ALBERT

Description: Background Allogeneic hematopoietic stem cell transplantation is an effective method for treatment of hematological malignancies, while GVHD and graft rejection are main complications, which seriously affect patients' survival rates and quality of life. Aim Establishing allo-transplantation mice model with mRFP and GFP transgenic mice, to simulate clinical hematopoietic stem cell transplantation and explore the mechanism of stem cell homing and GVHD. Methods 1) Thirteen C57BL/6 GFP transgenic mice, used as recipients, were irradiated with 7 Gy. Each mouse was injected through caudal vein with 2*106 bone marrow cells isolated from FVB mRFP transgenic mice. 2) Symptoms like weight loss, depilation, diarrhea were observed as GVHD manifestation while survival rates were evaluated. Routine blood test and FACS were performed at different time points to confirm hematopoiesis reconstitution. 3) Mice were perfused with paraformaldehyde under anesthesia to fix the tissue, while pathological examination and real-time PCR were performed for studying donor and recipient cells interactions in different organs. 4) Semi-solid decalcification was used to treat the femora before observing under confocal microscope directly or after making frozen section, three-dimensional reconstruction were made to observe the cellular interaction, especially for cells within the bone marrow. Result 1) Depilation, wrinkled skin, hunchback and sharp decline of weight were observed in 8/13 mice. Routine blood test implicated hematopoietic reconstitution. FACS showed 86.1%±7.8% mRFP+ cells in peripheral blood of recipients. 2) mRFP+ cells were found distributing throughout the body's organs. mRFP+ Lymphocyte infiltration and inflammatory exudate were seen especially in the small intestine, lung, liver and skin (Fig.1). GFP+ cells were found surrounding mRFP+ cells in the bone marrow of the femora decalcified with semi-solid decalcification. Their interactions can be further observed clearly in bone marrow microenvironment in three-dimensional reconstruction by confocal microscope (Fig.2). Discussion Owing to RFP on donors' cells and GFP on recipients' cells, together with our novel protocol named semi-solid decalcification, we can visually observe the donor and recipient cells' location, ratio and cellular interaction, as well as morphological changes. Within various tissues especially for such tissues as bone marrow and lung, the details between cells can be studied lively by fluorescence microscope and confocal microscope. In recipient mice with GVHD, donor cells can be found in various target tissues such as intestine, lung, liver and skin. Gene marked cells with fluorescence protein can benefit morphological, immunological, cytogenetic and molecular studies in recipients after HSCT. Conclusion The allo-transplantation model with mRFP and GFP transgenic mice is powerful in study of Stem Cell Homing and Donor-Recipient Cellular Interaction. The cellular interaction can be easily observed by three-dimensional reconstruction after semi-solid decalcification, especially for bone marrow and lung. Disclosures: No relevant conflicts of interest to declare.

Description: INTRODUCTION: Circulating tumor DNA (ctDNA), a portion of circulating cell-free DNA (cfDNA), is released from tumor cells into the circulatory system, which contains mutations corresponding to the patient's tumour alleles. Detection of ctDNA may noninvasively signal the presence of minimal residual disease (MRD) and predict prognosis. In recent years, droplet digital PCR (ddPCR) has emerged as a sensitive and effective tool for detection of point mutations in cfDNA. In this work, by application of ddPCR, we monitored the mutated TP53 ctDNA levels in serial plasma samples of lymphoma patients with identified TP53 hotspot mutations in their tumor biopsies. These results may give clues about the prognostic implications of different mutation locations and therapeutic strategies. METHODS: Lymphoma-focused next-generation sequencing were performed in tumor biopsies from over 200 lymphoma patients. A total of 134 sequential plasma samples from 32 lymphoma patients with TP53 hotspot mutations were subsequently monitored. cfDNA was extracted from a median sample volume of 4.8 ml (range 3.5-7.0 ml) of plasma. Specific MGB probes were designed for each TP53 mutation site and were then validated by tumor biopsies as well as healthy plasma samples as positive and negative controls respectively. Patient samples were considered to be mutation-positive if the mutant concentration in the sample was higher than the 95% confidence interval of the assay-specific