[The Expression Level and Diagnostic Value of Serum Free Light Chain in B-Cell Non-Hodgkin Lymphoma].

OBJECTIVE: To investigate the expression level and the diagnostic value of serum free light chain in B-cell non-Hodgkin's lymphoma (B-NHL). METHODS: We retrospectively analyzed the results of serum free light chain (sFLC) of 394 newly treated B-NHL patients in our hospital from January 2014 to December 2021 and compared the secretion levels of sFLC among different subtypes of B-NHL. The value of sFLC secretion levels in the diagnosis of WM was evaluated using ROC. RESULTS: Increased proportion of sFLC, abnormal ratio of sFLC (κ / λ) and the secretion levels of sFLC (κ+λ) were different in different B-NHL subtypes, Waldenstrom's macroglobulinemia (WM) had the highest proportion of elevated sFLC(82.68%) and abnormal sFLC(κ/ λ)(87.0%), the proportion of FL(18.0%) and DLBCL patients(12.8%) with elevated sFLC was lower (P<0.05). The expression levels of sFLC can helpful in the diagnosis of WM (AUC=0.874，P<0.001， 95% CI: 0.779-0.970). At the same time, higher sFLC levels and sFLC cloning patterns predicted the possibility of bone marrow infiltration of lymphoma. CONCLUSION: The serum free light chains is common in patients with B-NHL. The elevated level and type of free light chain are associated with the type of lymphoma, and the patients with bone marrow infiltration have higher sFLC（κ+ λ） expression level.

[Clinical Characteristics and Bone Marrow Histopathology Features in Essential Thrombocythaemia Patients with Different Gene Mutation in China].

OBJECTIVE: To investigate the clinical characteristics, laboratorial and bone marrow pathological features of primary thrombocytopenia (ET) patients with different mutations of CALR, JAK2 and MPL genes. METHODS: The chinical data of 120 cases of ET in Jiangsu provincial people's hospital/ The First Affiliated Hospital of Nanjing Medical University from January 2015 to December 2017 were collected and analyzed, including 76 cases with JAK2 gene mutation, 40 cases with CALR gene mutation, 2 cases with MPL gene mutations, 2 cases without gene mutation. RESULTS: Among the ET patients, compared with the JAK2 gene mutation, CALR gene mutation showed statistically significant deareament of white blood cells and hemoglobin (P=0.001, P=0.01) and the male platelets in CALR group showed significant increament (P=0.04). Fourthermore, the average number of megakaryocytes and its cluster numbers in each hight power field of vision showed statistically significant decreament in CALR group as compared with JAK2 group (P=0.001, P=0.001), and thrombotic events in CALR group were signicantly lower than those in JAK2 group (7.5% vs 18.4%) (P=0.03). CONCLUSION: Mutations of CALR, JAK2 have different clinical characteristics and blood pathological changes of Chinese ET patients, and their clinical significance is worth to explore.

[Analysis of Clinical and Laboratory Features of Patients with Acute Myeloid Leukemia(AML)-M1/M2 with Cuplike Nuclei Morphology].

