Hepatitis B virus core protein variations differ in tumor and adjacent nontumor tissues from patients with hepatocellular carcinoma.

OBJECTIVES: To characterize the mutation pattern of a hepatitis B virus (HBV) core protein (HBcAg) derived from hepatocellular carcinoma (HCC) and adjacent nontumor tissues. METHODS: HBV core gene fragments (nt. 1901-2365) were amplified from 98 HBV-related HCC tissues and 33 adjacent nontumor tissues. The deduced amino acids (AAs) of the core gene were aligned with the prototype sequences of HBV genotypes B and C. RESULTS: In total, there were 54 positions that showed polymorphism at the deduced AA level. The mutations were predominantly located in three major (codons 83-87, 95-104 and 130-135) and three minor (codons 21-38, 59-63 and 151-155) mutation-clustering regions (MCRs). The substitution rate in MCRs was significantly higher than in mutation-devoid regions (p < 0.001). The most frequently occurring mutations in rank were codon P130T (38.8%), I97L (37.8%) and S87G (23.5%). In addition, there were 7 patients that showed internal deletions in the middle of HBcAg with sizes ranging from 34 to 59 AAs. Unexpectedly, the core genes isolated from tumor tissues had fewer mutations compared with those isolated from adjacent nontumor tissues from the same patients (p < 0.05). CONCLUSIONS: Accumulation of naturally occurring mutations in certain restricted segments of HBcAg may be related to the development of HCC.

Time point-dependent concordance and prognostic significance of flow cytometry and real time quantitative PCR for measurable/minimal residual disease detection in acute myeloid leukemia with t(8;21)(q22;q22.1).

BACKGROUND: Flow cytometry (FCM) and PCR are reliable methods for assessing minimal residual disease (MRD) in acute myeloid leukemia with t(8;21)(q22;q22.1). The aim of this study was to analyze the concordant rate of these two methods and their prognostic significance. METHODS: PCR and FCM were simultaneously used for MRD analysis at four different time points on 450 BM samples from 124 patients with AML with t(8;21)(q22;q22.1). The four monitoring time points included post-induction (first), after the first consolidation (second) and the second consolidation (third), and at the end of chemotherapy or before Allo/Auto stem cell transplantation (fourth). RESULTS: The concordant rates of the two methods were 33.06%, 25.81%, 49.59%, and 75.31%, respectively, and the main discordant cases were FCM-/PCR+ cases. At all monitoring time points, the MRD level ≥ 10-4 by FCM indicated a poor 3-year Relapse-Free Survival (RFS) (p < 0.001). More than 2-log MRD reduction by PCR after induction and more than 3-log reduction by PCR after the first consolidation remained the significant predictors of better RFS (p < 0.001). After the second consolidation, the negative MRD by PCR (<10-5) was also associated with improved RFS (p = 0.002). A > 1-log increase in PCR can effectively predict recurrence after molecular remission (p < 0.001). In the multivariate analysis, MRD≥0.01% by. FCM and less than 2-log MRD reduction by PCR after induction remained the significant predictors of poor RFS (p < 0.05). CONCLUSIONS: FCM+ always indicates a poor prognosis. Sequential monitoring by PCR is of significance for evaluating prognosis. Our findings suggest a complementary role of two analyses in optimizing risk stratification in clinical practice.

[A new prognostic stratification for patients with acute myeloid leukemia].

