Diagnosis, treatment and genetic analysis of an elderly patient with Gaucher's disease characterized by marked multiple bone destruction.

Gaucher's disease is a rare autosomal recessive genetic disease. Due to the decrease or lack of glucocerebrosidase (GBA) activity in lysosome caused by the mutation of GBA gene, its substrate glucocerebroside is detained in lysosome, resulting in clinical manifestations of liver, spleen, kidney, bone, hematopoietic system and even nervous system involvement. Here, we report a case of elderly patient presenting marked multiple bone destruction, with childhood medical history of splenectomy and "osteomyelitis". The patient has a significantly enlarged liver, accompanied by anemia, thrombocytopenia and osteopenia. Laboratory studies show this patient has low blood GBA activity and high glucosyl sphingosine level and increased chitotriosidase activity. Genetic testing revealed a homozygous missense variant NM_001005741.2 c.770A>G (p.Asp257Gly) in the patient's GBA gene. After 6 months of enzyme replacement therapy, the patient's platelets returned to normal, anemia improved, and liver volume decreased. Further detections show that the mother and brothers of the patient have heterozygous mutations at this locus, which is consistent with Mendelian inheritance law. Although this variant has not been reported in literatures or database, both clinical manifestations, characteristics of enzymology and biomarkers, and the effect of enzyme replacement therapy support the diagnosis of Gaucher's disease. The Asp257Gly variant is therefore assessed as a clinical pathogenic variant. This study expands the spectrum of the GBA gene variants. The diagnosis and treatment process of this case also provide reference for the early identification, diagnosis and early treatment of this kind of patients.

Albumin-to-Fibrinogen Ratio as an Independent Prognostic Parameter in Untreated Chronic Lymphocytic Leukemia: A Retrospective Study of 191 Cases.

PURPOSE: Chronic lymphocytic leukemia (CLL) is one of the most frequent type of B-cell chronic lymphoproliferative disorders and chronic inflammation takes part in the development of CLL. However, there has been no valid immune biomarker to predict the prognosis of untreated CLL patients. MATERIALS AND METHODS: In this retrospective study, we analyzed the clinical correlations and prognostic value of albumin-to-fibrinogen ratio (AFR) detected at diagnosis in 191 CLL patients. RESULTS: The cut-off value of AFR was 9.7 calculated by X-tile. Patients who were more than 65 years old were often accompanied by low level of AFR (p < 0.001). Survival analysis showed that patients with low level of AFR had shorter overall survival (OS) than patients with high level of AFR (p < 0.001). Multivariate analysis illustrated that AFR had a negative impact on OS (p=0.003) and was independent of parameters involved in CLL international prognostic index and other prognostic markers such as CD38 and ZAP-70. CONCLUSION: These data provide a comprehensive view of AFR and shows that AFR at diagnosis is an adverse prognostic factor in untreated CLL patients.

Hyperfibrinogenemia is a poor prognostic factor in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphomas worldwide. Previous studies indicated that hyperfibrinogenemia was a poor predictor in various tumors. The purpose of our study was to evaluate the prognostic effect of hyperfibrinogenemia in DLBCL. Data of 228 patients, who were diagnosed with DLBCL in our hospital between May 2009 and February 2016, were analyzed retrospectively. The Kaplan-Meier method and Cox regression were performed to find prognostic factors associated with progression-free survival (PFS) and overall survival (OS). Receiver operator characteristic (ROC) curve and the areas under the curve were used to evaluate the predictive accuracy of predictors. Comparison of characters between groups indicated that patients with high National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) score (4-8) and advanced stage (III-IV) were more likely to suffer from hyperfibrinogenemia. The Kaplan-Meier method revealed that patients with hyperfibrinogenemia showed inferior PFS (P < 0.001) and OS (P < 0.001) than those without hyperfibrinogenemia. Multivariate analysis showed that hyperfibrinogenemia was an independent prognostic factor associated with poor outcomes (HR = 1.90, 95% CI: 1.15-3.16 for PFS, P = 0.013; HR = 2.65, 95% CI: 1.46-4.79 for OS, P = 0.001). We combined hyperfibrinogenemia and NCCN-IPI to build a new prognostic index (NPI). The NPI was demonstrated to have a superior predictive effect on prognosis (P = 0.0194 for PFS, P = 0.0034 for OS). Hyperfibrinogenemia was demonstrated to be able to predict poor outcome in DLBCL, especially for patients with advanced stage and high NCCN-IPI score. Adding hyperfibrinogenemia to NCCN-IPI could significantly improve the predictive effect of NCCN-IPI.

Clinical features of multiple myeloma patients with isolated extramedullary relapse.

