.8%, respectively (P = 0.464, Figure 1b; P=0.336, Figure 1c; P=0.329, fig. 1D). 　 　
3. Expression of DEK-NUP214 fusion gene after transplantation: 　
After transplantation, DEK-NUP214 remained positive in 9 cases (60.0%). However, its expression level decreased by about 500 (5.7-5,663) times within one month after transplantation. Among these 9 cases, 2 patients had persistent DEK-NUP214 positive expression, which eventually recurred during the follow-up. Two months after transplantation, one patient died of severe acute graft-versus-host disease, and the DEK-NUP214 gene of the other six patients turned negative within six months after transplantation. The 3-year CIR of DEK-NUP214 positive group was higher, which was 27.1 16.5%. There was no recurrence in DEK-NUP214 negative group (P=0.187, Figure 2A). Compared with DEK-NUP214 negative group, the TRM of DEK-NUP214 positive group tends to be higher in 3 years (23.8 14.8% and 16.7 15.2% respectively, P=0.674, Figure 2B). The 3-year DFS and 3-year OS between the two groups were 55.6 16.6% and 83.3 15.2 (P = 0.229, Figure 2C) and 53.3 17.3% vs.83.3 15.2% (P = 0.245, Figure 2D), respectively. 　 　 　 　
4. Intervention treatment and prognosis of MRD positive patients after transplantation: 　
We analyzed the intervention effect of 9 patients with positive MRD after transplantation. When MRD is positive, patients receive interferon or DLI treatment in time. Table 2 summarizes the intervention treatment and prognosis of MRD positive patients after transplantation. Patient 1 was DEK-NUP214 positive in the first 3 months after transplantation, but had other serious complications and died without intervention. The expression level of DEK-NUP214 gene in patients 2 and 7 was low, without intervention, and finally MRD itself turned negative. Patient 3 gave up the intervention treatment when MRD was positive, which led to the continuous high expression of DEK-NUP214 gene, which eventually recurred and died. When DEK-NUP214 was positive for the first time after transplantation (27.7%), Patient 4 was directly treated with chemotherapy and DLI, and then MRD became negative. Patient 5 died of infection early after transplantation. Patients 6 and 8 received interferon therapy and MRD became negative. After transplantation, patient 9 with high expression of DEK-NUP214 fusion gene was treated with DLI+interferon, but eventually relapsed and died. 　

Third,Discussion and analysis

Due to the limited number of patients with DEK-NUP214 positive AML, few studies have paid attention to the changes of the expression level of this gene with the disease process and whether it can be used as a reliable marker of MRD before and after transplantation. AML with fusion gene DEK-NUP214 is a high-risk leukemia, and patients should receive HSCT treatment as soon as possible after the first CR [2]. Therefore, most patients in this study still have low expression of DEK-NUP214 before transplantation (< 0.1%). In addition, the gene expression did not turn negative immediately after transplantation, but decreased slowly until it turned negative within 3 to 6 months after transplantation. Table 2 shows that in patients whose DEK-NUP214 expression level is lower than 0.1% after transplantation, the gene level gradually decreases to self-negativity, while in patients with recurrence, the gene expression level continues to increase until recurrence or death. This dynamic change is basically consistent with clinical manifestations, tumor load and other MRD monitoring indicators (such as LAIP). The expression level higher than 0.1% implies a high risk of recurrence after transplantation. 0.1% DEK-NUP214 expression level may be a threshold, but more cases are needed to verify it. 　 　
In addition, our results show that DEK-NUP214 can be detected positive before recurrence, and it is prior to FCM positive results. Ommen HB et al. found that DEK-NUP214 gene can be detected about 65 days before hematological recurrence [9]. Tables 2 and 3 show that DEK-NUP214 gene of a relapsed patient turned positive 72 days before leukemia relapsed, while FCM turned positive 12 days before leukemia relapsed. Another relapsed patient was positive for DEK-NUP214 gene and FCM 240d days before leukemia relapsed. Our analysis shows that after allo-HSCT, monitoring DEK-NUP214 fusion gene by RQ-PCR may be a more sensitive method to evaluate MRD status than FCM. Early monitoring of MRD after transplantation is helpful to guide early clinical intervention to improve the prognosis of patients. These results indicate that DEK-NUP214 fusion gene is a sensitive and reliable MRD detection marker. 　 　