OBJECTIVE: To evaluate the impact of the percentage of residual blasts in bone marrow at the end of induction chemotherapy (T1) or during myelosuppression phase (T2) on prognosis of de novo acute myeloid leukemia (AML) (non M(3)) in 105 cases. To refine AML risk-stratification by combining the percentage of residual blast cells (T1 or/and T2) with cytogenetic data based the South West Oncology Group (SWOG) criteria. METHODS: The data of 105 de novo AML (non M(3)) patients hospitalized between January 1st 1999 and February 1st 2008 were retrospectively reviewed. Results were analyzed with SPSS15.0 software. RESULTS: (1) Patients were divided into two subgroups by a cutoff of 5% residual bone marrow blasts at T1 or T2 time point. Patients with percentage of residual bone marrow blast cells < 5% had better complete remission (CR) rate, relapse-free survival (RFS) and overall survival (OS) than the patients with percentage > or = 5% at T1 or T2. The percentage of residual bone marrow blast cells at T1 was correlated with that at T2. (2) The prognosis of patients with intermediate karyotypes with percentage < 5% at T1 or T2 was similar to that of the patients with favorable karyotypes. The patients with intermediate karyotypes and percentage of residual bone marrow blasts > or = 5% at T1 or T2 are defined as a subgroup with prognosis similar to that of patients with unfavorable karyotypes. (3) COX regression analysis showed that the percentage of residual bone marrow blasts at T1 or T2 is an independent prognostic factor of AML. The percentage of residual bone marrow blasts at T1 may be more helpful in prognostication than that at T2. CONCLUSION: AML patients with percentage of residual bone marrow blasts < 5% after induction chemotherapy (T1 or T2) have better CR rate, RFS, OS than the patients with percentage > or = 5% at the same time point. Combination of cytogenetics and percentage of residual bone marrow blasts at T1 or T2 is helpful to divide patients with intermediate karyotypes into two subgroups with different prognosis. Thus, a better decision of treatment strategy can be designed.

The immunophenotypic characteristics and flow cytometric scoring system of acute myeloid leukemia with t(8;21) (q22;q22); RUNX1-RUNX1T1.

INTRODUCTION: The translocation t(8;21) is one of the most frequent chromosome translocations in AML. Molecular (cyto)genetics is regarded as the gold standard for diagnosis. However, due to the complicated variety of AML-related genetic abnormalities, comprehensive screening for all of these abnormalities may not be cost-effective. Therefore, a flow cytometric (FC) scoring system was generated in this study for rapid screening and diagnosis of t(8;21)AML. METHODS: The immunophenotypic characteristics of leukemic cells and neutrophils in cases with t(8;21) AML or other subtypes of AML were analyzed to find a method for the flow diagnosis of t(8;21) AML. RESULTS: In this study, we picked six FC features pointing to the diagnosis of t(8;21) AML: The blasts show high-intensity expression of CD34; aberrant expression of CD19, cCD79a, and CD56 in myeloblasts; co-expression of CD56 in neutrophils, especially in immature neutrophils; and a maturity disturbance in granulocytes. A six-point score was devised using these features. By ROC analysis, the AUC was 0.952, and the sensitivity, specificity, PPV, and NPV were 0.86, 0.90. 0.91, and 0.84 when the score was ≥3 points. The score was then prospectively validated on an independent cohort, and the AUC of the ROC curve for the validation cohort was 0.975. When the cutoff value was set at 3, the obtained sensitivity and specificity values were 0.91 and 0.94, respectively. CONCLUSIONS: The FC score described can be used for the identification and rapid screening of t(8;21) AML.

A modified busulfan and cyclophosphamide preparative regimen for allogeneic transplantation in myeloid malignancies.

BACKGROUND: Busulfan/cyclophosphamide (Bu/Cy) is commonly used as a standard conditioning regimen without total body irradiation for patients with hematological myeloid malignancies undergoing hematopoietic stem cell transplantation (HSCT). OBJECTIVE: To develop a new myeloablative conditioning regimen incorporating fludarabine (Flu) and cytarabine (Ara-c). SETTING: A tertiary blood disease hospital in Tianjin, China. METHODS: A Bu/Cy preparative regimen was used, modified by Flu 90 mg/m(2) and Ara-c 6 g/m(2) in 57 unselected patients (median age 37 years) with hematological myeloid malignancies. The patients were to receive allogeneic hematopoietic stem cell transplantation (allo-HSCT). Thirteen patients had high-risk leukemia, fifty patients had HLA matched sibling donors while seven patients had HLA mismatched sibling donors. Cy was given 50 mg/kg/day for 2 days while Bu was given 3.2 mg/kg/day intravenously for 3 days. MAIN OUTCOME MEASURE: Post-transplant donor chimerism, relapse tendency and minimal residual disease. RESULTS: Extramedullar toxicity was relatively limited; the incidence of treatment-related mortality (TRM) within 100 days was 3.5 %. The incidence of grade II-IV, grade III-IV acute graft versus host disease (GVHD) and chronic GVHD of the evaluable patients were 21.1, 8.8 and 36.4 %, respectively. With a median follow up of 59 (13-96.5) months, TRM and relapse rate (RR) at eight years were 24.1 ± 5.8 and 14.7 ± 4.8 %, respectively. Disease free survival at eight years was 67.9 ± 6.2 % for the entire group, 60.0 ± 8.9 % for patients with AML, 77.3 ± 8.9 % for patients with CML, 70.0 ± 6.5 and 42.9 ± 18.7 % or matched sibling and mismatched sibling HSCT respectively. CONCLUSION: The new regimen was associated with a low relapse rate, low incidence and severity of graft versus host disease and satisfactory survival for patients with myeloid malignancies.

