Substrate diversity of NSUN enzymes and links of 5-methylcytosine to mRNA translation and turnover.

Maps of the RNA modification 5-methylcytosine (m5C) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m5C site calling pipeline. With the resulting union list of 6,393 m5C sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed tRNA:m5C methyltransferases NSUN2 and NSUN6 as the main mRNA m5C "writers," but further showed that the rRNA:m5C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m5C sites and footprints of RNA-binding proteins, we identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. We found that lack of NSUN2 in HeLa cells affected both steady-state levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m5C writers and readers and their effect on mRNA function.

Epigenetic inactivation of the 5-methylcytosine RNA methyltransferase NSUN7 is associated with clinical outcome and therapeutic vulnerability in liver cancer.

BACKGROUND: RNA modifications are important regulators of transcript activity and an increasingly emerging body of data suggests that the epitranscriptome and its associated enzymes are altered in human tumors. METHODS: Combining data mining and conventional experimental procedures, NSUN7 methylation and expression status was assessed in liver cancer cell lines and primary tumors. Loss-of-function and transfection-mediated recovery experiments coupled with RNA bisulfite sequencing and proteomics determined the activity of NSUN7 in downstream targets and drug sensitivity. RESULTS: In this study, the initial screening for genetic and epigenetic defects of 5-methylcytosine RNA methyltransferases in transformed cell lines, identified that the NOL1/NOP2/Sun domain family member 7 (NSUN7) undergoes promoter CpG island hypermethylation-associated with transcriptional silencing in a cancer-specific manner. NSUN7 epigenetic inactivation was common in liver malignant cells and we coupled bisulfite conversion of cellular RNA with next-generation sequencing (bsRNA-seq) to find the RNA targets of this poorly characterized putative RNA methyltransferase. Using knock-out and restoration-of-function models, we observed that the mRNA of the coiled-coil domain containing 9B (CCDC9B) gene required NSUN7-mediated methylation for transcript stability. Most importantly, proteomic analyses determined that CCDC9B loss impaired protein levels of its partner, the MYC-regulator Influenza Virus NS1A Binding Protein (IVNS1ABP), creating sensitivity to bromodomain inhibitors in liver cancer cells exhibiting NSUN7 epigenetic silencing. The DNA methylation-associated loss of NSUN7 was also observed in primary liver tumors where it was associated with poor overall survival. Interestingly, NSUN7 unmethylated status was enriched in the immune active subclass of liver tumors. CONCLUSION: The 5-methylcytosine RNA methyltransferase NSUN7 undergoes epigenetic inactivation in liver cancer that prevents correct mRNA methylation. Furthermore, NSUN7 DNA methylation-associated silencing is associated with clinical outcome and distinct therapeutic vulnerability.

Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.

Cloning and Functional Characterization of SpZIP2.

Zinc (Zn)-regulated and iron (Fe)-regulated transporter-like proteins (ZIP) are key players involved in the accumulation of cadmium (Cd) and Zn in plants. Sedum plumbizincicola X.H. Guo et S.B. Zhou ex L.H. Wu (S. plumbizincicola) is a Crassulaceae Cd/Zn hyperaccumulator found in China, but the role of ZIPs in S. plumbizincicola remains largely unexplored. Here, we identified 12 members of ZIP family genes by transcriptome analysis in S. plumbizincicola and cloned the SpZIP2 gene with functional analysis. The expression of SpZIP2 in roots was higher than that in the shoots, and Cd stress significantly decreased its expression in the roots but increased its expression in leaves. Protein sequence characteristics and structural analysis showed that the content of alanine and leucine residues in the SpZIP2 sequence was higher than other residues, and several serine, threonine and tyrosine sites can be phosphorylated. Transmembrane domain analysis showed that SpZIP2 has the classic eight transmembrane regions. The evolutionary analysis found that SpZIP2 is closely related to OsZIP2, followed by AtZIP11, OsZIP1 and AtZIP2. Sequence alignment showed that most of the conserved sequences among these members were located in the transmembrane regions. A further metal sensitivity assay using yeast mutant Δyap1 showed that the expression of SpZIP2 increased the sensitivity of the transformants to Cd but failed to change the resistance to Zn. The subsequent ion content determination showed that the expression of SpZIP2 increased the accumulation of Cd in yeast. Subcellular localization showed that SpZIP2 was localized to membrane systems, including the plasma membrane and endoplasmic reticulum. The above results indicate that ZIP member SpZIP2 participates in the uptake and accumulation of Cd into cells and might contribute to Cd hyperaccumulation in S. plumbizincicola.

