Phase II study of novel orally PI3Kα/δ inhibitor TQ-B3525 in relapsed and/or refractory follicular lymphoma.

This registration study assessed clinical outcomes of TQ-B3525, the dual phosphatidylinositol-3-kinase (PI3K) α/δ inhibitor, in relapsed and/or refractory follicular lymphoma (R/R FL). This phase II study (ClinicalTrials.gov NCT04324879. Registered March 27, 2020) comprised run-in stage and stage 2. R/R FL patients after ≥2 lines therapies received oral 20 mg TQ-B3525 once daily in a 28-day cycle until intolerable toxicity or disease progression. Primary endpoint was independent review committee (IRC)-assessed objective response rate (ORR). Based on results (ORR, 88.0%; duration of response [DOR], 11.8 months; progression-free survival [PFS], 12.0 months) in 25 patients at run-in stage, second stage study was initiated and included 82 patients for efficacy/safety analysis. Patients received prior-line (median, 3) therapies, with 56.1% refractory to previous last therapies; 73.2% experienced POD24 at baseline. At stage 2, ORR was 86.6% (71/82; 95% CI, 77.3-93.1%), with 28 (34.2%) complete responses. Disease control rate was 95.1% due to 7 (8.5%) stable diseases. Median time to response was 1.8 months. Among 71 responders, median DOR was not reached; 18-month DOR rate was 51.6%. with median follow-up of 13.3 months, median PFS was 18.5 (95% CI, 10.2-not estimable) months. Median overall survival (OS) was not reached by cutoff date; 24-month OS rate was estimated as 86.1%. Response rates and survival data were consistent across all subgroups. Grade 3 or higher treatment-related adverse events were observed in 63 (76.8%) cases, with neutropenia (22.0%), hyperglycemia (19.5%), and diarrhea (13.4%) being common. TQ-B3525 showed favorable efficacy and safety for R/R FL patients after ≥2 lines prior therapies.

[Preliminary Study on the Relationship between the Developmental Stage of Multiple Myeloma Disease and the Results of Bone Marrow Whole Exome Sequencing].

OBJECTIVE: To investigate the genetic results of whole exome sequencing of bone marrow from new onset multiple myeloma (MM) patients to analyze the process of genetic clonal evolution in MM patients. METHODS: Genomic DNA was extracted from bone marrow samples of 15 MM patients and the whole exomes sequencing was performed using next generation sequencing technology. Using own buccal cells as germline controls, combinated with clinical information, the mutation profile of genes from high-risk asymptomatic myeloma to symptomatic myeloma were analyzed, and genes that may be associated with the efficacy and side effects of bortezomib were screened. RESULTS: Except for two patients in whom no peripheral neuropathy was observed after a short treatment period, other patients peripheral neuropathy developed of various degrees during treatment with bortezomib containing chemotherapy, and the vast majority of patients achieved remission after receiving this bortezomib-related chemotherapy regimen. All patients had comparable levels of the inherited mutations number, but the somatic mutations was correlated with disease evolution. CONCLUSION: different gene "mutational spectra" exist in myeloma patients at different stages and are associated with progression through all stages of the disease.

Immunohistochemical evaluation and prognostic value of monocarboxylate transporter 1 (MCT1) and 4 (MCT4) in T-cell non-Hodgkin lymphoma.

Tumor cells often exhibit the Warburg effect, wherein, they preferentially undergo glycolysis over oxidative phosphorylation for energy production. Monocarboxylate transporter 1 (MCT1) and 4 (MCT4) are critical symporters mediating lactate efflux and preventing intracellular acidification during tumor growth. Numerous studies have focused on inhibiting MCT1 or MCT4 in various cancers. However, its role in T-cell lymphoma (TCL) is not yet investigated owing to the low incidence of TCL. This study was designed to investigate the expression of MCT1/MCT4 in patients with TCL and determine their prognostic value in this cancer. We performed immunohistochemistry to evaluate the expression level of MCT1/MCT4 in 38 TCL tissue samples and then compared their expression among different TCL subgroups, which were formed based on different clinical characteristics. Survival analysis was performed to evaluate the relationship between MCT1/MCT4 expression and both overall survival (OS) and progression-free survival (PFS). Our results revealed that MCT1 and MCT4 expression was significantly increased in TCL tissues compared to the control group. In addition, increased MCT1 expression associated with the female sex, advanced disease stage, increased serum LDH, Ki-67 at ≥ 50%, and intermediate or high-risk groups as categorized by the International Prognostic Index (IPI) score. We also found that increased MCT1 expression may be associated with reduced OS and PFS. In conclusion, MCT1 and MCT4 are overexpressed in patients with TCL and may predict poor prognosis. MCT1 inhibition might be a novel treatment strategy for TCL, and further preclinical trials are required.

