To explore the genetic and clinical features of a rare t(1;12)(q21;p13) in a patient with myelodysplastic syndrome (MDS). A 53-year-old male was diagnosed as high-risk MDS, and died in a short period. A complete cytogenetic analysis of bone marrow by conventional G-banding karyotyping was performed at the time of initial evaluation. On the basis of chromosome karyotype, interphase and metaphase fluorescence in-situ hybridization (FISH) were carried out to further confirm the abnormal karyotypes. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to determine ETV6/ARNT fusion gene status. G-banding revealed karyotype 47, XY, +8, der(12) t(1;12)(q21;p13). FISH with the centromere 8 probe verified the trisomy 8, and the ETV 6 break-apart probe suggested heterozygous loss of ETV6 allele located in short arm of chromosome 12. Subsequently, the painting probe of whole chromosome 12 further confirmed the part break of short arm of chromosome 12, and the 1q21/1p36 probe yielded three signals of 1q21 and two signals of 1p36. The results of FISH were in accordance with the karyotype completely. No ETV6/ARNT fusion gene was detected by PCR. T(1;12)(q21;p13) is a rare abnormal karyotype, and the limited reports cannot supply definite clinical significance. Rapid deterioration of our case suggests this translocation of chromosome might have a poor effect on the survival of MDS.

Purpose Though mutations of the calreticulin (CALR) gene have been identified in essential thrombocythemia patients, the detailed mechanisms for CALR mutations have not been completely clarified. Our study is aimed at characterizing alteration of protein expression in ET patients with mutated CALRdel52 and further recognizing possible involvement of signaling pathways associated with CALR mutations. Patients and Methods Protein pathway array was performed to analyze the expression levels of proteins involved in various signaling pathways in peripheral blood neutrophils from 18 ET patients with mutated CALRdel52, 20 ET patients with JAK2V617F mutation and 20 controls. Results We found 20 proteins differentially expressed in ET patients with mutated CALRdel52 compared with healthy controls. These proteins were associated with molecular mechanisms of cancer in ingenuity pathways analysis (IPA) network. We identified top ten canonical pathways which including apoptotic pathways and cellular cytokine pathways might participate in pathogenesis of ET with mutated CALRdel52. Additionally, there were 8 proteins found to be dysregulated differently between ET patients with mutated CALRdel52 and those with JAK2V617F mutation. These proteins might be related to the unique signaling pathways activated by CALRdel52 mutation which were different to JAK/STATs pathway by JAK2V617F mutation. Conclusion Our study demonstrated that numerous alterations of signaling proteins and pathways in ET patients with mutated CALRdel52. These findings could help to gain insights into the pathological mechanisms of ET.

目的 分析2例早期前T淋巴母细胞白血病/淋巴瘤(early T-cell precursor acute lymphoblastic leukemia/lymphoma,ETP-ALL/LBL)患者的临床及实验室资料,结合文献复习,提高对该病的认识.方法 回顾性分析我院诊治的2例ETP-ALL/LBL患者病例资料,并进行文献检索分析.结果 2例患者均为中年,女性,以淋巴结肿大为主要表现起病.淋巴结活检病理诊断为T-淋巴母细胞淋巴瘤(T-LBL),流式细胞术免疫分型符合ETP-ALL/LBL(病例1为胸水标本,病例2为淋巴结标本).病例1先后给予VDCLP方案及Hyper-CVAD方案化疗,病例2给予BFM-90方案化疗,2例患者化疗后均无效,因疾病进展死亡.结论 ETP-ALL/LBL具有独特的免疫表型特点,病理诊断时注意加做早期标记和髓系抗体;ETP-ALL/LBL常规化疗效果较差,复发率高,预后差,强化疗及早期行造血干细胞移植可以改善预后.