N-3 polyunsaturated fatty acids protect esophageal epithelial cells from acid exposure.

Our previous study showed that n-3 PUFAs inhibited inflammation in rats with esophagitis. This study aimed to observe the protective effect of n-3 PUFAs against acid damage to esophageal epithelial cells (Het-1A cells) and to explore its mechanism. The level of malondialdehyde (MDA) was increased by acid exposure, while that of superoxide dismutase (SOD) was decreased. In groups with different ratios of n-6/n-3 PUFAs, the expression levels of nuclear factor E2 related factor 2 (Nrf2) and SOD were increased with increasing proportions of n-3 PUFAs and were highest in the 1:1 group. Compared with those in the 9:1 group, the expression of NOD like receptor pyrin domain-containing protein 3 (NLRP3) and caspase-1 and the pyroptosis rate in the 1:1 group were decreased. Intervention with an Nrf2 agonist increased the expression of Nrf2 and decreased the expression of NLRP3 and caspase-1 and the pyroptosis rate. However, inhibiting Nrf2 expression led to the opposite result. In conclusion, n-3 PUFAs protected esophageal epithelial cells from acid damage by upregulating Nrf2 expression, which disrupted oxidative stress and NLRP3 inflammasome activation and inhibited pyroptosis.

Comparison of Pull and Introducer Techniques for Percutaneous Endoscopic Gastrostomy.

Objective: To compare indications, success rates and complications of pull [P] and introducer [I] techniques for percutaneous endoscopic gastrostomy (PEG). Methods: In this retrospective study, inpatients who underwent primary PEG tube insertion between January 2015 and February 2020 at the Endoscopy Center of the First Affiliated Hospital of Fujian Medical University were included. Results: A total of 103 inpatients were included in this study (P group, n = 67; I group, n = 36). The rates of tube replacement within first six months in the P and I groups were 1.5% and 11.1%, respectively (P = 0.049). The most common primary indication of PEG was malignancy. The proportion of patients with esophageal cancer was significantly lower in the P group (24.4% vs 54.2%, P = 0.015). No significant difference was found in the overall, major, or minor complications between the two groups. In patients with esophageal stenosis, the pull method was a risk factor for complications (P = 0.03; odds ratio [OR] = 12, 95% confidence interval [CI]: 1.164-123.684). Logistic regression analysis showed that the risk factors for major and minor complications were the admission-to-gastrostomy interval (OR = 1.078, 95% CI: 1.016-1.145, P = 0.014) and lack of antibiotic use (OR = 4.735, 95% CI: 1.247-17.979, P = 0.022), respectively. Conclusion: Both PEG techniques have high clinical success rates. The introducer technique is more suitable for patients with esophageal stricture, which has lower minor complications, but higher rate of tube replacement compared to the pull technique. Use of antibiotics may reduce minor complications following PEG. Early PEG insertion may help to reduce post-PEG major complications.

Effect of Endoscopic Appendix Intubation and Irrigation on Acute Uncomplicated Appendicitis.