[Expression of cMPO in 502 cases of acute myeloid leukemia (AML) and its diagnosis significance in AML subtypes].

OBJECTIVE: To investigate the myeloperoxidase (cMPO) expression pattern by flow cytometry (FCM) in patients with acute myeloid leukemia (AML) and its role in classifying AML. METHODS: Eight- color multiparametric FCM with CD45/SSC gating was used to determine the cMPO expression in 502 AML patients. RESULTS: The positive rate of cMPO in all patients was 58.0%, in which the proportion of normal positivity, dim positivity and partial positivity was 21.5%, 34.1% and 2.4%, respectively. The remaining case (42.0%) were all negative. In AML with t (15;17）(q22;q12)/PMLRARα, the positive rate was the highest (100%) and the intensity was similar to that of the normal granular leukocytes, followed by AML with t (8;21（q22;q22/RUNX1-RUNX1T1, the positive rate was 91.4% and the intensity was mostly dim. AML with minimal differentiation and acute megakaryoblastic leukemia were all cMPO negative. The positive rates of cMPO in the remaining subtypes were between 22.7% and 76.2%. CONCLUSION: The positive rate and intensity of cMPO were significantly different among different subtypes of AML.

Leptin signaling enhances cell invasion and promotes the metastasis of human pancreatic cancer via increasing MMP-13 production.

Emerging evidence has suggested that leptin, an adipokine related to energy homeostasis, plays a role in cancer growth and metastasis. However, its impact on pancreatic cancer is rarely studied. In this study, we found that leptin's functional receptor Ob-Rb was expressed in pancreatic cancer cell lines. Treatment with leptin enhanced the migration and invasion of pancreatic cancer cells but did not affect the proliferation of human pancreatic cancer cells. Leptin up-regulated the expression of matrix metalloproteinase-13 (MMP-13) via the JAK2/STAT3 signaling pathway. The overexpression of leptin was shown to significantly promote tumor growth and lymph node metastasis in a subcutaneous model and an orthotopic model of human pancreatic cancer, respectively. Furthermore, in human pancreatic cancer tissues, the expression of Ob-Rb was positively correlated with the MMP-13 level. The increased expression of either Ob-Rb or MMP-13 was significantly associated with lymph node metastasis and tended to be associated with the TNM stage in patients with pancreatic cancer. Our findings suggest that leptin enhances the invasion of pancreatic cancer through the increase in MMP-13 production, and targeting the leptin/MMP-13 axis could be an attractive therapeutic strategy for pancreatic cancer.

Enriched environment inhibits mouse pancreatic cancer growth and down-regulates the expression of mitochondria-related genes in cancer cells.