This study sought to analyze the clinical features and prognosis of multiple myeloma with isolated extramedullary relapse and with the absence of systemic progression. The clinical features and outcome were retrospectively analyzed in six multiple myeloma patients. These patients had secretory multiple myeloma at diagnosis. When relapsed, the dissociation between medullary and extramedullary response was detected. The serum or urine monoclonal component was extremely low or absent. The plasma cells in bone marrow were <5%. All patients received new targeted therapies (thalidomide or bortezomib) before extramedullary relapse. It is difficult to achieve second remission for them. Even in those showing response, the duration of response was extremely short. The median of overall survival from diagnosis and from extramedullary relapse was 19 months and 6 months, respectively. The overall survival was significantly shorter compared to the patients without extramedullary involvement (84 months, P=0.001). These patients exhibited a special and rare relapse pattern. Patients with this relapse pattern were resistant to current therapies, including novel targeted agents and associated with poor prognosis.

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome.

Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

Assessment of clonality in T-cell large granular lymphocytic leukemia: flow cytometric T cell receptor Vß repertoire and T cell receptor gene rearrangement.

The usefulness of flow cytometric variable ß-chain repertoire (FC-Vß) and T-cell receptor gene rearrangement (TCR-GR) analyses for differentiating T-cell large granular lymphocytic leukemia (T-LGLL) from reactive T-large granular lymphocyte (T-LGL) lymphocytosis has been insufficiently studied to date. In this study, we analyzed the diagnostic value of TCR-GR and FC-Vß analysis in T-LGLL, and compared these results. In our study, FC-Vß analysis was positive in all cases of T-LGLL, and clonality assessment of FC-Vß had equal sensitivity and specificity to GeneScanning analysis but was more sensitive than heteroduplex analysis. Suspected T-cell clonality can best be addressed by evaluating two TCR targets (TCRß and TCRγ), either in parallel or consecutively. Signal transducer and activator of transcription 3 (STAT3) mutation may provide a diagnostic tool for classifying some cases of T-LGL lymphocytosis as true T-LGLL. Our results further demonstrate a significant correlation of STAT3 mutation with pure red cell aplasia, neutropenia, hepatomegaly, ß2-microglobulin and anemia.

Haploidentical hematopoietic stem cell transplantation following myeloablative conditioning regimens in hematologic diseases with G-CSF-mobilized peripheral blood stem cells grafts without T cell depletion: a single center report of 38 cases.

Many Chinese patients with hematologic diseases, who need allogeneic hematopoietic stem cell transplantation (HSCT), lack a human leukocyte antigen-matched donor. To save these patients and to avoid collecting donor bone marrow graft, we adopted haploidentical peripheral blood HSCT with granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood stem cells as the grafts without ex vivo T cell depletion. Thirty-eight patients were enrolled, and they received myeloablative preconditioning. Thirty-five patients attained a successful neutrophil and platelet recovery. The median time for the neutrophil recovery was 16 days (range of 10-23 days), and the median time for the platelet recovery was 19 days (range of 10-66 days). During the follow-up at a median time of 33.1 weeks (range of 1.1-412.6 weeks), eleven (28.9 %) patients developed aGVHD grade I-II and seven (18.4 %) patients developed aGVHD grade III-IV. The incidence of cGVHD was 27.6 %, and nine (23.7 %) patients died within the first 100 days after transplantation. The cumulative survival proportions at 1 and 2 years were 52.51 ± 8.57 % and 43.76 ± 9.11 %, respectively. These results suggested that the G-CSF-primed peripheral blood stem cell grafts, without in vitro T cell depletion, could be an appropriate stem cell source for Haplo-HSCT.

Angiogenic factors in chronic lymphocytic leukemia.

Angiogenesis is a complex process controlled by the balance of a large number of regulating factors, the pro- and anti-angiogenic factors. Dysregulation of angiogenesis occurs in various pathologies and is one of the hallmarks for cancer. Recent emphasis on the microenvironment's influence in chronic lymphocytic leukemia (CLL) progression and drug resistance nurtures the interest in angiogenesis. Researchers have already identified a variety of angiogenic factors involved in the CLL, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), angiopoietin-2(Ang-2), thrombospondin-1 (TSP-1), as well as extracellular proteinases such as matrix metalloproteinase-9 (MMP-9). Besides modulating neovascularization, angiogenic factors also participate in the regulation of pro-survival effects of CLL cells. However, the precise mechanism involved still needs to be elucidated further. At present, the levels of some angiogenic factors are regarded as prognostic markers of the progression of CLL, although it is not widely used. Several anti-VEGF agents are currently under clinical trial. Advances in the understanding of the bases of angiogenesis regulators will be benefit for the comprehension of CLL pathogenesis and help to conquer the disease.