OBJECTIVE: To investigate the efficacy and safety of endoscopic appendix intubation and irrigation (EAI) on acute uncomplicated appendicitis. METHODS: This prospective non-randomized study examined 169 patients with suspected acute uncomplicated appendicitis at The First Affiliated Hospital of Fujian Medical University from October 2015 to 2017. Patients were divided into three groups: endoscopic appendix intubation and irrigation (EAI, n = 18), laparoscopic appendectomy (LA, n = 87), and antibiotic alone (A, n = 64). The treatment success rate, duration of hospitalization, medical costs, operation time, duration of abdominal pain, fasting time, complications, and recurrence were analyzed. RESULTS: The three groups had no significant differences in baseline characteristics (age, gender, Alvarado score, white blood cell count, and neutrophil count; all P > 0.05). Compared to the LA group, the EAI group had shorter durations of the operation, fasting, and abdominal pain; less use of oral and intravenous antibiotics; and lower medical costs (all P < 0.05). Compared to the A group, the EAI group had shorter durations of abdominal pain and hospitalization, and less use of intravenous antibiotics (all P < 0.05). The EAI group had no complications, but 3 patients (3.4%) in the LA group had surgery-related complications. CONCLUSION: EAI is a safe and effective treatment for acute uncomplicated appendicitis. Patients who received EAI had shorter durations of abdominal pain and hospitalization than those who received LA or conservative antibiotic treatment. TRIAL REGISTRATION NUMBER AND AGENCY: ChiCTR-IPN-15006565, Chinese Clinical Trial Registry.

[Clinical features of childhood refractory cytopenia].

OBJECTIVE: To study the clinical features of patients with refractory cytopenia of childhood (RCC). METHODS: The clinical data of 1 420 children (0-14 years old) with an initial diagnosis of non-severe aplastic anemia between January 1990 and June 2013 were retrospectively analyzed. Bone marrow cell morphology and histopathology were re-evaluated, and the patients were re-classified using the criteria proposed in the 2008 edition of the World Health Organization classification of RCC in hematopoietic and lymphoid tumor tissues. The clinical outcomes were followed up every 3-6 months. RESULTS: Among all the 1 420 cases, 152 (10.7%) were reassessed as RCC. Patients with RCC had a lower level of hemoglobin and a higher percentage of fetal hemoglobin than those with non-severe aplastic anemia. Of the patients with RCC, 21.5% showed abnormal karyotypes at diagnosis. The median follow-up period for all patients was 36 months (ranging from 1 to 283 months). The rates of complete response, partial response, and no response to cyclosporine and androgen treatment in RCC patients were 19.0%, 26.7%, and 54.3%, respectively. The 5- and 10-year prospective overall survival rates of RCC patients were 87.9% and 72.4%, respectively. The 5- and 10-year prospective clonal evolution rates were 15.3% and 20.0%, respectively. The 2-year prospective incidence of newly diagnosed karyotype abnormality after the initial diagnosis was 3.6%. The 5- and 10-year prospective leukemia transformation rates were 10.0% and 20.0%, respectively. CONCLUSIONS: RCC shows clinical features similar to adult myelodysplastic syndrome. Children with RCC have a poor prognosis, an increased risk of transformation to leukemia, and a low response rate to cyclosporine treatment.

[Expression of SIRT1 in bone marrow biopsy tissues and its clinical significance among children with acute myeloid leukemia].

OBJECTIVE: To determine the expression level of silent mating-type information regulation 2 homologue 1 (SIRT1) in bone marrow biopsy tissues among children with acute myeloid leukemia (AML) and analyze its relationship with the prognosis of AML patients. METHODS: A retrospective analysis was performed on the clinical data of 54 children who were diagnosed with AML between July 2009 and April 2012 and who underwent bone marrow biopsy at diagnosis. The expression of SIRT1 in bone marrow was measured by immunohistochemistry. The 54 patients were divided into two groups according to the expression of SIRT1: SIRT1-negative (n=10) and SIRT1-positive (n=44). The SIRT1-positive group was further divided into three subgroups: SIRT1(+) (n=8), SIRT1(++) (n=7) and SIRT1(+++) (n=29) according to the expression levels of SIRT1. Cox multivariate regression analysis was used to determine the unfavorable factors for long survival in children with AML. RESULTS: The SIRT1(+++) subgroup had a significantly higher mortality than the SIRT1-negative group (P<0.05). Compared with the SIRT1-negative group, the SIRT1-positive group had a significantly lower 2-year overall survival rate (P<0.05) and a significantly lower 2-year event-free survival rate (P<0.05). Cox multivariate regression analysis showed that positive expression of SIRT1 was an unfavorable factor for long-term survival in children with AML, with a risk coefficient of 2.071 (95% CI: 1.017-4.219; P=0.045). CONCLUSIONS: SIRT1 is overexpressed in some of pediatric AML patients, and the overexpression of SIRT1 is associated with poor prognosis.