[Expression and Significance of PD-1, PD-L1 and CTLA-4 in the Bone Marrow of Patients with Multiple Myeloma].

OBJECTIVE: To study the expression and significance of PD-1, PD-L1 and CTLA-4 tumor-associated antigens in multiple myeloma. METHODS: Bone marrow specimens from 122 patients with multiple myeloma were collected and divided into new-onset group (NDMM), complete remission group (CRMM) and relapsed and refractory group (RRMM) according to the disease progression stage. The proportion of CD4+ T lymphocytes, CD8+ T lymphocytes, Treg cells and plasma cells in the specimens and the expressions of PD-1, PD-L1 and CTLA-4 were detected by multi-parameter flow cytometry. RESULTS: There was no significant difference in the proportion of CD8+T and Treg cells among the three groups (P>0.05), while the proportions of CD4+T cells and PC in NDMM group were significantly higher than those in the CRMM group (P<0.05), the ratios of CD4+ to CD8+T in the NDMM and RRMM groups were significantly higher than those in the CRMM group (P<0.05). The expressions of PD-1, PD-L1 and CTLA-4 in CD8+ T cells was no significant difference among NDMM, CRMM and RRMM groups (P>0.05). While the expressions of PD-1, PD-L1 and CTLA-4 in CD4+ T cells and PC in the NDMM group were significantly lower than that in the CRMM group (P<0.05). There was significantly difference among the three groups in the expression of PD-1 in Treg cells, of which the NDMM group was significantly lower than that of the CRMM group (all P<0.05). The expressions of PD-1 and CTLA-4 in PC were significantly higher than those in CD8+ T, CD4+ T and Treg cells (P<0.05), the expression of PD-L1 in CD8+ T cells was significantly higher than that in CD4+ T and Treg cells (P<0.05). CONCLUSION: There is a correlation between the immune status of multiple myeloma and the expressions of PD-1, PD-L1 and CTLA-4 in plasma cells and lymphocyte subsets in vivo.

A phase I dose-escalation study of neoantigen-activated haploidentical T cell therapy for the treatment of relapsed or refractory peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a type of highly heterogeneous non-Hodgkin lymphoma with a poor prognosis and lack of effective targeted therapies. Adoptive T-cell therapy has been successfully used in the treatment of B-cell malignancies. We first used adoptive transfer of haploidentical T cells activated by patient-specific neoantigens in vitro to treat an elderly patient with refractory angioimmunoblastic T-cell lymphoma (AITL) in 2017, and the patient achieved long-term complete remission (CR). Here we report on early results from this first-in-human phase 1 clinical trial that aims to assess the safety and tolerability of neoantigen-activated haploidentical T cell therapy (NAHTC) for relapsed/refractory PTCL. Clinical trial registration: http://www.chictr.org.cn/index.aspx, identifier [ChiCTR1800017440].

[Application of CD138 Immunomagnetic Bead Sorting Combined with Fluorescence in Situ Hybridization in Multiple Myeloma].

