[Detection and Diagnostic Values of JAK2, CALR, MPL Gene Mutations in 208 Cases of BCR/ABL1 Negative Chronic Myeloproliferative Diseases].

OBJECTIVE: To detect the JAK2, CALR and MPL gene mutations in patients with BCR/ABL1 negative chronic myeloproliferative diseases（BCR/ABL1-CMPD）and to evaluate their diagnostic value. METHODS: Two hundred and eight cases of BCR/ABL1-CMPD comprising of 146 cases of essential thrombocythemia（ET）, 37 cases of polycythemia vera（PV）and 25 cases of primary myelofibrosis（PMF）from March 2012 to December 2015 were enrolled in the BCR/ABL1-CMPD, while 124 cases of secondary thrombocythemia and 73 cases of secondary polycythemia were enrolled in the control group. The genomic DNA and total RNA Were isolated from bone marrow or peripheral blood, then the exons 12 to 20 of JAK2 gene, exon 10 of MPL gene and exons 3 to 9 of CALR gene were analyzed by using DNA sequencing. RESULTS: among 146 ET patients, the JAK2, CALR or MPL mutations were found in: 138 cases（94.5%）including 86 cases with JAK2V617F mutation（58.9%）and 2 cases（1.4%）with exon 12 of JAK2 mutations. CALR mutations were detected in 41 cases（28.1%）, among them type 1（c.1092_1143del）in 22 cases, type 2（c.1154_1155insTTGTC）in 11 cases, and type 5（c. 1091_1142del）, type 8（c.1104_1137del）, type 41(c.1107_1137del）, type 42(c.1125_1125del）in one case respectively. In addition, 4 cases were detected withother mutations of the CALR gene（c.1107_1115del, c.1111_1144 del, c.1101 A>C, c.1112_1117del）. Moreover, 9 cases harbored MPL mutations（6.2%）. Secondly, 31 patients were detected with JAK2V617F mutation（83.8%）in 37 cases of PV, and JAK2 exon 12 mutations were found in 2 cases（5.4%）. Besides, CALR mutations were detected in 2 cases（5.4%）, including 1 case of type I, the other of novel mutation of CALR. Thirdly, 19 in 25 cases of PMF were detected with JAK2V617F mutation（76%）, 2 cases with CALR mutations（8%）. 4 patients（16%）, JAK2, CALR or MPL mutations were not detected, but among them 3 cases were found harboring other genetic abnormalities. Fourthly, no mutations of JAK2, MPL and CALR genes were detected in 124 patients with secondary thrombocytosis and 73 cases with secondary polycythemia. CONCLUSION: Combined detection of JAK2, CALR and MPL gene mutations can cover the vast majority of patients with BCR/ABL1-negative myeloproliferative neoplasms. For higher frequencies of the mutations of CALR in ET patients, CALR mutation can be used as a new diagnostic marker in ET patients with JAK2 and MPL wild type.

Non-malignant T-cells lacking multiple pan-T markers can be found in lymph nodes.

In order to observe and ascertain the properties of a sub-group of T cells in the lymph node (LN) from seven patients who did not suffer from T cell lymphoproliferative disorders (T-LPDs), the expression levels of several pan-T markers were evaluated by multiparameter flow cytometry (FC) and the clonality of these T-cells was evaluated by both FC analysis and PCR assessment. It turned out that multiple pan-T-cell markers such as CD2, CD5 and CD7 were found to be lost in these T cells. The majority of them were positive for TCRαß, only a minority of them being positive for TCRγδ. A subset of these T-cells were positive for CD4 or CD8 or dual-negative for CD4 and CD8. Oligoclonality was detected in one case by FC, while clonal TCR rearrangement was detected in three cases. Absence of multiple pan-T-cell markers could be found in benign T cells in LNs.

[Application of Cytogenetic Test for Diagnosis of Bone Marrow Involvement in Patients with Non-Hodgkin's Lymphoma].

OBJECTIVE: To investigate the role of cytogenetic analysis in the detection of bone marrow (BM) involvement in patients with non-Hodgkin's lymphoma (NHL). METHODS: The bone marrow samples of 74 patients with NHL were detection by using morphology, cytogenetic test, flow cytometry and molecular biological assay. The detected results of morphology, cytogenetic test, flow cytometry and molecular biological assay alone and thier combined detection were compared, the detective rate and consistencies of the 4 methods were analyzed. RESULTS: The detection rates of BM involvement by using morphology, cytogenetic, flow cytometry, and molecular biological assays were 21.6%, 17.6%, 23.0% and 33.8% respectively. The detective rate was enhanced to 44.6% by combining the 4 methods. Cytogenetic test showed the result consistent with the other methods. CONCLUSION: Although cytogenetic test shows a lower detective rate than the other methods, but in some patients the cytogenetic test can detect the abnormality of bone marrow which can not be detected by other methods alone, the combination test of 4 detection methods can enhance the detectable rate of BM involvement.