2016年更新版《WHO造血和淋巴组织肿瘤分类》中[1],将伴有骨髓原始红细胞≥30％,原始髓系＜20％,既往有放化疗史,定义为治疗相关伴红系明显增生的急性髓系白血病(therapy-related acute myeloid leukemia,T-AML)的范畴.在急性红白血病(acute erythroleukemia,AEL)分类中,相比于2008年的标准[2],仅保留了纯红细胞白血病(pure erythroid leukemia,PEL),诊断标准为骨髓红系＞80％,原始红细胞≥30％,骨髓或外周血非红原始细胞＜20％、无放化疗史、无WHO重现性细胞遗传学异常.但如何诊断骨髓有核红＞80％,原始红细胞≥30％,既往伴有化疗史的患者,在更新分类中并未阐明.本文报道1例骨髓有核红＞80％,原始红细胞≥30％,既往伴有化疗史的患者.分析患者临床、实验室特征及治疗过程,文献复习T-AML与PEL特征及诊断.

Background: Somatic mutations in WT1 and TET2 were separately perceived as contributors to hematopoietic disorders and thought to have a mutually exclusive effect in acute myeloid leukemia (AML). Here, we report a case in which WT1 and TET2 mutations co-existed at different stages in an AML patient with t(9;11)(p21.3;q23.3), and without abnormal WT1 expression, which did not synchronize with the patient's tumor state. Hence, the origins and prognostic value of these two mutations were investigated. Methods: Bone marrow (BM) and buccal mucosal cells were obtained from a 27-year-old male AML patient, and next-generation sequencing (NGS), targeting multiple genes, was performed after DNA extraction. Single nucleotide variant (SNV) and insertion-deletion (Indel) associated with AML were identified in these two samples by using SAM tools. Then, peripheral blood or buccal mucosal cells were obtained from the patient’s relatives, and NGS targeting multiple genes was applied. Besides, the relationship between overall survival (OS) and alteration of these two genes in AML patients was analyzed by using cBioPortal and UALCAN databases, respectively. Results: SNV of WT1 (NM_024426:exon7:c.1109G>C；p.Arg370Pro) and TET2 (NM_001127208:exon11:c.5530G>A；p.Asp1844Asn) were present in the BM of the patient, and these two mutations were also observed in his buccal mucosal cells, which suggested that they were germline mutations. Variation analysis of samples from other relatives indicated that WT1 (NM_024426:exon7:c.1109G>C;p.Arg370Pro) and TET2 (NM_001127208:exon11:c.5530G>A;p.Asp1844Asn) were also present in his father and mother, respectively. The patient’s brother also carried TET2 (NM_001127208:exon11:c.5530G>A；p.Asp1844Asn) mutations. Bioinformatic analysis suggested that mutations in the WT1 and TET2 genes are both associated with a poor prognosis of AML, and abnormal TET2 expression is associated with a poorer prognosis than abnormal WT1 expression. Conclusion: It is firstly report that WT1 p.Arg370Pro and TET2 p.Asp1844Asn mutations co-existed in an AML patient with t(9;11)(p21.3;q23.3) and no WT1 abnormal expression, which highlight the importance of the identification and prognostic evaluation of germline mutations in clinical practice for AML.

OBJECTIVE:To explore the clinical value of PKC412 (midostaurin) in treatment of AML patients with FLT3-.METHODS:The bone marrow or peripheral blood were collected and heparinized from 21 newly diagnosed FLT3- AML patients, then the mononuclear cells from bone marrow or peripheral blood were isolated by density-gradient method. The sensitivity of leukemia cells to PKC412 of 8 concentration in vitro was detected by ATP-bioluminescence-tumor chemosensitivity assay (ATP-TCA), and the relationship among sensitivity results in vitro, risk stratification and therapeutic efficacy was analyzed.RESULTS:The leukemia cells of 21 patients with AML displayed different sensitivities to PKC412 in vitro. The rate of sensitivity in vitro was 42.9%, and sensitive concentration in vitro were between 1 µmol/L and 5 µmol/L. There was no significant relationship between risk stratification and sensitivity results of PKC412 in vitro. There was also no significant relationship between clinical efficacy and sensitivity results of PKC412 in vitro. The survival of patients in low-risk and intermediate-risk groups was better than that of patients in high-risk groups (P=0.015).CONCLUSION:PKC412 can be one of the effective therapeutic method for AML patients without FLT3 mutation. The sensitivity of leukemia cells to PKC412 may become a prognostic marker for evaluating clinical efficacy of PKC412, which is independent of other factors.