OBJECTIVE: To compare the effects of direct fluorescence in situ hybridization (D-FISH) detection without sorting and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) on cytogenetic analysis of patients with multiple myeloma (MM). METHODS: FISH test results of 229 patients with initial MM were retrospectively analyzed. The patients were divided into two groups, 140 patients were tested with D-FISH and 89 patients with MACS-FISH. The combination probe was designed as P53, D13S319, RB1, 1q21, and IgH. Cytogenetic detection results were compared between the two groups. RESULTS: The total detection rate of cytogenetic abnormalities in D-FISH group was 52.9%, and that in MACS-FISH group was 79.8%. There was a significant difference in the cytogenetic abnormality rate between the two groups (P=0.020). The abnormal genes with the highest detection rate in the two groups were 1q21 and IgH, respectively, while the lowest was P53. There was no significant difference in the percentage of P53 positive cells (positive rate) between the two groups, while D13S319, RB1, 1q21, and IgH showed significant difference in positive cell rate (P=0.0002, P<0.0001, P=0.0033, P=0.0032). There was no significant correlation between the proportion of plasma cells (PC) detected by bone marrow morphology and cytogenetic abnormality rate in the D-FISH group, while there was a correlation between the proportion of PC detected by flow cytometry and cytogenetic abnormality rate (r=0.364). The PC proportion detected by bone marrow morphology and flow cytometry in the MACS-FISH group had no correlation with the cytogenetic abnormality rate and positive cell rate of the 5 genes mentioned above. Additionally, the PC proportion detected by bone marrow morphology and flow cytometry showed significant difference (P<0.0001). CONCLUSION: CD138 immunomagnetic bead sorting combined with FISH technology can significantly improve the abnormality detection rate of MM cytogenetics.

A novel germline HAVCR2 (TIM-3) compound heterozygous mutation is related to hemophagocytic lymphohistiocytic syndrome in EBV-positive peripheral T-cell lymphoma (NOS) with down-regulated TIM-3 signaling.

Recently, it have been reported that Hepatitis A Virus-Cellular Receptor 2(HAVCR2,encoding T-cell immunoglobulin and Mucin-Containing Protein 3[TIM3]) mutations are associated with severe hemophagocytic syndrome(HLH) in subcutaneous panniculitis-like T-cell lymphoma(SPTCL),and there are also frequent mutations in sporadic SPTCL, suggesting the individuals harboring HAVCR2(TIM-3) germline mutations are highly susceptible to familial or sporadic SPTCL. Here, we identify a novel germline compound heterozygous mutation of TIM-3 gene,c.245A>G (p.Tyr82Cys) and c.265C>T(p.Arg89Cys) variations in a single familial case with EBV-positive peripheral T-cell lymphoma(NOS),accompanied HLH;we also detected Tyr82Cys germline mutation in TIM-3 gene in one sporadic patient with cutaneous T cell lymphoma. We screened the distributive frequencies for TIM-3 mutations in healthy controls(n=87), B-(n=79) or T-cell lymphoma(n=25) not SPTCL, and the results showed that the mutation was found in two out of 25 patients with T-cell lymphoma but was not detected in 79 patients with B-cell lymphoma nor in a group of 87 controls. The mRNA expression of TIM-3 on primary cells and transfected HEK293 cells reduced significantly, indicating Tyr82Cys and Arg89Cys mutations is a loss-of function mutations on TIM-3,resulting in a weakened TIM-3 signaling. Our results suggest Tyr82Cys TIM-3 germline mutations are not only limited in SPTCL, and also occurred in other types of T-cell lymphoma, especially complicated HLH. TIM-3 mutations may be an predisposing factor for T-cell lymphoma and molecular marker for auxiliary diagnosis in T cell lymphoma,especially complicated with HLH.

CHST15 gene germline mutation is associated with the development of familial myeloproliferative neoplasms and higher transformation risk.

Herein, we describe the clinical and hematological features of three genetically related families predisposed to myeloproliferative neoplasms (MPNs). Using whole-exome sequencing, we identified a c.1367delG mutation(p.Arg456fs) in CHST15 (NM_001270764), a gene encoding a type II transmembraneglycoproteinthat acts as a sulfotransferase and participates in the biosynthesis of chondroitin sulfate E, in germline and somatic cells in familial MPN. CHST15defects caused an increased JAK2V617F allele burden and upregulated p-Stat3 activity,leading to an increase in the proliferative and prodifferentiation potential of transgenic HEL cells. We demonstrated that mutant CHST15 is able to coimmmunoprecipitate the JAK2 protein,suggesting the presence of a CHST15-JAK2-Stat3 signaling axis in familial MPN. Gene expression profiling showed that the FREM1, IFI27 and C4B_2 genes are overexpressed in familial MPN, suggesting the activation of an "inflammatory response-extracellular matrix-immune regulation" signaling network in the CHST15 mutation background.We thus concluded that CHST15 is a novel gene that predisposes to familial MPN and increases the probability of disease development or transformation.