Psycho-social stress has been suggested to influence the development of cancer, but it remains poorly defined with regard to pancreatic cancer, a lethal malignancy with few effective treatment modalities. In this study, we sought to investigate the impacts of enriched environment (EE) housing, a rodent model of "eustress", on the growth of mouse pancreatic cancer, and to explore the potential underlying mechanisms through gene expression profiling. The EE mice showed significantly reduced tumor weights in both subcutaneous (53%) and orthotopic (41%) models, while each single component of EE (inanimate stimulation, social stimulation or physical exercise) was not profound enough to achieve comparative anti-tumor effects as EE. The integrative transcriptomic and proteomic analysis revealed that in response to EE, a total of 129 genes in the tumors showed differential expression at both the mRNA and protein levels. The differentially expressed genes were mostly localized to the mitochondria and enriched in the citrate cycle and oxidative phosphorylation pathways. Interestingly, nearly all of the mitochondria-related genes were down-regulated by EE. Our data have provided experimental evidence in favor of the application of positive stress or of benign environmental stimulation in pancreatic cancer therapy.

[IL-18 single nucleotide polymorphisms in hematologic malignancies with HLA matched sibling donor allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To explore the impact of interleukin-18 (IL-18) single nucleotide polymorphisms on outcomes of hematologic malignancies with HLA-matched sibling donor hematopoietic stem cell transplantation (allo-HSCT). METHODS: Single- nucleotide polymorphisms in IL-18 promoter was detected by PCR-sequence-specific primer analysis (PCR-SSP) in 93 recipients and their HLA matched sibling donors. Hematopoietic reconstitution, incidences of graft versus host disease (GVHD) and infections, transplant related mortality (TRM), and disease free survival (DFS) were analyzed. RESULTS: In comparison with -137 G/C+C/C donor genotype, patients with -137 G/G donor genotype had shorter duration of neutrophil recovery [15(11-23) days vs 17(11-24) days, P=0.01], higher incidence of extensive chronic GVHD (20.6% vs 3.3%, P=0.029), but no difference in the interval of platelet recovery [20(11-46) days vs 20(7-38) days, P=0.844]. The incidence of extensive chronic GVHD in -607 C/C donor genotype (31.6%) was significantly higher than that (10.8%) in C/A + A/A donor genotype (P=0.024). Recipients with -607 C/C genotype also had higher incidence (33.3%) of extensive chronic GVHD than those with C/A+A/A genotype (10.7%, P=0.016). There were no differences in acute GVHD, TRM, and DFS between different genotypes. CONCLUSION: IL-18 -137 G homozygous genotype in donor facilitated neutrophil reconstitution, but increased the risk of extensive chronic GVHD in patients with allo-HSCT.

[Treatment outcome of childhood standard-risk and median-risk acute lymphoblastic leukemia with CCLG-2008 protocol].

OBJECTIVE: To estimate the significance of the adjustment of acute lymphoblastic leukemia (ALL) risk group by monitoring minimal residual disease(MRD). METHOD: Totally 285 children ALL patients who were diagnosed and systematically treated according to CCLG-2008 in Institute of Hematology and Blood Diseases Hospital, CAMS and PUMC, from April 2008 to August 2011 were prospectively selected. Among these cases, 62.8% (n = 179) were boys and 37.2% (n = 106) were girls and the median age was 5.3(0.5-14.0). The patients who were at high-risk group initially were excluded. The grouping of cases: the patients were divided into two groups according to the dates of initial diagnosis. Group I had 126 patients who were initially diagnosed between April 2008 and December 2009 in whom therapeutic regimen was not adjusted by reassignment of risk group by MRD. Group II had 159 patients who were initially diagnosed between January 2010 and August 2011 whose therapeutic regimen was adjusted by reassignment of risk group by MRD at specific time (33rd day of induction chemotherapy and 12 weeks after the beginning of chemotherapy). MP-FCM Coulter FC-500 was used in the detection of MRD. RESULT: Among these 285 patients, 94.0% (n = 268) were diagnosed as B-lineage acute lymphoblastic leukemia and 6.0% (n = 17) were T-lineage acute lymphoblastic leukemia. In group I, 61.9% (n = 78) patients belonged to low-risk group, 38.1% (n = 48) median-risk; in group II, before the adjustment, the rates of the low-risk group and median-risk group were 68.6% (n = 109) and 31.4% (n = 50) , respectively, while after the adjustment they were altered to 53.5% (n = 85) and 39.6% (n = 63) , furthermore 6.9% (n = 11) patients went into the high-risk group. Both groups were followed up for 2.5 years after their diagnoses, the disease of 7.4% (n = 21) patients relapsed, and the rates of two groups were 12.7% (n = 16) and 3.1% (n = 5) respectively, P = 0.009. The rate of serious infection (such as sepsis, pulmonary infection) of all these patients was 32.3% (92/285) , there was no significant difference between the two groups [28.6% (36/126) vs.35.2% (56/159) , P = 0.392]. The mortality of all these patients was 6.7% (19/285) , and that of group I was higher than that of group II [10.3% (13/126) vs. 3.8% (6/159) , P = 0.044]. The 2.5 years overall survival (OS), event-free survival (EFS) and disease-free survival (DFS) of group I were all lower than those of group II in Kaplan-Meier survivorship analysis (all P < 0.05). The two groups were followed up for 2.5 years after their diagnoses, after elimination of the confounding influence of sex, age, FAB subtype, WBC count, ratio of blast cells in bone marrow at diagnosed, chromosome karyotype and fusion gene, reassignment of risk group by MRD was used to calculate the OS, EFS and DFS of ALL patients (all P < 0.05). After the adjustment the risk group was more significant in the assessment of prognosis. CONCLUSION: The reassignment of risk group in low and median risk groups children with acute lymphoblastic leukemia by MRD did not increase the rate of serious infection but could reduce the relapse rate and mortality, and was beneficial to increase the patients' OS, EFS and DFS.