目的 研究自身免疫性溶血性贫血(autoimmune hemolytic anemia,AIHA)患者外周血滤泡辅助性T细胞(T follicular helper cells,Tfh细胞)数量的变化,探讨Tfh细胞在AIHA发病机制中的作用.方法 采用流式细胞术的方法检测分析26例AIHA患者及30例正常人(对照组)外周血B细胞、成熟B细胞及CD4+T细胞比例,Tfh细胞数量.结果 AIHA患者外周血CD19+B细胞百分比为(17.46±16.65)％,高于对照组(9.12±4.60)％(P=0.011);成熟B细胞百分比为(87.33±7.80)％,明显高于对照组(76.52±13.90)％ (P =0.001).AIHA患者CD4+T细胞为(25.47±11.75)％,较正常对照组(34.36 ±7.21)％明显减低(P =0.001).AIHA患者Tfh细胞比例为(6.72±5.38)％,明显高于对照组(1.52±1.05)％(P=0.000).结论 Tfh细胞数量的扩增参与了AIHA病理过程,并在其发病过程中起重要作用.

目的 初步探索三磷酸腺苷生物荧光肿瘤体外药敏检测(ATP bioluminescence-tumor chemosensitivity assay,ATP-TCA)技术在急性髓系白血病(acute myeloid leukemia,AML)中的临床应用价值.方法 采集18例初治AML患者的骨髓或外周血,分离单个核细胞,应用ATP-TCA技术,观察体外白血病细胞对化疗药物的敏感性,并分析体外药敏结果、危险度分层及临床疗效三者之间的关系.结果 18例初治AML患者的白血病细胞在体外对8种化疗药物显示出不同的敏感性,有效率最高的是阿柔比星,无效率最高的是吡柔比星.在14例可进行疗效评价的初治AML患者中观察到蒽环类药物体外敏感性与临床疗效之间的完全相符率为78.6％,不同危险度分层患者之间的临床疗效(P=0.012)和生存时间(P=0.018)均有显著差异,高危组患者生存时间最短.结论 AML体外药敏检测结果与临床治疗反应之间具有相关性趋势,ATP-TCA可能是开展AML个体化化疗的一种有效的体外药物筛选方法,尤其是老年AML.

Somatic mutation of calreticulin (CALR) has been found in about 25% patients with essential thrombocythemia (ET), where is the second most common mutated gene after JAK2V617F.Recent data have shown that thrombopoietin receptor MPL function is essential in mutant CALR-mediated cellular transformation. However, the mechanisms for CALR mutant-driven MPN have not been fully elucidated.

Objective To explore the meaning of midkine (MK) levels in serum in different development stage of acute leukemia,and to explore the relationship between MK and WT1.Methods The levels of the MK in serum of 86 cases of acute leukemia and 30 cases of normal people were detected by ELISA.Real-time quantitative PCR (RQ-PCR) method was used to determine the expression of WT1 at mRNA level in 15 AML patients.Results The MK level in serum in the new diagnosed group was higher than that in the complete remission group and the normal control group [7.52 (5.44,10.55) ng/ml vs 3.52 (1.56,5.20) ng/ml vs 2.44 (1.89,3.12) ng/ml].There' s no statistical difference between midkine level in new diagnosed acute B cell leukemia (B-ALL) group and acute myeloid leukemia (AML) group [7.88 (5.78,15.78) ng/ml vs 6.25 (4.59,16.33) ng/ml].The clear correlation was found between the level of serum MK and quantities of marrow WT1 gene (r =0.529,P =0.043).Conclusions The level of MK in serum of acute leukemia patients is increased at the time of new diagnosis and decreased at complete remission.ELISA may be a way to measure the status of AL.The location of MK gene is adjacent to WT1 gene and MK' s clinical significance is similar to WT1' s,furthermore,there is a clear correlation between MK in serum and WT1 of marrow in quantities.