[Treating chronic persistent bronchial asthma children with abnormal myocardial enzyme spectrum by Yupingfeng powder: an efficacy observation].

OBJECTIVE: To observe the clinical efficacy of treating chronic persistent bronchial asthma (CPBA) children with abnormal myocardial enzyme spectrum (AMES) by Yupingfeng Powder (YP) combined routine therapy. METHODS: From January 2010 to December 2012, 156 CPBA children patients with AMES were randomly assigned to the treatment group (80 cases) and the control group (76 cases). All patients received routine treatment (inhaled corticosteroids and/or leukotriene regulator). Besides, those in the treatment group took YP. The treatment duration was 3 months. The scores of children asthma control test (C-ACT), pulmonary function (FEV,% and PEF%), myocardial enzyme spectrum were observed before and after treatment, and 3 months before and after treatment. The myocardial enzyme spectrum of 40 healthy children at the baby clinics during the same period were recruited as the control. RESULTS: Compared with the control group, creatine kinase isoenzyme (CK-MB), creatine kinase(CK), and lactate dehydrogenase (LDH) increased in the two treatment groups (P <0.01), but there was no statistical difference in AST (P >0.05). Compared with before treatment in the same group, CK-MB, CK, LDH, and AST decreased in the treatment group after treatment and 3 months after treatment (P <0.01). CK-MB, CK, LDH, and AST decreased in the control group 3 months after treatment (P <0.01, P <0.05).Compared with after treatment, CK decreased in the control group 3 months after treatment (P <0.01). C-ACT score, FEV(1),%, and PEF% all increased in the two groups after treatment and 3 months after treatment (P <0.01, P <0.05). Compared with after treatment in the same group, CK decreased in the control group 3 months after treatment (P <0. 01). Compared with the control group in the same period, post-treatment CK-MB and CK decreased (P <0. 01, P <0. 05), while post-treatment C-ACT score, FEV, %, and PEF% increased (P <0.05) in the treatment group (P <0.05). CONCLUSION: YP could strengthen specific and non-specific immunity of the organism, and improve clinical symptoms and the level of myocardial enzyme spectrum.

[Determination and clinical significance of serum surfactant proteins A and D in children with bronchiolitis].

OBJECTIVE: To study the variation and clinical significance of serum levels of surfactant proteins A (SP-A) and D (SP-D) among children with different degrees of bronchiolitis. METHODS: Seventy children with bronchiolitis were divided into acute (n=42) and recovery phase groups (n=28). According to the severity of symptoms, the acute phase group was further divided into severe (n=12) and mild subgroups (n=30). Another 26 children who were hospitalized in the same period due to non-infectious diseases and had not undergone surgery were used as the control group. Competitive enzyme-linked immunosorbent assay was performed to measure serum levels of SP-A and SP-D in each group. RESULTS: The acute phase group had significantly higher serum levels of SP-A and SP-D compared with the recovery phase (P<0.01) and control groups (P<0.01). Compared with the control group, the recovery phase group had elevated levels of SP-A and SP-D (P<0.01). Within the acute phase group, serum levels of SP-A and SP-D in the severe subgroup were significantly higher than in the mild subgroup (P<0.01). CONCLUSIONS: Serum levels of SP-A and SP-D are significantly elevated in children with acute bronchiolitis, and severe cases have higher serum levels of SP-A and SP-D than mild cases. Even after the relief of clinical symptoms, serum levels of SP-A and SP-D remain high. These findings suggest that serum levels of SP-A and SP-D might be useful biomarkers for evaluating the severity of bronchiolitis among children.