[Immunosuppressive therapy using antithymocyte globulin and cyclosporin A with or without human granulocyte colony-stimulating factor in children with acquired severe aplastic anemia].

OBJECTIVE: To compare the efficacy and safety of four different regimens for pediatric severe aplastic anemia (SAA) with immuno-suppressive therapy (IST) with or without combined human granulocyte colony-stimulating factor (G-CSF). METHOD: The authors retrospectively analyzed 105 children with SAA treated with IST with or without G-CSF in the hospital from February 2000 to September 2010. Regimen A, without G-CSF in the whole treatment, was used to treat Group A patients, n = 27; Regimen B, G-CSF, was initiated in Group B, n = 24, before the IST until hematologic recovery; Regimen C, G-CSF, was used together with the IST for Group C patients, n = 24, until hematologic recovery; Regimen D,G-CSF was used for Group D, n = 30, after the end of IST until hematologic recovery. The response rate, relapse rate, mortality, infection rate, infection-related death rate, risk of evolving into MDS/AML, survival rate, factors affecting the time of event-free survival and so on. RESULT: (1) The response (CR+PR) rates 4, 6, 12 and 24 months after IST of the whole series of 105 SAA children were 50.5% (7.6%+42.9%) , 60.0% (21.9%+38.1%) , 67.6% (38.1%+29.5%) and 69.5% (40.0%+29.5%) respectively. The 2-year survival rate was 90.5%; the follow-up of the patients for 13 years showed that the whole survival rate was 87.6%. (2) The differences of the response rates 4, 6, 12 and 24 months after IST of the 4 groups were not significant (P > 0.05). (3) No significant differences were found in the mortalities 4, 6, 12 and 24 months among the 4 groups (P > 0.05). (4) Of the 105 patients, 4 children had relapsed disease in the period of time from 6 to 24 months after IST. All the four patients belonged to the groups with G-CSF. (5) The use of G-CSF could not decrease the infection period before IST (day) (P = 0.273), and it had no impact on the infection rate after IST (P = 0.066). It did not reduce the rates of septicemia and infectious shock. And to the infection-related death rate no significant conclusion can be made. (6) Follow up of the patients for 13 years, showed that 2 had the evolution to MDS/AML in the 105 patients and the two children belonged to the groups with G-CSF. (7) Kaplan-meier curve analysis did not show any differences in the survival rates of the four groups. (8) Cox regression analysis showed that the use of G-CSF had no benefit to the patients' long term survival. While the age of diagnosis and the infection history before IST were significantly related to the patients' long term survival. CONCLUSION: The use of G-CSF did not contribute to the early response and could not reduce the infection rate, infection-related death rate and the patients' long term survival. There were no significant differences in the survival rates of the four groups. Attention should be paid to the risk of the evolution to MDS/AML.