OBJECTIVE:To screen signaling pathway proteins in myelodysplastic/myeloproliferative neoplasms-unclassifiable (MDS/MPN-U), and to explore the possible role of the differentially expressed signaling pathway proteins in pathogenesis of MDS/MPN-U.METHODS:Protein Pathway Array (PPA) was applied to analyze the differential expression levels of signaling pathway proteins in 10 patients with MDS/MPN-U and normal controls, and furthermore to identify the signaling pathways and network in which these proteins were analyzed by Ingenuity pathway analysis program.RESULTS:The expressions of 25 signaling proteins in MDS/MPN-U were significantly different, compared with the control group. Among them 15 proteins were upregulated in MDS/MPN-U patients, while 10 proteins were downregulated. These dysregulated proteins were involved in 10 major signaling pathways related with cell proliferation and immunity. The complicated interactive network was established by these proteins and pathways.CONCLUSION:The differentially expressed signaling proteins screened from the MDS/MPN-U patients by PPA might be helpful to reveal the pathogenesis of MDS/MPN-U and to discover the therapeutic targets.

This study was aimed to investigate the genetic characteristics of human acute lymphoblastic leukemia cell line Molt-4, and evaluate its application in measuring telomere length by Flow-FISH. Molt-4 cell line was cultured in suspension and subcultured regularly. Eight different passages of Molt-4 cells in exponential stage were selected.The growth curves were drawn by cell counting method, meanwhile calculating the population doubling times of cells,DNA ploidies were determined by flow cytometry,karyotypes were analyzed by G-banding and telomere lengths were measured by Southern blot. The results showed that the population doubling time of Molt-4 cell line was (1.315 ± 0.062) d, DNA ploidy index was (2.085 ± 0.0093) , and the telomere length was (32.05 ± 5.27) kb. There were no significant difference among different passages (P = 0.931,0.888 and 0.935 separately). The karyotypes showed that the chromosome numbers of Molt-4 cell line were from 91 to 99 in different metaphases, and the majority of them were hypertetraploid, and stable and recurrent structural abnormalities of chromosomes could be kept. It is concluded that the stable genetic characteristics and the longer telomere length of Molt-4 cell line makes it be a feasible control cells in measurement of telomere length by Flow-FISH.

Objective To explore the clinical diagnostic significance of cerebrospinal fluid (CSF)detection with flow cytome-try (FCM)in central nervous system leukemia (CNS-L).Methods The 1 9 5 cerebrospinal fluid (CSF)samples were ana-lyzed using four-color flow cytometry by CD45/SSC secondary gating strategy and detected by conventional cell morpholo-gy.Results The results showed that 24 (33.80%)patients had abnormal cells by flow cytometry and 15 (19.72%)pa-tients had abnormal cells by conventional cell morphology in 71 Non-Hodgkin’s lymphoma (NHL)patients,respectively. The difference was statistically significant (P=0.022).The results showed that 13 (22.41%)patients had abnormal cells by flow cytometry and 1 2 (1 8.9 7%)patients had abnormal cells by conventional cell morphology in acute lymphoma leuke-mia (ALL)patients,respectively.The difference was no statistically significant (P=1.000).The results showed that 4 (5.9 7%)patients had abnormal cells by flow cytometry and 4 (5.9 7%)patients had abnormal cells by conventional cell morphology in acute nonlymphocytic leukemia (ANLL)patients,respectively.The difference was no statistically significant (P=1.000).Conclusion Cerebrospinal fluid (CSF)detection with flow cytometry (FCM)may be helpful in the diagnosis of central nervous system leukemia in NHL.It has higher sensitivity and is an important supplement to CSF conventional cell morphology.