Diagnosis and treatment of multiple myeloma in Hunan Province. / 湖南省多发性骨髓瘤诊治现状.

OBJECTIVES: There is less clinical data on multiple myeloma (MM) in China, and the aim of this study was to collect and analyze the clinical data of newly diagnosed multiple myeloma (NDMM) patients in Hunan Province during 1 year, to understand the real clinical features and treatment outcome for Hunan Province patients with MM, and to strengthen the understanding of the standardized diagnosis process and treatment plan of MM. METHODS: The clinical data of 529 patients with NDMM in 12 large-scale general hospitals in Hunan Province from January 1 to December 31, 2019 were collected and analyzed, including baseline data, treatment regimens, duration of treatment, and adverse reactions. The clinical characteristics, treatment, and safety of patients were analyzed by SPSS 21.0. RESULTS: Among the 529 NDMM patients, the age was 33-90 (median 64) years and the male-female ratio was 1.38꞉1. The clinical features ranged from high to low were as follows: Bone pain (77.7%), anemia (66.8%), renal insufficiency (40.6%), hypercalcemia (15.1%). Typing: IgG 46.5%, IgA 24.6%, IgD 2.6%, IgM 0.8%, light chain 15.7%, double clone 3.0%, no secretion 0.6%, absence 6.2%. Staging: Durie-Salmon stage I, II, and III were 4.5%, 10.6%, 77.3%, respectively, and 40 cases (7.6%) missed this data. International Staging System (ISS) stage I, II, and III were 10.4%, 24.4%, and 47.6%, respectively, and 93 cases (17.6%) were missing. Revised International Staging System (R-ISS) stage I, II, and III were 5.5%, 27.0%, 23.1%, respectively, and 235 cases (44.4%) missed this data. Among the 98 NDMM patients in the Third Xiangya Hospital, Central South University, Durie-Salmon (DS) stage missing 2.0%, ISS stage missing 12.3%, and R-ISS stage missing 12.3%.Treatment: Among the 529 patients,475 received treatment, the rate of treatment was 89.8%; 67.4% of the patients were able to complete four courses of chemotherapy at induction phase, 90.3% of the patients received proteasome inhibitor based combination chemotherapy regimen more than once, 67.2% received immunomodulator based regimen more than once, and 59.8% of the patients received proteasome inhibitor and immunomodulator based combination chemotherapy regimen more than once. Curative: Overall response rate (ORR) and high quality response rate (HQR) of the 4-course group were better than those of the 2-course group (ORR: 85% vs 65%, P=0.006; HQR: 68.3% vs 24.0%, P<0.001). The HQR of the standard chemotherapy group was better than that of the non-standard chemotherapy group (65.1% vs 48.2%, P=0.035). Adverse reactions during treatment included hematologic toxicity (17.5%), peripheral neuropathy (24.8%), gastrointestinal adverse events (23.8%), pulmonary infection (25.9%), herpes zoster (4.6%), and venous thrombotic events (1.7%). CONCLUSIONS: In 2019, the missed diagnosis rate of MM patients was high, the medium age of diagnosis was older, and the accuracy of patient diagnosis was not high. There is a great difference among medical centers, especially in the stage and risk stratified, nearly half of NDMM patients are not diagnosed with R-ISS stage; the lack of cytogenetic data needs to be supplemented by follow-up studies. A high proportion of patients with NDMM present with bone pain and anemia.Patients received treatment have higher use of chemotherapy regimens containing proteasome inhibitors and/or immunomodulators, but there is a significant gap among different medical centers, and standardized treatment needs to be strengthened. The safety during chemotherapy is controllable.

A drug targeting 5-lipoxygenase enhances the activity of a JAK2 inhibitor in CD34+ bone marrow cells from patients with JAK2V617F-positive polycythemia vera in vitro.