false positive rate. The ctDNA sequential samples were quantitatively tracked using the ddPCR system QX200 (Bio-Rad Laboratories). RESULTS: 30 different TP53 mutations in 134 plasma samples from 32 lymphoma patients were monitored. All the mutations are hotspot in tumors according to COSMIC database, and are predicted to make damaging effect on TP53 protein by SIFT prediction. The average TP53 mutation frequency in tumor biopsies is 40.41% (2.30~92.21%). A total number of 30 specific MGB probes were designed for each TP53 mutation site. Their average false positive rate is 0.0004 copies/ul (0~0.002 copies/ul). In our results, 6 out of 20 (30%) patients with mutations in Loop3 and LSH motifs within the DNA binding domain of TP53 had a clearance of ctDNA, while 6 of 12 (50%) patients with mutations outside of the Loop3 and LSH motifs had a clearance of ctDNA. Literatures reported that patients with non-detectable ctDNA in plasma have negative minimal residual disease (MRD). So these results suggested that Loop3 and LSH motifs, which were reported to interact with DNA groove directly, are more critical than other structures. To investigate the association between different Ann Arbor stages and prognosis in TP53 mutated patients, we divided all the patients according to their stages. We found that patients with earlier Ann Arbor stages were more likely to have their ctDNA cleared (stage I/II: 83.33%; stage III/IV: 28%). In addition, we also analyzed the prognostic implication of cytogenetic abnormality. We found that, in patients with del (17p) as well as TP53 mutation, 7 of 8 had persistent detectable TP53 mutated ctDNA, only 1 of 8 (12.5%) had a clearance of ctDNA, While among the patients carrying TP53 mutations but not accompanied del (17p), 11 of 24 (45.83%) patients had a clearance of ctDNA. These results suggested that it is more difficult to achieve remission in patients with no wide-type TP53 allele compared to those have one wide-type TP53 allele. We further evaluated CAR-T cell immunotherapy and chemotherapy alone to 30 refractory recurrent TP53 mutated lymphoma patients. After CAR-T cell immunotherapy, 11 of 21 (52.38%) patients have non-detectable TP53 mutated ctDNA, while only 2 of 9 (22.22%) patients who received chemotherapy alone have non-detectable ctDNA. It suggested that CAR-T cell immunotherapy is more effective than chemotherapy alone to clear TP53 mutated ctDNA. CONCLUSIONS: Circulating tumor DNA is a promising biomarker to noninvasively monitor tumor load and quantify MRD. By applying droplet digital PCR, ctDNA could be measured and monitored sensitively, specifically and fast. In this work, we designed and validated 30 TP53 probes and tracked 134 sequential plasma from 32 patients. We found that it is easier to clear ctDNA for earlier stages of lymphoma patients with TP53 mutations. CAR-T cell immunotherapy is more effective than chemotherapy alone to continuously clear ctDNA. DISCLOSURE: No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

Description: Introduction DNA methyltransferase (DNMT) family play an important role in the development and growth of lives, encoding enzymes that catalyze the addition of a methyl group to the cytosine residue of CpG islands. With the increase in methylation, the downstream genes are often associated with reduced expression. In this family, DNMT3a occupies the essential position to implement the de novo methylation. Timothy J. Ley and many other scientists found that in M4 and M5 acute myeloid leukemia (AML), around 20% patients suffered from DNMT3a mutation (most are R882H mutation), always associating with adverse prognosis. But what's the reason for adverse prognosis? Additionally, our formal Meta analysis showed that the de novo AML patients with DNMT3a mutation have higher platelet counts, WBC and RBC counts. To shed some light on the possible causal relation between the increasing in platelet count and poor prognosis led by DNMT3a mutation, we transduced the MK cell lines with genes null-mCherry (null), DNMT3a wild type-mCherry (DNMT3aWT) and DNMT3a R882H mutation type-mCherry (DNMT3aMT) respectively, trying to figure out the possible role that the mutation plays in the megakaryopoiesis and thrombopoiesis. Also, we tested several drugs that may target the mutation. Methods The SFFV-null-IRES-mCherry, SFFV-DNMT3aWT-IRES-mCherry and SFFV-DNMT3aMT-IRES-mCherry plasmids were constructed by Dr. Qianli Jiang, modified from LEGO-iC plasmids. MK cell lines (chrf-288-11, meg-01) were flow-through transduced with the lentivirus produced by packaging plasmids and those above. All the fluorescence positive cells have been doubly sorted by flow cytometry. Cell ploidy was analyzed by flow cytometry using Propidium Iodide (PI); colony forming unit (CFU-MK) were enumerated 14d after being plated with TPO and IL-3; cell proliferation were tested by CCK-8; apoptosis was measured via flow cytometry with PI and Annexin V-FITC; CD41a and CD61 were tested with flow cytometry. The drug tests including Decitabine, Dasatinib and Rituximab were analyzed using CCK-8 test and cytomorphologic tests. Results With CCK8 test of chrf-288-11 and meg-01, DNMT3aMT proliferates faster than the null and DNMT3aWT (P