目的 探讨热变性对外周血细胞亚群标记效果的影响,从而建立稳定的流式-荧光原位杂交技术流程(flow cytometry-fluorescence in situ hybridization,Flow-FISH).方法 采集10例健康人肝素抗凝新鲜外周血标本,分别以Alexa Fluor(@) 647标记的CD3、CD19、CD14抗体标记T淋巴细胞、B淋巴细胞和单核细胞,高温变性后,采用流式细胞术分析热变性前后细胞系列特异性抗原表达的变化;以Fluor(@) 647-CD3标记外周血T淋巴细胞,以4种浓度的Bis[sulfosuccinimidyl] suberate(BS3)交联剂进行抗原抗体交联,比较不同浓度BS3的交联效果.结果 高温变性后,Alexa Fluor(@) 647标记的CD3和CD19的荧光强度减弱,但仍能有效区分出T淋巴细胞和B淋巴细胞;Alexa Fluor(@) 647-CD14的荧光强度无变化,单核细胞清晰可辨;4种浓度交联剂BS3作用后,CD3的标记效果基本一致.结论 经过热变性后,Alexa Fluor(@) 647标记的CD3、CD19、CD14抗体仍能够有效标记出血细胞中的不同细胞亚群,耐热性好,可以用于Flow-FISH技术中;交联剂BS3的适宜作用浓度为2.5 mmol/L.

目的 初步探讨热变性对外周血细胞在流式细胞术分析结果中的影响,从而提高流式-荧光原位杂交(Flow-FISH)技术的应用.方法 收集5例非骨髓造血干细胞疾病患者肝素抗凝外周血标本,以CD45-Alexa Fluor(R)647标记细胞表面抗原,高温变性后,采用流式细胞术分析热变性前后外周血有核细胞的散射光信号和荧光信号变化.结果 热变性后,外周血粒细胞的侧向散射光明显缩小;单核细胞不易通过散射光被区分和设门.所有细胞CD45表达强度均减弱,以淋巴细胞为著;通过侧向散射光和CD45设门虽能大致区分各群细胞,但不及未热变性细胞清晰.结论 热变性后,外周血细胞在流式细胞术中的散射光信号和荧光信号均发生了变化,按照常规FSC/SSC和CD45/SSC方法设门进行细胞亚群分析的结果不精确,利用系列特异性荧光抗体标记是一种有前景的方法.

Objective:To explore the significance of the number of CD34 + cells in the peripheral blood from patients with low-risk myelodysplastic syndromes(MDS).Methods :Marrow cells were collected from sixty three patients with pancytopenia dysplastic changes in one or more hematopoietic lineages and blast cells less than 10%.The number of CD34 + cells in the peripheral blood from patients were analyzed using four-color flow cytometry by secondary gating strategy.The sensitivity and specificity of the number of CD34 + cells were determined.Results :1.The results showed that the rate of peripheral blood CD34 + cells by FCM in MDS group(0.43 ± 0.66) % was significantly higher than that of in Non-MDS(0.02 ± 0.02) %,(P = 0.000).Of 43 MDS patients,the rate of the peripheral blood CD34 + cells was found increase in 39 cases(90.7%).Of 20 non-MDS cases,the rate of peripheral blood CD34 + cells was found increase in 2 cases(10.0%).The rate of peripheral blood CD34 + cells in MDS patients was higher than that of in non-MDS patients,(χ2 = 39.116,P = 0.00).2.The results showed that 7(16.3%) patients have abnormal peripheral blood blasts by morphology and 39(90.7%) patients have abnormal rate of peripheral blood CD34 + cells by FCM in 43 MDS patients,respectively.The difference was statistically significant(χ2 = 47.861,P = 0.000).3.When taking abnormal rate of peripheral blood CD34 + cells as the diagnositic trial,the sensitivity was 90.7%,the specificity was 90.0%,the positive predicted value was 95.1%,the negative predicted value was 81.8%.Conclusion:In low-risk MDS,abnormal increase of peripheral blood CD34 + cells was an important characteristic comparing with non-MDS.It will be helpful for the diagnosis and differential diagnosis of patients with low-risk MDS.

