[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.　 　 　　

　　China News Service, Yibin, June 22 (Reporter He Yuqing) The China Seismological Network officially determined that a 5.4-magnitude earthquake occurred at 22: 29 on June 22 in Gongxian County, Yibin City, Sichuan Province (28.43 degrees north latitude and 104.77 degrees east longitude) with a focal depth of 10 kilometers.

　　"I saw at the resettlement site in Gongxian County during the earthquake that there were two buildings about 30 stories high swaying and shaking very badly." Huang Jian, director of Yibin detachment of Sichuan Armed Police Corps, said that after the earthquake, all the people of Yibin detachment who participated in the disaster relief in Gong County quickly assembled, and sent teams to the resettlement sites to check whether there were any villagers injured and to investigate the communities around the resettlement sites.

　　Yu Dong, a staff member of One Foundation in xunchang, Gong County, recalled that the earthquake that happened tonight was the strongest since the Changning earthquake in Gong County. After the earthquake, everyone ran into the street, and people are going home or returning to resettlement sites.

　　The earthquake felt strong in Chengdu, Luzhou, Leshan, Meishan and Chongqing.

　　At 22: 55 on June 17th, an earthquake of magnitude 6.0 occurred in Changning County, Yibin City, Sichuan Province. As of 16: 00 on the 21st, nearly 2,000 aftershocks were recorded. (End)

　　"Female Durban" preached that feudal cultures such as male superiority and female inferiority were shut down. The picture shows some scenes of students in class (video screenshot)

　　Recently, several videos of undercover filming minors "Female Durban" have been circulated on the Internet. In the class of parent-child summer camp held by Zhejiang Wenzhou Traditional Culture Promotion Association, the teacher put forward the contents of "never fighting back, never swearing back, never divorcing" and so on. The content of the lecture has aroused extensive discussion among netizens. Many netizens believe that the teacher in class is actually promoting content that violates social morality under the banner of inheriting traditional culture, which is extremely inappropriate. Yesterday afternoon, Beijing Youth Daily reporter learned from Wenzhou Civilization Office that several departments have formed a joint investigation team to investigate this matter. At present, Wenzhou Civilization Office and Civil Affairs Bureau have interviewed the person in charge of Wenzhou Traditional Culture Promotion Association and ordered them to immediately stop running classes and close training points.

　　After combing, the reporter of Beiqing Daily found that similar "female Durban" had appeared before, and the "female Durban" in Fushun, Liaoning Province, which triggered a heated discussion last year, was also asked to close and stop enrolling students. Among the minors "Female Durban" in Wenzhou, some teachers are from Fushun Traditional Culture Education School, which was previously shut down. In the face of the farce that "Female Durban" was repeatedly shut down but reappeared, many netizens questioned why "Female Durban" was repeatedly banned.

　　The teaching content of Wenzhou "Female Durban" is controversial.

　　Recently, the minor "Female Durban" in Wenzhou City, Zhejiang Province has continuously attracted the attention of netizens. The classroom video filmed by undercover online shows that the teacher’s content in the course of teaching has some contents that promote male superiority and female inferiority, requiring women not to fight back, scold back, submit to it, and resolutely not divorce.

　　Some underage students said in class sharing that she dressed herself in fashion and exposure, which was equivalent to teaching others to insult her. "Three refinements make one poison, which only hurts unclean women."

　　The lecturer’s remarks in the classroom triggered discussion among netizens. Many netizens thought that the contents such as "men are superior to women" and "belittling women" not only promoted feudal culture such as men are superior to women, but also did not conform to the current values of equality between men and women.

　　The reporter of Beiqing Daily learned that the online "Female Durban" was run by the Wenzhou Traditional Culture Promotion Association of Zhejiang Province. The classroom content came from the parent-child summer camp held during this summer vacation. The girls recruited ranged in age from 5 to 18. They came from all over the country and were "educated" accompanied by their parents.

　　After the controversy over the "female Durban" of minors, Wenzhou established a joint investigation team of cities and counties composed of the Municipal Civilization Office, the Civil Affairs Bureau, the Municipal Education Bureau and wencheng county. The survey results show that the content of online video comes from the parent-child summer camp held by Wenzhou Traditional Culture Promotion Association in Zhoudun Village, Zhourang Town, wencheng county from August 10 to August 24, 2018. Some teachers of this class come from Fushun Traditional Culture Education School, and some courses do use the teaching content that is contrary to social morality. At present, the Municipal Civilization Office and the Civil Affairs Bureau have interviewed the person in charge of the Wenzhou Traditional Culture Promotion Association and ordered them to immediately stop running classes and close training points.