Description: Objective Acute hemorrhagic cystitis (HC), a severe complication of hematopoietic stem cell transplantation (HSCT), being considered mainly as a result of cyclophosphamide (CTX), seriously affects the quality of life of patients. Mesna, whose half-life is about 70min, is widely used to prevent HC. Aim of this research is to explore the effect of preventing HC in HSCT by continuous intravenous injection of mesna using micro pump on HC in HSCT. Methods (1) 359 patients who underwent allogenic HSCT in Nanfang hospital from January 2003 to December 2012 were recruited into this study (227 male and 132 female). Conditioning regimens were BuCy or TBI-Cy, in which CTX were given 60mg/kg·d,d-3,-2; or GIAC, in which CTX were given 1.8g/m2, d-5, -4. Intravenous injection of mesna was used to prevent HC continuously using micro pump (continuous group, n=250) or intermittently (intermittent group, n=185). Graft versus host disease (GVHD) prevention regimen was cyclosporine A+MTX for HLA-matched sibling donors and cyclosporine A+MTX+ATG for unrelated donors or HLA partial-matched related donors. (2) Both groups received the same daily dose of mesna, which is about 150% of CTX daily dosage. In the intermittent group, 25% of mesna’s daily dosage was injected at 0h, 3h, 6h and 9h after the use of CTX at each time-point; while in the continuous group, 25% of mesna’s daily dosage was injected before the use of CTX, with the rest dosage being continuously injected intravenously for 24hs using micro-infusion pump (25% daily dosage of mesna dissolved in 40ml 0.9% sodium chloride lasting for 8h was given, q8h), from the first dose of CTX till 48hs after the last injection of CTX. Incidences and grades of HC in the two groups were followed up and analyzed. (3)The mesna concentration in urine of two groups was detected by High Performance Liquid Chromatography (HPLC), with the comparison of the trough concentrations and the peak concentration. Results (1) Within 30d after transplantation, HC occurs in 30 of the 160 (18.75%) cases in the intermittent group vs. 16 of 199(8.04%) in the continuous group (P=0.00077). Within 60d after transplantation, HC occurs in 45 of 160 (28.13%) cases in the intermittent group (17cases of I°, 18cases of II°, 8 cases of III°, 2 cases of IV°) with the mean occurrence time being +18.16d (-5-+41d); while only 24 of 199 (12.06%) cases (15 cases of I°, 6 cases of II°, 3 cases of III°, 0 cases of IV°) in the continuous group with the mean time of +26.58d (+2d-+58d). There were statistical significances of the incidence within 60d(P=0.000123) and occurrence time(P=0.018), however there was no statistical significances of grade/severity (P=0.057) of HC between the two groups. (2) Logistic regression analysis shows that within 30d after transplantation, the HC occurrences relates with the way of using mesna (P=0.004), with continuous mesna injection being a protective factor (OR=0.299, 95% CI=0.130-0.684); while age, sex, 24h mean liquid intake, 24h mean excretion, 24h mean urinary volume (each P〉0.2) and HLA matching (P=0.099) were unrelated factor. Within 60d after transplantation, the HC occurrences relates with the way of using mesna (P=0.001)and GVHD (P=0.007); continuous mesna injection is a protective factor (OR=0.296, 95% CI=0.146-0.600) and HLA matching is a risk factor (OR=2.422, 95%CI=1.280-4.580). (3) Althrough 73 samples were detected by HPLC for mesna in urine, there were no significant difference of peak and trough concentration between groups. Discussion Early occurrence of HC is mostly related to high dose of cyclophosphamide, while late occurrence of HC can also be related with GVHD and infections. The continuous injection of mesna is a better way for the prevention of HC in HSCT patients, due to its short half-life of mesna. While injection with micro-infusion pump can reduce liquid intake, especially suitable for those who have heart or kidney dysfunctions. Conclusion Continuous intravenous injection of mesna is efficient to prevent HC in hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.