Dysregulated expression of signaling proteins may contribute to the pathophysiology of essential thrombocythemia (ET). This study aimed to characterize protein expression in ET and to correlate the dysregulated proteins with phenotypes and prognosis of ET patients. The expression of 128 proteins in peripheral blood neutrophils from 74 ET patients was assessed and compared with those from 29 healthy subjects and 35 polycythemia vera (PV) patients using protein pathway array. Fifteen proteins were differentially expressed between ET patients and normal controls. These dysregulated proteins were involved in the signaling pathways related with apoptosis and inflammation. Our results showed a significant overlap in protein expression between ET patients with JAK2V617F mutation and PV patients. In addition, nine proteins were associated with JAK2V617F mutation status in ET patients. Furthermore, estrogen receptor beta (ERβ) and Stat3 were independent risk factors for subsequent thrombosis during follow-up on multivariable analysis. Our study shows a broad dysregulation of signaling protein in ET patients, suggesting their roles in ET pathogenesis. The expression levels of ERβ and Stat3 could be promising predictors of subsequent thrombosis in ET patients.

Objective To investigate the characteristics of immunophenotype in patients with myelodysplastic syndrome(MDS) using the samples of peripheral blood(PB).Methods The antigen expression of myeloblasts,granulocytes and monocytes in samples of peripheral blood from 38 patients with MDS and 24 patients with Non-MDS were detected by multicolor flow cytometry with four color and CD45/SSC gating.Results The abnormal immunophenotypes were existed in myeloblasts,granulocytes and monocytes in samples of PB from MDS group.The results were showed that 16 patients(42.1%) have clustered distribution of CD34+ cells on PB myeloblasts in MDS group and 3 patients(12.5%) in Non-MDS group(P0.05).The abnormal expression of CD11b+ cells on PB myeloblasts in MDS group(26.3%) was higher than that of in Non-MDS group(2.6%)(P0.05).The numbers of SSC and the Geometric mean fluorescent intensity(G-MFI) for CD10 on granulocytes in MDS group(7.4±2.8,4.0±3.4) were significantly lower than that of in Non-MDS(9.5±2.7,8.2±6.6)(P0.01) respectively.Conclusioren The abnormal Immunophenotypes can be found in patients with MDS using PB.It may be helpful to distinguish MDS from Non-MDS.

This study was aimed to investigate the feasibility of flow cytometry-fluorescence in situ hybridization (Flow-FISH) in measuring the telomere length of bone marrow cell subgroups in patients with myelodysplastic syndrome (MDS). Seven newly diagnosed patients with low-risk MDS and seven nutritional anemia patients who were matched with age and sex, were enrolled in this study. Heparinized bone marrow were sampled. Taking Molt-4 cell line as internal control cells, leukocytes isolated from whole bone marrow were labeled with CD34-Alexa Fluork ® 647, then denatured by high temperature and hybridized with FITC-conjugated telomere probe. The DNA was counterstained and the relative telomere length (RTL) of nucleated cells and CD34(+) cells in bone marrow were measured by four-color flow cytometry. The results showed that CD34(+) cells could be gated for the measurement of RTL in both groups, undergoing the denaturation and hybridization. Primary analysis indicated that the RTL of bone marrow CD34(+) cells in MDS patients was significantly shorter than that of bone marrow nucleated cells (P = 0.001), and the RTL of both CD34(+) cells and nucleated cells in bone marrow of MDS patients were significantly shorter than that of control group (P = 0.020, 0.002). It is concluded that the application of Flow-FISH in the measurement of RTL of certain cell subgroup is feasible by labeling the cell with thermostable fluorescence-conjugated antibody, and this technique is worthy to be investigated further.