OBJECTIVE: To analyze the clinical and laboratory features of acute myeloid leukemia (AML) with cuplike nuclei morphology. METHODS: One hundred and seventy patients diagnosed with AML (M1andM2) between December 2009 and December 2016 were included in the study. Bone marrow smears were prepared for morphologic alanalysis, the immunophenotype was analyzed by flow cytometry and the RHG-banding was for conventional cytogenetic assay (CCA) ,gene mutation was detected by sequencing. RESULTS: Among the 170 AMLpatients, 67 were diagnosed as M1 and 103 patients was diagnosed as M2, 43 patients(25.3%) defined as cuplike nuclei-positive, among them 38patients (88.4%) were M1 while only 5 patients (11.6%) were with M2(P<0.01). No significant value about sex(P> 0.05) between cuplike nuclei-positive and -negative group, while older patients were found in cuplike nuclei-positive group (P<0.05). Higher peroxydas (POX) ratio (P<0.05) and integration (P<0.05) were found in cuplike nuclei- positive group. Furthermore, the patients with cuplike nuclei-positive lack the expressions of CD34 (P<0.01) and HLA-DR(P<0.01) while no other immunophenotype markers were found. Among the 152 patients (89.4%) for genetic analysis ,83.8% karyotype of the cuplike nuclei-positive group were normal while only 54.8 of negative group was normal by CCA. Molecular biology analysis showed that the patients in cuplike nuclei-positive group have significantly highe rNMP1 (P<0.01) and FLT3(P<0.01) mutations as compared with the negative group. Furthermore, the relationship of the ratio o fcuplike nuclei and the type of gene mutations were investigated, and no significant associations were found. However, it was found that the patients with FLT3 mutation displayed more biological nuclear invagination than the patients with NPM1 mutations (P<0/01). CONCLUSION: AML patients with positive cuplike nuclei have characteristic morphological changes, typical immunophenotype with HLA-DR- and CD34-, normal karyotype accompanied by NPM1 and FLT3 mutations.

[Distribution of Type 9 T Helper Cells and Expression of Transcriptional Factors in Acute Myeloid Leukemia].

OBJECTIVE: To investigate the distribution of T helper (Th9) cells and its relationship with clinical characteristics of patients with acute myeloid leukemia (AML), and to analyze the activating levels of different transcriptional factors in Th9 cells. METHODS: The peripheral blood specimens of 102 AML patients and 83 healthy persons as controls were collected, then the T cells of peripheral blood in AML patients and controls were isolated by using CD3 magnetic beads, the mRNA expression of IL-9 was detected by real-time quantitative PCR, the Th9(CD4+IL-9+) cell levels in diffrent stages and activating level of Th9 coexpression with IL-9 were detected by flow cytometry. RESULTS: The mRNA expression of IL-9 in peripheral blood of AML M2 and M3 patients was significantly higher than that in control groups (P<0.01), at same time the CD4+IL-9+ cell rate was significantly higher than that in control group also(P<0.01). The results of dynamically monitoring the distribution of Th9 cells in AML-M2 and M3 patients showed that the Th9 cell rate and the mRNA expression of IL-9 in newly diagnosed M2 and M3, and relapsed M2 groups were significantly higher than those of M2 and M3 in remission (P<0.01); the detection results of IL-9- co-expression with transcriptional factors (SMAD3+, IRF-1+ and IRF-4+) indicated that the percantage of Th9 pSMAD3+ cells in peripheral blood of newly diagnosed and relapsed M2 and M3 patients was significantly higher than that in M2 and M3 patients in remission (P<0.01); on the contrary, the percentage of Th9 IRF-1+ cells in peripheral blood of M2 and M3 patients in remission was significantly higher than that in newly diagnosed M2 and M3 patients (P<0.01). CONCLUSION: The distribution of T helper cells in peripheral blood of AML-M2 and M3 patients significantly increases, moreover, correlates with disease status. The prediction of Th9 cell functions should be performed in combination with it transcriptional factors which have inmportant significance for microenvironment of tumors in AML patients.

Molecular mechanisms of synergistic induction of apoptosis by the combination therapy with hyperthermia and cisplatin in prostate cancer cells.