Janus kinase 2 (JAK2) inhibitors, the first targeted treatments for myeloproliferative neoplasms (MPNs), provide substantial benefits, including a marked reduction in splenomegaly and MPN-associated symptoms. However, these drugs rarely induce molecular remission in patients with MPNs. Zileuton, a 5-lipoxygenase (5-LO) inhibitor, has been demonstrated to selectively deplete hematopoietic stem cells (HSCs) expressing a JAK2 point mutation (JAK2V617F) in mouse models of JAK2V617F-induced polycythemia vera (PV). To determine the potential activity of 5-LO inhibitors in combination with JAK inhibitors against human PV HSCs, the present study first analyzed 5-LO expression in CD34+ bone marrow cells from patients with JAK2V617F-positive PV using western blotting and reverse transcription-quantitative PCR, and then examined the effect of zileuton combined with ruxolitinib on colony formation using a colony formation assay. Furthermore, cell cycle and apoptosis in CD34+ cells from patients with PV and healthy volunteers were determined by flow cytometry. In the present study, 5-LO expression was upregulated in CD34+ cells from patients with PV compared with in CD34+ cells from healthy volunteers. Higher levels of leukotriene B4, a product of the 5-LO signaling pathway, were detected in patients with PV compared with in healthy volunteers. Zileuton treatment suppressed the colony formation of CD34+ cells from patients with PV in a dose-dependent manner. Furthermore, zileuton and ruxolitinib exerted their anticancer effects by suppressing hematopoietic colony formation, inducing apoptosis and arresting the cell cycle of human CD34+ cells from patients with PV. The combination of these two drugs exerted a more beneficial effect than either agent alone. Based on these data, zileuton enhanced the antitumor activity of low-dose ruxolitinib in hematopoietic progenitor cells from patients with PV, providing conceptual validation for further clinical applications of combination treatment with ruxolitinib and zileuton for patients with PV.

Breakpoint mapping of a t(9;22;12) chronic myeloid leukaemia patient with e14a3 BCR-ABL1 transcript using Nanopore sequencing.

BACKGROUND: The genetic changes in chronic myeloid leukaemia (CML) have been well established, although challenges persist in cases with rare fusion transcripts or complex variant translocations. Here, we present a CML patient with e14a3 BCR-ABL1 transcript and t(9;22;12) variant Philadelphia (Ph) chromosome. METHODS: Cytogenetic analysis and fluorescence in situ hybridization (FISH) was performed to identify the chromosomal aberrations and gene fusions. Rare fusion transcript was verified by a reverse transcription-polymerase chain reaction (RT-PCR). Breakpoints were characterized and validated using Oxford Nanopore Technologies (ONT) (Oxford, UK) and Sanger sequencing, respectively. RESULTS: The karyotype showed the translocation t(9;22;12)(q34;q11.2;q24) [20] and FISH indicated 40% positive BCR-ABL1 fusion signals. The RT-PCR suggested e14a3 type fusion transcript. The ONT sequencing analysis identified specific positions of translocation breakpoints: chr22:23633040-chr9:133729579, chr12:121567595-chr22:24701405, which were confirmed using Sanger sequencing. The patient achieved molecular remission 3 months after imatinib therapy. CONCLUSIONS: The present study indicates Nanopore sequencing as a valid strategy, which can characterize breakpoints precisely in special clinical cases with atypical structural variations. CML patients with e14a3 transcripts may have good clinical course in the tyrosine kinase inhibitor era, as reviewed here.

A small molecular compound CC1007 induces cross-lineage differentiation by inhibiting HDAC7 expression and HDAC7/MEF2C interaction in BCR-ABL1- pre-B-ALL.