Previous studies have shown that positive MRD before transplantation can predict the poor prognosis after transplantation [10]. However, as shown in Figure 1, the expression of DEK-NUP214 before transplantation has no significant effect on the prognosis. This is because most patients (n=12, 80.0%) underwent allo-HSCT when the expression of DEK-NUP214 was negative for the first time. Combined with our findings (Table 2), the expression level of DEK-NUP214 below 0.1% has no significant effect on prognosis. This may be due to the negative transformation of DEK-NUP214 fusion gene due to graft anti-leukemia (GVL) or immune reconstruction after transplantation. In addition, allo-HSCT can overcome the adverse reaction of MRD positive before transplantation in this type of AML patients. Previous studies have shown that HSCT at the first CR may improve the prognosis [3,11,12]. Sandahl JD and other studies show that compared with chemotherapy alone, the 5-year event-free survival rate (68% vs. 18%) and overall survival rate (68% vs. 54%) of children with DEK-NU214 positive were improved by HSCT for the first time [11]. Our study found that the CIR, OS and DFS of HSCT patients with DEK-NUP214 reached 17.5%, 60.5% and 61.5% respectively. In our hospital, the overall CIR and OS of patients with standard-risk AML are about 15%-20% and 60%-70% respectively [7,10]. Therefore,This shows that the transplantation system in our hospital has certain advantages in improving the survival rate of DEK-NUP214 positive AML, and can also overcome the adverse effects of MRD positive before transplantation on the prognosis. 　 　
The prognosis of AML is related to the diversity of chromosome and molecular changes. Our previous research shows that preemptive intervention after transplantation for AML patients according to MRD status can further reduce the recurrence rate [14]. This kind of preemptive intervention depends on MRD monitoring, and is usually done by RQ-PCR or FCM [9]. Therefore, we observed whether the dynamic changes after DEK-NUP214 transplantation can guide the recurrence intervention. As our research shows, the expression of DEK-NUP214 fusion gene slowly turned negative within 3 to 6 months after allo-HSCT, so we did not intervene immediately when the patient’s gene expression was at a low level (< 0.1). Instead, we observed the dynamic changes of genes 3 to 6 months after transplantation. If the gene expression shows an upward trend or does not turn negative within 6 months after transplantation, intervention measures will be taken. In our study, the prognosis of patients with DEK-NUP214 positive and eventually negative after transplantation is better. 　 　
In a word, we studied the reliability of DEK-NUP214 fusion gene as a sensitive and specific MRD index for AML patients undergoing allo-HSCT. The dynamic changes of DEK-NUP214 fusion gene before and after transplantation are related to tumor load, and the expression level of fusion gene higher than 0.1% implies high recurrence risk after transplantation. In addition, allo-HSCT can overcome t(6; 9) Adverse effects on AML patients. Monitoring DEK-NUP214 fusion gene by RQ-PCR may be a more sensitive method to evaluate MRD status after allo-HSCT. Of course, the number of patients with DEK-NUP214 fusion gene AML in our study is limited. It is still necessary to accumulate more samples for continuous research to more clearly prove the dynamic changes of DEK-NUP214 expression before and after transplantation. 　 　
Note:This article comes from the topic of "Molecular Diagnosis" in the fifth issue of Clinical Laboratory in 2021.　 　 　　

eightmoonthirtyOn the afternoon of, Beijing Sport University.2022The mobilization meeting of students’ military training was held in Yingdong track and field. Zhou Qidi, member of the Standing Committee of the Party Committee and vice president of the school, attended the conference, and responsible comrades of relevant functional departments and colleges, all freshmen counselors, all military training instructors and2022Grade-6 students attended the conference. The conference was presided over by Zhang Lili, Minister of Student Affairs Department (Armed Forces Department).

　

　

The military training mobilization meeting opened in the sound of the solemn national anthem.

　

　

Huang Shize, deputy director of the training department of Beijing Guoxing Shengrong National Defense Education and Training Base and head of the military training regiment, solemnly took over the flag of the military training regiment from Zhou Qidi on behalf of the military training regiment.

　

　

Sun Jiahao from China Wushu Institute spoke on behalf of all the students participating in the training and called on all of them.2022Students of Grade I carry forward the school motto of "civilize their spirit and be brutal with their physique", train carefully, strictly observe discipline and temper their character, devote themselves to military training with high morale and full enthusiasm, and present their youth to the 20th National Congress of the Communist Party of China.

　

　

Pang Xu, a military training instructor, spoke on behalf of all military training instructors. He said that all military training instructors will show their rigorous training style and first-class and excellent military quality to ensure the high standard and high quality completion of this military training task.