The application of CD73 in minimal residual disease monitoring using flow cytometry in B-cell acute lymphoblastic leukemia.

The expression of CD73 by flow cytometry (FC) in bone marrow (BM) specimens of B-cell acute lymphoblastic leukemia (B-ALL) with or without minimal residual disease (MRD) was studied, and its advantages were evaluated using the MRD assay. This study also detected the expression profile of CD73 in hematogones and mature B cells in BM specimens of 18 healthy donors. Results showed that the mean value of CD73 expression in MRD-positive B cells was 6-fold greater than that in the MRD negative ones. Also, 41.82% MRD-positive B-ALL cases expressed high CD73 and the sensitivity of CD73-based MRD detection reached 10(-4). Since the expression of CD73 increases with the maturation of normal B cells, it is better to mix it with CD34, CD10 and CD20 in one tube to prevent the disturbance of mature B cells. CD73 is recommended as an optional MRD marker for B-ALL patients by using FC.

[Significance of CD37 expression in malignant B cells].

The aim of this study was to clarify the clinical significance of CD37 expression in B cells from B acute lymphoblastic leukemia (B-ALL) and B cell non-Hodgkin's lymphoma (B-NHL). The expression level of CD37 on B cells from bone marrow samples of normal controls (n = 19), B-ALL patients [including untreated cases (n = 5) and cases with minimal residual disease (MRD, n = 15)] and B-NHL patients (n = 25) whose bone marrow was involved by lymphoma cells, was detected by multiple parameter flow cytometry. The results indicated that the B cells from both untreated cases and cases with MRD lowly expressed CD37 (1.04 ± 0.24 and 1.50 ± 0.89), the normal precursor B cells (control cases) also lowly expressed CD37 (1.64 ± 0.52). There was no difference of CD37 expression level between 3 groups of cases(P > 0.05). Meanwhile the normal mature B cells and B-NHL cells highly expressed CD37 (14.23 ± 7.84 and 14.53 ± 10.93), but there was no difference of CD37 expression between them (P > 0.05). The comparison of CD37 expression level in normal B cells of development stages showed that the progenitor B cells lowly expressed CD37 (0.88 ± 0.17), the CD37 expression of precursor B cells was enhanced (2.44 ± 0.69), while the CD37 expression level of mature B cells was highest. It is concluded that the low expression of CD37 is not the characteristic of B- ALL cells. The expression level of CD37 increases gradually during the mature process of B cells, i.e, the expression level of CD37 does not associate with benignity or malignancy of B cells.

[Cyclosporine A based therapy for myelodysplastic syndrome].

To determine the efficacy and tolerance to cyclosporine A (CsA) based therapy in patients with myelodysplastic syndrome (MDS), 16 patients with MDS consisting of 10 refractory anemia (RA) and 6 refractory anemia with accessory blasts less than 10% (RAEB-1) were analyzed. Five patients had hypocellular bone marrows and 11 patients had normocellular or hypercellular marrows. The dose of CsA was 2.5-5.5 mg/(kg.d) for 2 weeks to 2 years (mean 8 months). Two out of 16 patients were treated with CsA alone, 14 patients were treated with CsA, recombinant human erythropoietin, androgens, 1, 25 dihydroxy vitamin D(3) or two or three of them combination with CsA. Treatment responses were classified according to the International Working Group (IWG) criteria as complete remission (CR), partial remission (PR), hematological improvement (HI) and no response (NR). Patients who obtained CR, PR or HI were defined as responders. The results showed that HI was observed in 12 patients, PR in 2 patients and NR in 2 patients. Total response rate was 87.5%. Response rates shown in neutrophil lineage, platelet and erythroid lineage were 83.3%, 66.7% and 60%, respectively; their shortest time required to obtain some hematologic improvement after initiation of CsA therapy was 2 weeks, 1 month and 1 month, respectively. Of 13 patients being transfusion-dependent before treatment, 3 patients did not need transfusion any more and 5 showed the reduced transfusion requirements after CsA therapy. In 10 patients with RA, 9 responded to CsA. Of 6 patients with RAEB, 1 patient had no response and died of RAEB-t and 5 patients had transient responses. One of the latter transformed to CMML and two relapsed. The total response rate decreased to 50% in the patients with CsA therapy lasting more than 3 months at the end of following-up. The adverse effects included hirsutism, hyperplastic gingiva, reversible hepatic and renal dysfunction. In conclusion, the usefulness of CsA based therapy for MDS-RA and RAEB-1 with any marrow cellularity is useful, the CsA dose of 3-5 mg/(kg.d) is safe and efficacious.