Histone deacetylase 7 (HDAC7), a member of class IIa HDACs, has been described to be an important regulator for B cell development and has a potential role in B cell acute lymphoblastic leukemia (B-ALL). CC1007, a BML-210 analog, is designed to indirectly inhibit class IIa HDACs by binding to myocyte enhancer factor-2 (MEF2) and blocking the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of these targets. In this study, we investigated the anticancer effects of CC1007 in breakpoint cluster region-Abelson 1 fusion gene-negative (BCR-ABL1-) pre-B-ALL cell lines and primary patient-derived BCR-ABL1- pre-B-ALL cells. CC1007 had obvious antileukemic activity toward pre-B-ALL cells in vitro and in vivo; it also significantly prolonged median survival time of pre-B-ALL-bearing mice. Interestingly, low dose of CC1007 could inhibit proliferation of BCR-ABL1- pre-B-ALL cells in a time-dependent manner not accompanied by significant cell apoptosis, but along with cross-lineage differentiation toward monocytic lineage. From a mechanistic angle, we showed that HDAC7 was overexpressed in BCR-ABL1- pre-B-ALL cells compared to normal bone marrow samples, and CC1007 could reduce the binding of HDAC7 at the promoters of monocyte-macrophage-specific genes via inhibition of HDAC7 expression and HDAC7:MEF2C interaction. These data indicated that CC1007 may be a promising agent for the treatment of BCR-ABL1- pre-B-ALL.

Highly Stable and Stretchable Conductive Films through Thermal-Radiation-Assisted Metal Encapsulation.

Stretchable conductors are the basic units of advanced flexible electronic devices, such as skin-like sensors, stretchable batteries and soft actuators. Current fabrication strategies are mainly focused on the stretchability of the conductor with less emphasis on the huge mismatch of the conductive material and polymeric substrate, which results in stability issues during long-term use. Thermal-radiation-assisted metal encapsulation is reported to construct an interlocking layer between polydimethylsiloxane (PDMS) and gold by employing a semipolymerized PDMS substrate to encapsulate the gold clusters/atoms during thermal deposition. The stability of the stretchable conductor is significantly enhanced based on the interlocking effect of metal and polymer, with high interfacial adhesion (>2 MPa) and cyclic stability (>10 000 cycles). Also, the conductor exhibits superior properties such as high stretchability (>130%) and large active surface area (>5:1 effective surface area/geometrical area). It is noted that this method can be easily used to fabricate such a stretchable conductor in a wafer-scale format through a one-step process. As a proof of concept, both long-term implantation in an animal model to monitor intramuscular electric signals and on human skin for detection of biosignals are demonstrated. This design approach brings about a new perspective on the exploration of stretchable conductors for biomedical applications.

RNF8 is responsible for ATRA resistance in variant acute promyelocytic leukemia with GTF2I/RARA fusion, and inhibition of the ubiquitin-proteasome pathway contributes to the reversion of ATRA resistance.

BACKGROUND: GTF2I-RARA is a newly identified RARA fusion gene in variant acute promyelocytic leukemia (APL) patients with t(7;17)(q11;q21). Clinical manifestation in the patient showed that it is a sort of ATRA-insensitive oncogene and is different from the classic PML-RARA in terms of therapeutic reaction. METHODS: To reveal the functional characteristics and regulating mechanism of the GTF2I-RARA fusion gene, we established a GTF2I-RARA-transfected HL60 cell model and examined its sensitivity to ATRA by western blot, MTT assay, flow cytometry, and Wright-Giemsa staining. Coimmunoprecipitation and confocal microscopy were used to examine the binding of GTF2I-RARA and transcriptional corepressors. We also performed ChIP-seq to search for potential target genes. Immunoprecipitation, ubiquitination assay, western blot, luciferase assay, and real-time PCR were used to analyze the effects of RNF8 on RARA. Flow cytometry and Wright-Giemsa staining were used to study the effect of MG132 and ATRA on the GTF2I-RARA-transfected HL60 cell model. RESULT: We confirmed resistance of GTF2I-RARA to ATRA. Compared with PML-RARA, GTF2I-RARA has a higher affinity to HDAC3 under ATRA treatment. Using the ChIP-sequencing approach, we identified 221 GTF2I-RARA binding sites in model cells and found that the RING finger protein 8 (RNF8) is a target gene of GTF2I-RARA. RNF8 participates in disease progression and therapy resistance in APL with the GTF2I-RARA transcript. Elevated RNF8 expression promotes the interaction between RARA and RNF8 and induces RARA Lys-48 linkage ubiquitylation and degradation, resulting in attenuated transcriptional activation of RARA. CONCLUSION: Our results suggest that RNF8 is a key GTF2I-RARA downstream event. Using the combination of MG132 and ATRA to treat GTF2I-RARA-HL60 cells, a synergistic effect leading to GTF2I-RARA-HL60 cell differentiation was confirmed. Taken together, the targeting of RNF8 may be an alternative choice for treatment in variant APL with GTF2I-RARA fusion.