[Expression of survivin in patients with acute myeloid leukemia].

Objective of this study was to detect the expression of Survivin in acute myeloid leukemia (AML) and investigate the relationship of its expression levels with clinical variates and its correlations with BCL-2 ,Bcl-xL and MCL-1. The expression of Survivin, BCL-2, Bcl-xL and MCL-1 were measured by immunohistochemistry in bone marrow biopsy of 63 newly diagnosed AML patients, and the relationship between its expression level and clinical parameters (age, sex, WBC count, diagnosis, prognosis), especially fusion protein AML1/ETO was investigated. The results showed that the expression level of Survivin in newly diagnosed AML patients was higher than that of normal controls (P < 0.01), the expression levels of Survivin did not correlate with age, sex, and WBC counts of patients and so on. There was no significant difference of Survivin expression between different NCCN prognosis groups, either between patients with AML1/ETO or FLT3-ITD mutation and the other patients. Survivin positive patients were got to have lower CR rate but higher relapse rate, however that was not significant in statistics. Indeed, the cumulative survivin rate of Survivin positive patients were lower than that of Survivin negative patients (P = 0.04). Finally, positive correlation between Survivin and MCL-1 was also observed (r = 0.639, P = 0.000). It is concluded that overexpression of Survivin are closely related with occurrence and development of acute leukemia, and may be used as an indicator of prognosis evaluation. In addition, Survivin and MCL-1 may have a relationship of cooperation or interaction.

[Retrospective analysis of treatment strategy and prognosis in childhood myelodysplastic syndromes from China and Japan].

This study was aimed to retrospectively analyse the prognosis of childhood myelodysplastic syndromes (MDS-RCC ) from China and Japan. Two hematologists and one pathologist from China and Japan constituted a diagnosis group. According to the criteria of 2008 WHO, 33 children with MDS-RCC from 50 chinese children diagnosed as acquired bone marrow failure syndrome from 2009 to 2011, and 74 Japanese children with MDS-RCC in a prospective registration group conducted by the Japanese Society of Pediatric Hematology were enrolled in this study. The outcome of total 107 childhood MDS-RCC treated with different treatment strategies was analyzed retrospectively. The results indicated that: (1) the 3 and 5-year overall survival rates (OS) for all patients were 93.8% and 79.7% respectively. (2) All 107 patients with MDS-RCC were further subclassified into 2 groups: transfusion dependent group and transfusion independent group. The 3 and 5-year overall survival rates (OS) for transfusion dependent group were 89.9% and for transfusion independent group were 70.6% respectively, the 5-year overall survival rate (OS) for transfusion independent group was 100.0%. (3) Treatment strategy: patients from transfusion dependent group were treated with immunosuppression therapy (IST), in which CsA combined with or without ATG and patients were treated with HSCT. The 5-year overall survival rate (OS) was 100% for IST group. The 3 and 5-year overall survival rates (OS) for HSCT patients were 82.9% and 30.6%, respectively. All the patients from transfusion independent group were alive till the last follow up. (4) Compared with patients from our hospital and Japan, the 5-year overall survival rate (OS) for patients in our hospital was 100.0%, the 3-and 5-year overall survival rates (OS) for patients in Japan were 93.7% and 75.0%, respectively. It is concluded that children with MDS-RCC are seldom progressive. The observation and wait strategy is applicable for patients with MDS-RCC who have no transfusion dependent. IST therapy is recommended to those who have transfusion-dependent.

[Clinicopathologic features and differential diagnosis of splenic B-cell marginal zone lymphoma involving bone marrow].