Prostate Cancer has become the second leading cause of male cancer-related incidence and mortality in United States. Hyperthermia (HT) is known to serve as a powerful tool in treatment of prostate cancer in clinical. The combination treatment with HT and cisplatin has a synergistic effect to inhibit prostate cancer progression and demonstrates better clinical effectiveness than HT or chemotherapy alone. But molecular mechanisms of this phenomenon have not been illuminated clearly. In this study, we used MTS assay to examine cell viabilities of PC-3, LNCaP, DU-145 and RM-1 cells after treated by HT and cisplatin. Then colony formation of PC-3 and DU-145 cells after treated with HT and cisplatin were photographed. To investigate whether the combination therapy would enhance apoptosis of PC-3 and DU-145 cells, we used Western blot analysis to detect expression level of proteins on apoptosis-regulated signaling pathway in PC-3 and DU-145 cells. Our results showed that the combination treatment decreased cell viabilities and colony formation of prostate cancer cells in a dose-dependent manner and this combination therapy enhanced apoptosis of PC-3 and DU-145 cells via activation of Caspase-3 and cleavage of PARP. We also found that the combination therapy could down-regulate the anti-apoptotic Bcl-2 and IAP family proteins. At last, the combination therapy activated AMPKα-JNK signaling pathway and inhibited Akt-mTOR-p70s6k signaling pathway to promote apoptosis of PC-3 and DU-145 cells. In conclusion, this study clearly elucidated how the combination therapy with HT and cisplatin promoted apoptosis of prostate cancer cells in synergy.

[Immunophenotypic Analysis of Patients with CD56⁺ Acute Myeloid Leukemia].

OBJECTIVE: To explore the immunophenotype characteristics of newly diagnosed patients with CD56⁺ acute myeloid leukemia (AML). METHODS: Combining with cytomorphology, four-color flow cytometry was used to analyze the immunophenotype of 342 AML patients with CD56⁺ or CD56⁻. RESULTS: In 342 AML patients, the CD56⁺ expression was found in 83 AML patients who accounted for 24.27% and included 10 cases of M1, 45 cases of M2, 5 cases of M3, 6 cases of M6 and 17 cases of M5. The statistical analysis showed that there was statistical difference between CD56⁺ and CD11b⁺ patients (P < 0.05), but there was no statistical difference between CD56⁺ and HLA-DR, CD34, CD38, CD13, CD33, CD15, CD117, CD14, CD64, CD2, CD7, CD5, CD3, CD4, CD10, CD19, CD20, CD22 (P > 0.05). CONCLUSION: AML with only CD56 positive always has poor prognosis, thus the prognosis of patients with CD56⁺ AML accompanied by other antigens still needs more research.

[Expression characteristics of CD200 in acute myeloid leukemia and its clinical significance].

This study was aimed to investigate the relationship between expression of CD200 antigen and clinical characteristics in AML patients and to analyse the value of CD200 in evaluation of AML prognosis. The CD200 and immunophenotypes were detected by flow cytometry, the chromosome karyotypes were determined by R banding, the FISH was used to measure the AML1/ETO, PML/RARa and inv(16), and PCR technique was used to detect the fusion genes AML1/ETO and PML/RARα. The results showed that the positive rate of CD200 antigen expression in 54 patients was 57.4% (31/54), the CD200 antigen expression between sex and age of patients was no significant different (P > 0.05). There was significant difference of CD200 expression between CD34 and CD117 (P < 0.05), but the difference of CD200 expression in chromosome karyotypes was no significant difference(P > 0.05). Moreover, there was significant difference of CD200 expression in CD34 and CD117 of CBF positive AML patients (P < 0.05). It is concluded that the CD200 antigen expression in AML may associate with a poor prognosis of patients.

[Mutation and expression of PAX5 gene in adult acute lymphoblastic leukemia].