Immunophenotypic characterization of sphere-forming cells derived from the human renal cell carcinoma cell line 786-O.

Cancer stem cells (CSCs) have been found in many different types of malignant tumors. In our previous study, we found that sphere-forming cells (SFCs) from the renal cell carcinoma (RCC) cell line SK-RC-42 are rich in CSCs. However, our previous reports are based on only one human RCC cell line, which makes it difficult to determine whether the findings from this cell line represent the general mechanisms in human RCC. Therefore, in this study, we attempted to evaluate whether the sphere culture method could enrich for CSCs from another human RCC cell line, 786-O, and to characterize their immunological phenotype. We discovered that a small population of 786-O cells was capable of growing as tumor spheres. The SFCs had many properties similar to CSCs, including greater ability to self-renew in vitro and in vivo, higher mRNA expression levels of several 'stemness' genes, stronger tumorigenicity and resistance to chemotherapeutic agents than monolayer adherent cells (MACs). The SFCs expressed low levels of MHC-I, HLA-DR, CD80, CD86, CD152 and CD154. Additionally, the SFCs had lower expression levels of Her2 and hTERT, FasL, Fas, the transcription factor forkhead box protein 3 (FoxP3) and activating natural killer cell receptors than did the MACs. In addition, both 786-O SFCs and MACs were weakly positive for B7-H4 expression, while the expression level of B7-H1 in 786-O SFCs was lower than that in MACs. Furthermore, 786-O SFCs and MACs both expressed substantial and comparable levels of membrane complement regulatory proteins (mCRPs). Finally, we found that 786-O SFCs triggered T cell apoptosis. These findings suggested that tumor spheres from 786-O cells are rich in CSCs. The immunological phenotype of the SFCs described in our study suggests that CSCs might play an important role in tumor immune evasion.

[Effect of Decitabine on Megakaryocyte Culture of Steroid-resistant ITP Patients].

OBJECTIVE: To investigate the effect of decitabine and plasma of ITP patients on in vitro cultrue of megakaryocytes in bone marrow of steroid-resistant ITP patients. METHODS: Bone marrow mononuclear cells were isolated from 20 steroid-resistant ITP patients, both methyl cellulose semisolid culture system (to observe and count the number of megakaryocytes colony-forming unit) and liquid culture system (to analysis the expression rate of CD41a(+) cells) were used for megakaryocyte cultrue. The experiments were divided into 4 groups according to the different components of the culture system, group A was control, group B was added with decitabine, group C with ITP plasma, group D with both decitabine and ITP plasma, and the rest of the culture components were the same in the 4 groups except the above-mentioned materials. Morphology of megakaryocytes was observed by inverted and light microscopy. The expression rate of CD41a⁺ cells in culture was analysed by flow cytometric. RESULTS: Different concentration of decitabine showed different effect on megakaryocyte growth of steroid-resistant ITP patients and the optimal concentration to differentiate into megakaryocyte for bone marrow mononuclear cells is 3.0 µmol/L. Compared with group A, both megakaryocyte colony forming units (CFU) and expression rate of CD41a⁺ cells in group B were statistically significantly higher (P < 0.05). As compared with group A, the megakaryocyte colony-forming units in group C decreased with statistically significant difference, while compared with group C, the megakaryocyte colony-forming units in group D obviously increased with statistically significant difference. CONCLUSIONS: Decitabine is able to induce bone marrow mononuclear cells of steroid-resistant ITP patients to differentiate into megakaryocyte and the optimal concentration is 3.0 µmol/L; ITP plasma is able to inhibit the megakaryocyte growth of steroid-resistant ITP patients.