Downregulated Dicer expression predicts poor prognosis in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Alterations in microRNAs (miRNAs) expression have been proposed to play a role in CLL pathogenesis. Dicer and Drosha are the main regulators of miRNA biogenesis, and deregulation of their expression has been indicated as a possible cause of miRNA alterations observed in various cancers. To investigate the role of Dicer and Drosha in CLL, we assessed the expression of Dicer and Drosha and their correlation with other prognostic factors, including Binet stages, immunoglobulin heavy chain variable gene (IGHV) mutation status, TP53 mutation status, ZAP-70 protein and CD38 expression level in 165 CLL patients by using real-time polymerase chain reaction methods. Patients with unmutated IGHV genes had significantly lower expression of Dicer than patients with IGHV mutations. The lower expression level of Dicer was also significantly associated with higher level of CD38 and ZAP-70, and more aggressive Binet stage. We also analyzed Dicer expression in different cytogenetic subgroups. Lower Dicer level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22.3) in contrast to higher level in good risk cytogenetics (deletion in 13q14 as the sole abnormality). Furthermore, the lower expression of Dicer in CLL shows a strong association with shorter overall survival (OS) (P = 0.0046) as well as with reduced treatment free survival (TFS) (P = 0.0006). By contrast, no differences in the expression of Drosha among these groups of patients were observed. Our data suggest that Dicer expression may play an important role in the progression and prognosis of CLL.

[Effects of human bone marrow mesenchymal stem cells on proliferation and apoptosis of K562 cells].

This study was aimed to compare K562 cell proliferation, cell cycle and apoptosis before and after adhesive culture with MSCs of the same and different counts, so as to investigate the effect of human bone marrow mesenchymal stem cells (MSCs) on the growth of K562 cells. Culture system of bone marrow MSCs and co-culture system of K562 cells and BMSCs in vitro were established. And K562 cell proliferation curves were drawn by co-cultured of K562 cells with different counts of BMSCs. Cell cycle and apoptosis were determined by flow cytometry. The results showed that compared with the control, the proliferation of K562 cells cultured with the same amounts of MSCs was inhibited. After co-culture with the different amounts of MSCs, MSCs exhibited a dose-dependent anti-proliferation effect on K562 cells. The percentages of K562 cells in G(1) phase and G(2) phase were higher than those of the control. It is concluded that the normal bone marrow mesenchymal stem cells can inhibit the proliferation, the progress of cell cycle and the rate of apoptosis of K562 cells. As the number of mesenchymal stem cells increased, their anti-proliferation effect on the K562 cells were enhanced in a certain range.

[Standard-dose of idarubicin in combination with continuous infusion of cytarabine as induction therapy in patients with acute myeloid leukaemia].

The objective of this study was to investigate the efficacy and toxicity of standard-dose idarubicin in combination with continuous infusion of cytarabine as induction therapy in patients with acute myeloid leukemia (AML). A total of 38 AML patients were enrolled, including 30 new diagnosed patients, 8 relapsed and refractory patients. Cytogenetic analysis was performed in all patients, 15 patients had cytogenetic aberrations including 4 complex abnormalities. All patients were treated with standard-dose idarubicin [12 mg/(m(2).d), days 1 to 3] and continuous infusion of cytarabine [100 mg/(m(2).d), days 1 to 7]. The results showed that after one course of induction therapy, the overall response rate was 89.5% (34/38), and 32 out of 38 (84.2%) patients achieved complete remission (CR), including 27 of 30 (90.0%) new diagnosed AML patients, 5 (62.5%) refractory and relapsed AML patients, all 4 patients with complex cytogenetic aberrations achieved cytogenetic CR. Out of 6 relapsed patients 2 showed as extramedullary relapse, 4 showed as bone marrow relapse. The median survival duration was > 22 months and median disease-free survival time was > 16 months. Myelosuppression and infections due to neutropenia were the most frequent adverse effects, severe nonhematologic toxicity and the early death were not observed in the patients. It is concluded that standard-dose of idarubicin combined with continuous infusions of cytarabine as the induction therapy is highly effective and well tolerated approach in patients with AML, this regimen provides an opportune moment for hematopoietic stem cell transplantation.

[Allogeneic peripheral blood stem cell transplantation for 75 cases of hematologic malignancies].