　

　

In his mobilization speech, Zhou Qidi put forward three hopes and requirements for all the teachers and students participating in the training: First, shoulder the historical mission and strengthen the confidence of progress. It is hoped that students will integrate their feelings of the motherland being thicker than water and sharing the same fate with the people throughout their studies and their career pursuit, and take military training as an opportunity to strengthen their physique, shape their character and be tough-willed. The second is to keep up with excellent examples and learn from the motivation of forging ahead. It is hoped that students will follow good soldier’s example, resolutely complete the training tasks of various training subjects, and cultivate a vibrant spirit, a sense of discipline that forbids behavior, an orderly living habit, and a collective concept of unity and cooperation. The third is to establish lofty ideals and forge practical skills. I hope that students will study hard, study hard, seek truth and learn, practice true skills, strive to be pioneers of the times, and write wonderful things belonging to the people of Beiti. Zhou Qidi emphasized that in the whole process of military training, the school will make overall arrangements for all the work to ensure the safety and order of the campus. At the same time, I also hope that students will firmly establish the consciousness of "the first person responsible for their own health" and strictly abide by the campus epidemic prevention policy.

　

　

Military training is an important part and compulsory course of university study and life, and it is an important measure for the school to fully implement the National Defense Education Law and promote students’ quality education.1955In 2000, according to the Military Service Law of People’s Republic of China (PRC) and the spirit of the CPC Central Committee, as one of the only two pilot colleges for military training for students in China, the school took the lead in carrying out military training among students and became one of the earliest colleges and universities in China to carry out military training education for students. Close to running a school70Over the years, the school has trained for the country.10More than 10,000 outstanding graduates have not only trained a number of outstanding talents for the sports field, but also delivered many outstanding talents for the China People’s Liberation Army, the Armed Police Force and the public security police team, and won the title of "Advanced Unit of Recruitment Work in Beijing" for eleven consecutive years.

It is reported that this year the school has1900More than 100 freshmen participated in military training, and the military training brigade was set up.fourA battalion,36A company. In addition to military training, the school will also carry out patriotic education activities and national defense education activities in light of actual conditions. selfeightmoonthirtyStarting from the day,2022Grade 10 students will be fully engaged in military training, opening a new chapter in high-spirited and colorful university life.

　

　

　

Original title: Keep You Safe: Warm heroism in intertextual narration

As a unique cultural phenomenon in human society, rumors map the power structure in a specific period and intervene in the operation of real society with virtual emotional shocks. The sinologist Kong Lifei’s "Calling the Soul" tells the operating mechanism of traditional social rumors; The director dissects the rumor production mode in the network age through intertextual narration that spans the cyberspace and the real society, and praises the warmth of ordinary people with heroic romantic narration.

Rumors of Intertextuality: Ethical Dilemma in Dual Narration

Wei Ping-an, the hero of the movie "Keep You Safe", lives by selling cemeteries and is down and out. His client, Han Lu, was slandered after his death. Wei Ping ‘an sees rough roads, but he also falls into new rumors. At the beginning of "Keep You Safe", the core conflict of the whole film was directly presented to the audience. Rumors that are constantly fermenting in cyberspace eventually overflow the virtual world, changing the real fate of the dead, and also holding Wei Pingan, a cemetery agent, pushing his career to the edge of a dangerous cliff.

The victims of online "yellow rumors" are the dead who can’t prove their innocence, and there are no relatives who defend the dead. They have also taken a stand by while connecting orphanages with millions of donations. Although the rumors that are hard to match will soon be "sealed by the coffin" due to the lack of discourse confrontation, and will soon be forgotten, the Internet memory mechanism of "flying geese and leaving a sound" can completely change the life image of rumor victims. It is precisely this that stimulated Wei Pingan, who could have stayed out of it. He jumped into the whirlpool of "yellow ballads" at the risk of his life, trying hard to distinguish right from wrong and to compete with the huge and secret network power.

Once involved in this grand "yellow rumor" carnival, Wei Ping-an is like a lantern bearer in the "dark forest law", replacing the late Han Lu as a new prey of online rumors &mdash; &mdash; Not only is his relationship with Han Lu distorted, but even his just "criminal record" that he was jailed for standing up for his friends will become a new rumor that cannot be said.

The intertextuality between Wei Ping-an and Wei Ping-an is the reality rumor impact that Wei Ping-an’s daughter Wei Mo faced. If Wei Mo-li, who witnessed the truth, wants to expose rumors, he will be reviled by rumor makers like Wei Ping-an and become a new "back-pot man" and "scapegoat".