OBJECTIVE: To study the clinicopathologic features and differential diagnosis of splenic B-cell marginal zone lymphoma (SMZL) involving bone marrow. METHODS: The clinical and pathologic features of 22 patients with SMZL were retrospectively studied. Immunophenotypic analysis was carried out by flow cytometry and immunohistochemistry. Immunoglobulin heavy chain rearrangement study was performed using polymerase chain reaction-based method. RESULTS: Villous lymphocytes were found in peripheral blood smears of 11/18 of the patients. In bone marrow aspirates, lymphocytosis (> 20%) was demonstrated in 15 cases (15/18) and villous lymphocytes in 6 cases (6/18). Flow cytometry showed CD19(+) CD20(+) FMC7(+) CD22(+) CD10(-) CD2(-) CD3(-) CD7(-) in 18 cases. Bone marrow biopsies of all the 22 patients revealed various degrees and patterns of neoplastic infiltration, as follows: mild (4 cases, 18.2%), moderate (11 cases, 50.0%) or severe (7 cases, 31.8%); intrasinusoidal (16 cases, 72.7%), interstitial (14 cases, 63.6%), nodular (11 cases, 50.0%) or diffuse (1 case, 4.5%). Reactive germinal center formation (CD23(+) bcl-2(-)) was found in 2 cases (91.0%). Immunohistochemical study showed the following results: CD20(+) PAX5(+) CD3(-) CD5(-) CD10(-) cyclin D1(-) CD23(-) CD43(-) Annexin A1(-) CD11C(-) CD25(-) in all the 22 cases, CD38(+) in 2 cases (9.1%) and CD138(+) in 2 cases (9.1%). CONCLUSIONS: Different and overlapping patterns of bone marrow involvement are observed in SMZL. As the histologic and immunophenotypic features are not specific to SMZL, distinction from other types of mature B-cell lymphomas is necessary.

[Clinical features of cytopenia with bone marrow hypoplasia in children: an analysis of 100 cases].

OBJECTIVE: To summarize the clinical features of cytopenia with bone marrow hypoplasia in 100 children and to investigate an effective treatment regimen for myelodysplastic syndrome (MDS) in children. METHODS: A retrospective analysis was performed on the clinical data of 100 children non-randomly selected from Japan and China who were diagnosed with cytopenia with bone marrow hypoplasia between 2006 and 2011. The data of patients from China were subjected to prognostic analysis. RESULTS: There was no significant difference in the proportion of MDS cases and acquired aplastic anemia (AA) cases between the Japanese and Chinese children. Of the 100 patients, there were 29 cases of acquired AA, 58 cases of refractory cytopenia of childhood (RCC) and 13 cases of refractory cytopenia with multilineage dysplasia (RCMD). There were significant differences in reticulocyte absolute value in peripheral blood and degree of bone marrow proliferation among the three patient groups (P<0.05). The patients from China were followed up for 16-70 months (median, 41 months). After being treated with cyclosporine (CsA) combined with stanozolol, the patients with AA had response rates of 25% and 75%, the patients with RCC had response rates of 47.1% and 82.4%, and the patients with RCMD had response rates of 60% and 60% respectively at 3 and 6 months after treatment. CONCLUSIONS: There are significant differences in reticulocyte absolute value in peripheral blood and degree of bone marrow proliferation among patients with RCC, RCMD and acquired AA. CsA combined with stanozolol has a good therapeutic efficacy in the treatment of acquired AA and hypoplastic MDS in children, but studies of more cases and a longer follow-up duration are needed.

[Hematopathologic features of T-cell large granular lymphocytic leukemia].