The aim of this study was to explore the clinical effect and complications of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) in hematologic malignancies through retrospective analysis of 75 patients (42 male, 33 female; aged from 13 to 72 years old) received allo-PBSCT from HLA matched (n=61) or haploidentical donors (n=14). 75 patients included 35 patients with chronic myeloid leukemia (CML), 30 patients with acute myeloid leukemia, 5 patients with severe aplastic anemia, 3 patients with acute lymphocytic leukemia, one patients with multiple myeloma and one patients with paroxysmal nocturnal hemoglobinuria. Conditioning regimens were (1) Cy/TBI or Bu/Cy; (2) Cy/TBI+Ara-C; (3) fludarabine+TBI/or (CTX+ATG). Minimal residual disease has been monitored regularly by PCR and FISH. Patients received cyclosporine A and methotrexate or ATG and anti-CD25 monoclonal antibody and mycophenolate mofetil for graft-versus-host disease (GVHD) prophylaxis. Relapsing patients after transplantation received DLI and/or chemotherapy. Patient with CML were treated with imatinib. The results showed that 74 patients had hematopoietic reconstitution, and eventually converted to full donor chimerism by FISH or PCR-STR. The median time for the initial hematopoietic reconstitution was 15 (5-25) days. 46 out of 75 patients were alive and median duration was 23 (2-61) months. Among 29 dead patients, 9 died of disease relapse, 7 died of III-IV grade of acute GVHD and 7 died of severe infection (2 patients developed interstitial pneumonia). 9 out of 14 patients received haploidentical transplantation were alive, and the time of event-free survival was 30 (6-53) months, the mean survival time of 5 died patients was 7 (2-17) months. 16 patients were infected by cytomegalovirus, 2 of them died of interstitial pneumonia. None of them suffered from veno-occlusive disease in the liver. It is concluded that allo-PBSCT is effective to treat refractory hematologic diseases, and DLI/or chemotherapy should be used in the patients relapsing after transplantation.

[Analysis of complex chromosomal aberrations in patients with myelodysplastic syndromes using multiplex fluorescence in situ hybridization combined with whole chromosome painting].

OBJECTIVE: To explore the value of multiplex fluorescence in situ hybridization (M-FISH) in combination with whole chromosome painting (WCP) in the detection of complex chromosomal aberrations (CCAs) in myelodysplastic syndromes (MDS). METHODS: M-FISH was used in seven MDS patients with R-banding CCAs to refine the complex chromosomal rearrangements, and to identify cryptic translocations and characterization of marker chromosomes. Dual-color WCP procedures were further performed in 7 cases to confirm some rearrangements detected by M-FISH. RESULTS: M-FISH confirmed all results of R-banding. The composition and origin of 6 kinds of marker chromosomes, 9 kinds of chromosomes with additional material undetermined and 5 kinds of derivative chromosomes undefined by conventional cytogenetics (CC) were defined after M-FISH analysis; four kinds of cryptic translocations overlooked by CC were found on derivative chromosomes and previously normal appearing chromosomes. In addition, M-FISH revealed some nonrandom aberrations: aberrations involving chromosome 17 and -5/5q- were the two most frequent aberrations. Some misclassified and missed chromosomal aberrations by M-FISH were corrected by WCP. CONCLUSION: M-FISH is a powerful molecular cytogenetic tool in clarification of CCAs. Complementary WCP helps us to identify misclassified and missed chromosomal aberrations by M-FISH. CC in combination with molecular cytogenetic techniques, such as M-FISH and WCP, can unravel complex chromosomal aberrations more precisely.

[Expression of P27(Kip1) and its clinical significance in acute and chronic leukemia].

To evaluate the expression of cyclin dependent kinase inhibitor P27(Kip1) in leukemia and to investigate its clinical significance, the P27(Kip1) protein in bone marrow or peripheral blood samples from 82 cases of leukemia was measured by Western blot and enhanced chemoluminescence (ECL). The results showed that the expression of P27(Kip1) protein in ALL was higher than that in ANLL (P = 0.033) and also that in CML (P = 0.008). P27(Kip1) expression in CLL was higher than that in CML too (P = 0.017). In acute leukemia, the effective rate (CR and PR) of initial chemical therapy in the group of P27(Kip1) > 0.655 was higher than that in the group of P27(Kip1) < or = 0.655, P = 0.041. For ANLL and ALL patients, the survival time in the group of P27(Kip1) > 0.655 was longer than that in the group of P27(Kip1) < or = 0.655, P = 0.0065. There were similar statistical significance for ANLL and ALL patients, P = 0.0271 and P = 0.0266 respectively. There was a negative correlation between chromosomal abnormalities and P27(Kip1) expression in ALL patients (r = -0.775, P = 0.04). The expression of P27(Kip1) protein appeared nothing to do with sex, age, white blood cell number, blast cell number in peripheral blood, serum LDH or uric acid. In conclusion, the expression level of P27(Kip1) protein is in relation to the effect of initial chemical therapy and survival time, so that the lower P27(Kip1) expression may associated with poor prognosis in acute leukemia.