　　The supervision mechanism of "female Durban" of thunder people needs to be improved.

　　The reporter of Beiqing Daily found that the "female Durban" of Wenzhou minors is not a case. Last year, there was a similar educational institution in Fushun, Liaoning Province, and there were similar quotations from Lei Ren in the teaching process, including "Women who order takeout and don’t brush dishes are disobedient" and "Women should not go up, but should be at the bottom &hellip; &hellip;” . At that time, after the video was exposed, the local education bureau issued a notice saying that there were problems in the teaching content of "Female Durban" in the school, demanding that it immediately stop running the school and dismiss the students.

　　During the investigation, Wenzhou Civilization Office found that some lecturers of the minor "Female Durban" came from Fushun Traditional Culture Education School, which had been shut down before.

　　In addition, the reporter of Beiqing Daily learned that the Wenzhou Traditional Culture Promotion Association had enrolled students many times before this incident was exposed. Recently, there were still closed classes for minors enrolling students all over the country. On the afternoon of December 8th, the 12th training center of Wenzhou Traditional Culture Promotion Association was still enrolling students. Teacher Yang of the training center said that the class was closed, mainly for underage girls, and they needed to attend classes in Wenzhou accompanied by their parents.

　　When the reporter of Beiqing Daily asked about the specific content of the course, Teacher Yang didn’t want to explain too much. She only stressed that this closed class is a public welfare free school, which will provide basic protection for food and shelter. In the training center, vegetables are mainly eaten, not meat. "The training center now advocates health preservation, and vegetables have better nutrition for the body."

　　According to a survey conducted by the reporter of Beiqing Daily, the organizers of "Female Durban" all passed the examination and approval of relevant departments in the name of public welfare mass organizations, but they were all related to education when actually conducting business.

　　According to public information, Wenzhou Traditional Culture Promotion Association is a social organization. In August 2011, it was recorded in Wenzhou Civil Affairs Bureau, and the competent business department was Wenzhou Civilization Office. Fushun Traditional Culture Education School, which was shut down before, was approved by Fushun Civil Affairs Bureau in April 2011, and Fushun Traditional Culture Research Association was formally established, which is a public welfare mass organization. Yesterday afternoon, the reporter of Beiqing Daily called Wenzhou Civilization Office and learned that the management departments involved in Wenzhou Traditional Culture Promotion Association include local education bureau and Civilization Office.

　　Some experts said that inappropriate teaching content in the organization is not easy to be found in actual supervision. Because there is no problem just from the names of courses reported by these organizations, what teachers really talk about in actual teaching can’t be supervised. In addition, experts believe that "female Durban" does not involve corporal punishment and illegal restraint of minors’ personal freedom, and it cannot be legally prohibited, which is one of the reasons why "female Durban" has been repeatedly banned.

　　Traditional culture education needs to standardize training qualifications.

　　"Female Durban" has been criticized frequently, but it has been repeatedly banned. In this regard, Professor Han Xing of the Teaching and Research Section of the National College of China Renmin University believes that there is an extreme in the voice of "equality between men and women" in today’s society, and some men and women who pursue sexual liberation are more indulgent. In family relations, some women do not bear family responsibilities, including the education of their children, but some people mistakenly blame this on the lack of "female morality".

　　Professor Han Xing told the reporter of Beiqing Daily that at present, in traditional culture education, folk lectures are rather chaotic. "For example &lsquo; Female Durban &rsquo; There are few formal scholars involved. Some people have only learned a little, some may be driven by economic interests to set up training courses, but there are deviations in understanding, some have deviations in understanding the application of traditional culture in ancient times, and some have mechanically copied it regardless of the changes of the times and social environment. "

　　Regarding the present situation of traditional culture education, Professor Han Xing thinks that the unified management is not enough at present, and it needs the supervision of education, culture and other relevant departments, such as the qualification management of training institutions and lecturers, and the standardization of training contents. "In some traditional culture courses, it is no problem to just look at the topic, but it may be biased when teaching the specific content. If you find something wrong after attending the training, you should quit in time."

　　Professor Han Xing said that the contents such as The Book of Filial Piety, The Story of Empress Dowager, which frequently appeared in the "Female Durban", are good in themselves and generally teach people to be kind, but the key lies in the lecturer’s understanding and how to teach. "Filial piety is a part of traditional culture, but kowtowing and washing feet for parents in public can easily become a performance. Learning traditional culture should be combined with the actual situation of society, aiming at problems, analyzing problems, finding out the root causes of problems, and solving them with the wisdom of traditional culture, but don’t exaggerate real problems too much. "

　　This edition/reporter Zhang Xiangmei

　　Intern Dai Youqing Ren Yingnan