[Correlation of single-cell gel electrophoresis and mitomycin C-induced chromosomal breakage for chromosomal instabiligy in children with Fanconi anemia].

OBJECTIVE: Fanconi anemia (FA) is characterized by bone marrow failure, congenital abnormalities and predisposition to neoplasia. Hypersensitivity of FA cells to the clastogenic effect of mitomycin C (MMC) provides a unique marker for the diagnosis before the beginning of hematological manifestations. The aim of this study was to evaluate the relationship between Single-Cell Gel Electrophoresis (SCGE) and mitomycin C-induced chromosomal breakage in children with FA. METHOD: Between January 2007 and June 2011, 248 children (< 15 years) with hypocytosis were included. Chromosomal breakage was induced by MMC 0 ng/ml, 40 ng/ml, and 80 ng/ml. SCGE was performed at the same time. We analyzed the results of the two methods and compared with each other. The receiver operating characteristic (ROC) curve was used to evaluate the parameters in SCGE. RESULT: Seventeen patients were diagnosed as FA and 231 as non-FA. Chromosomal breakage was found to be significantly higher in FA patients [(32.2 ± 4.8)%] than non-FA [(19.9 ± 3.0)%] and controls[(21.6 ± 4.8)%] when induced by MMC 80 ng/ml. The parameters of SCGE were significantly different between FA patients and non-FA or controls. All the parameters were rectilinearly correlated with MMC (P = 0.000). The most closely correlated parameter was the rate of comet cell (r = 0.848, P = 0.000). The results of ROC curves suggested the comet cell rate (0.999) was more important. CONCLUSION: SCGE might be used to discriminate between FA and non-FA individuals. The relationship between SCGE and MMC-induced chromosomal breakage was significant. The rate of comet cell was the important parameter.

[Role of IFN-γ + 874 genetic polymorphisms in allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To explore the impact of IFN-γ + 874 polymorphisms on the outcome in HLA matched sibling HSCT. METHODS: We used PCR-sequence-specific primer analysis (PCR-SSP) to analyze the polymorphisms of IFN-γ + 874 T/A in 80 recipient and donor pairs from October 2005 to March 2008. RESULTS: Recipients having donors who possessed IFN-γ + 874 A/A genotype had significantly earlier neutrophil recovery compared with those having donors with non-A/A genotype (15 (11 - 27) days vs 18 (12 - 30) days, P = 0.029). And IFN-γ + 874 A/A in both recipients and donors further facilitated neutrophil recovery compared with others (13 (11 - 25) days and 19 (12 - 31) days, P = 0.019). Besides, IFN-γ + 874 A/A in recipients increased the probability of grade II-IV acute graft versus disease (aGVHD) and cytomegalovirus viraemia compared with IFN-γ + 874 T/A or T/T genotype (20% vs 4% P = 0.041, 43.6% vs 16.0% P = 0.032), which lead to increased 5-year transplant-related mortality (TRM) (33.7% ± 6.8% vs 12.0% ± 6.5%, P = 0.050) and decreased 5-year event free survival (EFS) \[(58.2 ± 6.7)% vs (84.0 ± 7.3)%, P = 0.032\] compared with the latter. IFN-γ + 874 A/A in both recipients and donors also significantly increased the probability of grade II-IV aGVHD and cytomegalovirus viraemia compared with the other (21.7% vs 5.9%, P = 0.050; 45.7% vs 20.6%, P = 0.020), which caused increased 5-year TRM \[(31.6 ± 7.5)% vs (13.6 ± 6.5)%, P = 0.048\] and decreased 5-year EFS \[(56.8 ± 7.3)% vs (79.4 ± 6.9)%, P = 0.037\] compared with the other. CONCLUSION: In HLA-matched sibling HSCT setting, the presence of IFN-γ + 874 T allele in recipients or in both recipients and donors significantly decreased the risk of grade II-IV aGVHD and CMV infection and increased EFS. While IFN-γ + 874 A/A in donors or in both recipients and donors was associated with shorter duration to neutrophil recovery.