[The frequency of JAK2 V617F mutation, expression level of phosphorylated JAK/STATs proteins and their clinical significance in myeloproliferative disorders patients].

OBJECTIVE: To investigate the frequency of JAK2 V617F mutation in 145 myeloproliferative disorders (MPDs) patients, analyze the correlation between JAK2 V617F mutation and clinical features. METHODS: The JAK2 V617F mutation was detected by direct DNA sequencing of PCR product and allele-specific PCR respectively. The expression of JAK2, phospho-JAK2 and phospho-STAT5 proteins was determined by Western blot. The clinical data of MPDs patients with or without JAK2 V617F mutation was collected and analyzed for evaluating the clinical significance of JAK2 V617F mutation. RESULTS: 1) The frequency of JAK2 V617F mutation for PV, IMF, ET was 92%, 58%, 50% respectively. Compared with conventional DNA sequencing (PV 84%, IMF 44%, ET 39%, respectively), allele-specific PCR exhibited a higher sensitivity in JAK2 V617F mutation detection. 2) The expression levels of phospho-JAK2 and phospho-STAT5 in peripheral blood mononuclear cells (PBMNCs) were upregulated significantly in JAK2 V617F-positive patients than in JAK2 V617F negative patients. 3) Compared with the patients with no JAK2 V617F mutation, the JAK2 V 617F-positive patients' features were as follows: older age of onset, higher mean leukocyte counts, lower platelet counts and smaller spleen volume. Frequency of thrombosis events in PT, ET, IMF was 17%, 32%, 16% respectively for JAK2 V617F positive group, and 0% (PV), 16% (ET), 5% (IMF) for JAK2 V617F negative group. CONCLUSIONS: MPDs patients display higher frequency of JAK2 V617F mutation. JAK2 V617F mutation positive patients predispose to a thrombosis tendency.

[Analysis of angiogenesis related proteins and its implication in type-2 hereditary hemorrhagic telangiectasia].

OBJECTIVE: To detect the level of transforming growth factor-beta1 (TGF-beta1), TGF-beta2, vascular endothelial growth factor (VEGF) and platelet-derived growth factor receptor-alpha (PDGFRalpha) in plasma and peripheral blood leukocytes in a hereditary hemorrhagic telangiectasia type 2 (HHT-2) family, and explore the implication of angiogenesis related proteins in HHT-2 pathogenesis. METHODS: The diagnosis of the HHT-2 patient was based on clinical features and further confirmed by determining a C1231T mutation of activin receptor-like kinase 1 (ALK1) gene. Five other new members in this family were evaluated with ALK1 gene screening and clinical manifestation. Plasma level of TGF-beta1, TGF-beta2 or VEGF was measured by ELISA, and the expression of PDGFRalpha,TGF-beta1, and VEGF in peripheral blood leukocytes by flow cytometry combined with direct or indirect immunofluorescence. RESULTS: No C1231T mutation was detected in exon 8 of ALK1 gene in the 5 new members. Plasma TGF-beta1 and TGF-beta2 concentration in 3 affected HHT case was (16 954 +/- 3 709) ng/L and (11 548 +/- 2 611) ng/L, respectively, compared with that of normal control, the difference was not significant (P > 0.05). VEGF concentration in the 3 HHT patients, 6 unaffected family members and 6 normal controls was (179.2 +/- 22.0) microg/L, (149.8 +/- 22.7) microg/L and (132.9 +/- 21.0) microg/ L, respectively. Plasma VEGF level in HHT patients was significantly higher than that in normal subjects (P < 0.025). Peripheral leukocyte PDGFRalpha and VEGF in HHT patients and unaffected family members were markedly higher than that of normal control (P < 0.05 and P < 0.02), while TGF-beta1 distribution was similar in HHT patients and normal subjects. CONCLUSION: Compared with normal controls there is no difference in plasma TGF-beta1 concentration on peripheral leukocytes of HHT patients. Plasma VEGF concentration or leukocytes VEGF expression in HHT is significantly higher than that of normal subjects. Leukocytes PDGFRalpha expression in HHT is significantly higher than that of normal control. These changes may be associated with a compensable mechanism in HHT.