OBJECTIVE: To explore the hematopathologic features of T-cell large granular lymphocytic leukemia (T-LGLL). METHODS: A retrospective analysis of the clinical presentation, bone marrow morphology, immunophenotyping and T-cell receptor gene rearrangement status were performed in 19 patients with T-LGLL. RESULTS: Of 19 patients, the most frequent hematological abnormalities were anemia and neutropenia (16/19 and 17/19 patients, respectively). Large granular lymphocytes (LGLs) were observed in 17 of 19 peripheral blood smears and 15 of 19 bone marrow aspirate specimens. Lymphocytosis (> 0.2) was present in 17 of 19 patients in their bone marrow aspirate specimens. Bone marrow biopsy specimens revealed lymphocytosis in 16 cases, with a mild to moderate increase of lymphocytes observed in 12 cases (12/16). The pattern of lymphoid distribution was interstitial in bone marrow sections. Intravascular distribution was seen in 8 cases. Lymphoid nodules were present in 4 cases. Flow cytometery showed an immunophenotype of CD3(+) CD4(-) CD8(+) CD56(-) CD57(+) of the tumor cells in 13 cases. Of the other 6 cases, the immunophenotypes included CD8(-) (1 case), CD56(+) (2 cases) and CD57(-) (3 cases). Immunohistochemistry showed CD3+ (10/10), CD57+ (3/3), CD8+ (6/7), TIA-1+ (6/7), granzyme B+ (4/7), perforin + (1/7), CD4- (4/4) and CD56- (9/9). Clonal T-cell receptor γ gene rearrangement by PCR was detected in 12 cases (12/17). CONCLUSIONS: Hematopathologic features of most T-LGLL are distinct. Morphologic, immunophenotypic and molecular analysis of both peripheral blood and bone marrow specimens are essential and complementary in the diagnosis and differential diagnosis of T-LGLL.

[Analysis of clinical features and prognosis of primary myelodysplastic syndromes with myelofibrosis patients].

OBJECTIVE: To analyze the clinical features and prognosis of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients and to improve the cognition of MDS-MF. METHODS: Four hundred and sixty-six primary MDS patients with bone marrow (BM) biopsy were divided into two groups according to whether BM associated with fibrosis, the clinical features and prognosis of the two groups were analyzed retrospectively. RESULTS: 167 (35.8%) MDS cases revealed myelofibrosis, of which MF-1 123 cases (26.4%), MF-2 40 cases (8.6%), MF-3 4 cases (0.9%). The proportion of hepatosplenomegaly in MDS-MF group was significantly higher than in MDS without MF group, the difference had statistical significance (P = 0.031). The proliferation of BM biopsy in MDS-MF group was significantly more active than in MDS without MF group. The number of blasts, megakaryocytes and abnormal megakaryocytes in MDS-MF group were significantly higher than in MDS without MF group, the differences had statistical significance (P < 0.05). Among the 345 patients who had available results of cytogenetic analysis, 121 cases were MDS-MF patients, the proportion of middle and high-risk prognostic group according to IPSS karyotype prognosis groups in MDS-MF group were significantly higher than in MDS without MF group, the differences had statistical significance (P = 0.047). The median survival was 17 (1 - 60) months in MDS-MF group, and was 32 (1 - 62) months in MDS without MF group. The difference had statistical significance (P = 0.001). Myelofibrosis had independent prognostic significance by multi-variable analysis (P = 0.019). CONCLUSION: The myelofibrosis in MDS is main the proliferation of reticular fiber. The proliferation of reticular fiber is closely related with the number of blast cells, the proliferation and developmental abnormalities of megakaryocytes and the karyotype. The prognosis of MDS-MF patients is poor.

[Richter syndrome: clinical characteristics and treatment experiences].