The similar experience of Wei’s father and daughter directly points to the ethical dilemma in the operation of rumors. In the anonymous field of the internet, rumors are only the entrance to the carnival of words and imagination, and also the outlet for netizens to vent their pressure. The existence of rumors satisfies the spiritual pleasure of rumor producers, disseminators and onlookers. Once someone wants to deprive this pleasure, he will sacrifice himself to become a new "plaything". Internet rumors use this kind of "person-to-person" pyramid scheme to ensure their vitality and value, and also deter the intervention of rumors. In real life, rumors, through the words and physical threats of rumor makers, shield the opponents of rumors from the rumor field and are ready to swallow up the rumors at any time. Whether in virtual space or in real life, rumors are burdened with heavy mental pressure and suffer in the ideological struggle of watching and intervening.

As the film says: "When you open your mouth and say that a woman is a lady, whether she is or not, she is already." The production of rumors does not require cost, but to compete with rumors, it requires the rumors to constantly consume themselves until they fall into an ethical dilemma of self-doubt. Compared with the film directed in 2012, the breakthrough of "Keep You Safe" is to shift the focus from the victim of rumors to the rumor-maker, and to show the chain destructive power of rumors more integrally with the structure of intertextual narration, so that the audience has a deeper understanding and vigilance against rumors in the Internet age.

Romantic warmth: heroism in daily fireworks

Wei Ping-an is an elderly citizen with no special skills, who lives by selling graves in the cemetery with the help of his former friends. But it is such a humble little person who has shouldered the banner of fighting against the "yellow rumors" on the Internet without hesitation. After losing the battle for the right to speak in cyberspace, Wei Ping-an struggled with General Feng and Chairman Wolongshan, who were eager to move their graves, to gain time. On the other hand, he "hacked" Chairman Jin, the key person in the rumor spreading chain, and finally tracked down the clues of the rumor maker. Then, it was a thrilling adventure of "riding a thousand miles". After spending a lot of time, money and energy, Wei Ping-an ended the rumor with "one-man fighting".

Walking in the same direction as Wei Ping’s rumor-dispelling trip is the spiritual growth process of Wei Li, a female middle school student, from hesitation to firmness. She was bullied on campus because of rumors. Instead of falling into depression as her parents feared, she quickly revived with blood based on the belief of "doing what you think is right", which provided a firm value support for Wei Pingan, who is thousands of miles away.

In the process of breaking the two rumors, we did not focus on social organizations and schools as representatives of power discourse, but shaped little people like Wei’s father and daughter into civilian heroes against rumors. In this rumor war, as heroes, they have no weapons to rely on, only the warmth of caring for kindness in their hearts. The film’s display of Wei Ping ‘an and Wei’s inner tenacity is actually to pin the hope of dispelling rumors and stopping rumors on the audience and every ordinary person in real life.

The production and dissemination of rumors are based on people. To eradicate rumors, we must eradicate the psychological mechanism of producing rumors and spreading rumors. In the process of Han Lu’s "yellow ballads", "Mr. Perfect 421" certainly played a fundamental role, but the pursuit of self-exposure by a large number of netizens eventually turned this insignificant snowflake, which was originally submerged in the information ocean, into a snowball that crushed the real world. In the comparative narrative of spreading rumors and dispelling rumors, the film actually raises a sharp question: which producer or disseminator played a greater role in making rumors "rumors"?

At the end of the story, the late Han Lu arranged fireworks for Wei Ping ‘an in the form of a will. Naturally, this splendid fireworks is not a thank you to Wei Pingan for defending his reputation, but a thank you for the warmth he has always kept in his heart. In the fireworks all over the sky, Han Lu’s warmth to the world and Wei Ping’s warmth to Han Lu met across time and space. The heroic portrayal of Wei’s father and daughter in the film also declares a distinct value position: "Rumors stop at the wise" does not require everyone to be observant, as long as the warmth in their hearts is not lost in the information carnival, everyone can become a hero in the fireworks world.

In the "information cocoon" of the Internet and big data, the production and dissemination of rumors are easier and faster than at any time in history, and the difficulty of dispelling rumors has reached the peak of history. Everyone can be a gossip, and naturally they can also be rumors. "Keep you safe" uses a warm heroic narrative to praise the Wei father and daughter who "don’t believe in rumors and don’t preach them"; With the warmth of heroism, I look forward to everyone who is not afraid of "tempering". (Author: Associate Professor Du Xiaojie, School of Journalism and Communication, Nanning Normal University)