OBJECTIVE: To summarize the clinical characteristics of Richter syndrome and explore the methods of successful treatment and timely diagnosis. METHODS: Five patients with Richter syndrome in the last three years (from January 2009 to December 2011) were analyzed retrospectively at our hospital, including their clinical features and therapy before and after transformation. RESULTS: There were 4 males and 1 female with a median age on a diagnosis of chronic lymphocytic leukemia (CLL) at 47 (44 - 68) years. The median duration from a diagnosis of CLL to transformation was 52 (5 - 90) months. As for cytogenetic abnormalities, 3/4 patients had 17p deletion by fluorescence in situ hybridization (FISH). The clinical manifestations on transformation included regional enlargement of lymph node (n = 2) and systemic enlargement of lymph nodes (n = 3). All diagnoses were confirmed by lymph node biopsy and all transformed into diffuse large B cell lymphoma (classical transformation). The subgroups were germinal center B-cell like (GCB) (n = 3) and non-GCB (n = 1). After transformation, one patient underwent sibling allo-stem cell transplantation and survived 24 months until April 2012. Another patient with auto-stem cell transplantation relapsed and died 12 months later. One patient lost the treatment opportunity due to worsening condition. Another 2 patients gained partial remission after therapy and survived 20 and 8 months respectively. CONCLUSIONS: Richter syndrome may occur during a late stage of CLL. Such a high-risk cytogenetic abnormality as del17p may be correlated with transformation. Early identification and optimal therapy may extend the survival of Richter syndrome. Allo-stem cell transplantation remains a curable option.

[The role of bone marrow pathology in diagnosis and differential diagnosis of refractory cytopenia of children].

OBJECTIVE: To explore the diagnosis and differential diagnosis of refractory cytopenia of children (RCC) according to WHO classification, and discuss the relationship between the cytology reviewed by hematologists and histology reviewed by pathologists. METHODS: We selected 50 non-severe aplastic anemia cases from 2007 - 2010 in our hospital and collected clinical data. Experienced hematologists and pathologists evaluated bone marrow biopsy and smear respectively. RESULTS: Of 50 cases, 23 were male and 27 female (M:F = 1:1.17), the median age at diagnosis was 9 years (ranged from 3 to 14 years). 5 patients had disagreement of diagnosis between hematologists and pathologists. In 3 cases hematologists diagnosed as aplastic anemia (AA) and pathologists as RCC, 2 cases vice versa. The final diagnoses of 50 patients reached consensus between hematologists and pathologists were AA 16 cases, RCC 34 cases including 8 refractory cytopenias with multilineage dysplasia (RCMD) cases. All 16 cases AA showed severe hypocellularity. Only 4 cases (25.00%) RCC showed severe hypocellularity, 19 cases (73.08%) RCC showed mild hypocellularity and 3 cases (11.54%) RCC were normal hypocellularity. CONCLUSION: Our results suggests that RCC was not rare in China. The main feature of RCC was dysplasia because of absence of increased blast. RCC was easily confused with AA. The main points of differential were present dysplastic changes of megakaryocyte best appreciated by the hematologists and morphologists and abnormal location of hematopoietic easily observed by pathologists. Overall, cytology and histology were complementary in the investigation of RCC and AA, because of sometimes one might give information that not be given from the other.

Sequential development of diffuse large B-cell lymphoma in a patient with angioimmunoblastic T-cell lymphoma.

Lymphoma of different histologic type can occur in the same patient. Here, we describe a 64-year-old male patient with angioimmunoblastic T-cell lymphoma (AITL) who subsequently developed diffuse large B-cell lymphoma (DLBCL). At the time of initial diagnosis, histologic examination of a left inguinal lymph node of the patient and a monoclonal pattern of TCRß gene rearrangement showed typical features of AITL, and there was no evidence of a monoclonal B-cell population. Twenty-six months later, he had generalized lymphadenopathy and organs involvement by DLBCL. A monoclonal IgH gene rearrangement proved de novo development of secondary B-cell lymphoma and excluded relapse of a primary composite lymphoma. The in situ hybridization analysis showed Epstein-Barr-encoded RNA (EBER) sporadic positivity in sample collected from AITL but extensive positivity in the immunoblasts collected from DLBCL. Our observation supports the hypothesis that Epstein-Barr virus (EBV) is etiologically related to AITL in this case. Clonal expansion of EBV-associated DLBCL is a secondary event in AITL via EBV infection or reactivation.