PAX5 is an important transcription factor of paired-box(PAX) family. The aim of this study was to investigate the mutations and expression of PAX5 and its clinical significance in adult patients with acute lymphoblastic leukemia (ALL). Reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR were performed to detect the deletions of PAX5 and point mutations of PAX5 exon 2-10 in 101 cases of adult ALL and were confirmed by cloning and sequencing. In addition, quantitative PCR (qPCR) was performed to evaluate the expression of PAX5. Furthermore, the correlations of mutations and expression of PAX5 with clinical parameters were analyzed, and the prognostic significance was evaluated as well. The results showed that PAX5 mutations were observed in 8 of 101 (7.9%) patients with B-ALL. A total of 9 types of mutations were detected, including 4 types of deletions, 4 types of point mutations and 1 insertion mutation; percentage of patients with age ≥ 50 years was higher in PAX5 mutation group than in wide-type group (62.5% vs 21.5%,P = 0.031) . The statistical differences were observed in B-cell subtype, initial platelet count and immunophenotypes between high and low expression of PAX5 (P < 0.05) . In addition, patients with high expression of PAX5 had higher first complete remission rate (86.7% vs 62.5%, P = 0.030) and 6-month overall survival rate (75.0% vs 50.0%, P = 0.034) compared with patients with low expression of PAX5. It is concluded that deletion/insertion/point mutations and aberrant expression of PAX5 can be observed in adult patients with B-ALL. Mutations and aberrant expression of PAX5 correlated with clinical parameters and have important clinical significance.

[Mutation and expression of LEF1 in adult acute lymphocytic leukemia and their clinical significance].

Lymphoid enhancer factor 1 (LEF1) is a key transcription factor in Wingless-type (Wnt) pathway. The present study was aimed to explore the genetic mutation and expression of LEF1, and their clinical significance in adult patients with acute lymphocytic leukemia (ALL). Genomic DNA was amplified and sequenced to detect the mutation of LEF1 in 131 newly diagnosed adult patients with ALL. Quantitative PCR (qPCR) was performed to detect the expression of LEF1. Moreover, the correlations between mutations and expression of LEF1 with clinical characteristics were analyzed. The results showed that the frequency of LEF1 mutation in adult ALL was 3.1% (4/131) and all of them were point mutations located in exon 2 and 3; the median white blood cell count and median percentage of blasts at diagnosis were significantly higher in LEF1 high expression group than in low expression group (70.6 × 109/L vs 26.2 × 109/L)(P = 0.010); (81.0% vs 57.0%) (P = 0.014); in addition, the percentage of patients with Philadelphia chromosome positive and patients in high-risk group significantly increased in LEF1 high expression group compared with that in low expression group (66.7% vs 36.5%) (P = 0.038); (79.2% vs 56.2%) (P = 0.044). It is concluded that high expression of LEF1 may play an important role on development of adult ALL.

[Characteristics of NOTCH1 mutation in adult T-cell acute lymphoblastic leukemia].

This study was aimed to investigate the characteristics and clinical significance of NOTCH1 mutation in adult T-cell acute lymphoblastic leukemia (T-ALL). Exon 26/N-terminal region of the heterodimerization domain (HD-N) , exon 27/ C-terminal region of the heterodimerization domain (HD-C) , exon 28 and exon 34/ proline-glutamic acid-serine-threonine (PEST) domain of the NOTCH1 gene were amplified, cloned and sequenced in 42 adult patients with T-ALL to identify the frequency, position and type of NOTCH1 mutation, their correlations with laboratorial and clinical parameters, as well as their relevant prognostic significance. The results showed that the frequency of NOTCH1 mutation in this cohort of adult patients was 66.7% (28/42); A total of 45 types of NOTCH1 mutations were identified in present study, most of them were in HD-N (48.9%, 22/45) and PEST (40.0%, 18/45) domains. Mutation in amino acid 1575 (L1575P) was the top one type of mutation in HD-N (25.0%, 7/28), and amino acid 2443 was the most common mutation position in PEST domain (14.3%, 4/28). In newly diagnosed patients, white blood cell (WBC) >10×10(9)/L and blasts in bone marrow > 50% were predominant in patients with NOTCH1 mutation (91.7% vs 54.5%, P = 0.021 and 95.8% vs 57.1%, P = 0.006 respectively). Immunophenotyping analysis indicated that patients with CD10 positive were more in NOTCH1 mutation group than wild-type group (51.9% vs 0%, P = 0.006), whereas patients with CD15 and CD11b positive were less in NOTCH1 mutation group (5.3% vs 42.9%, P = 0.047 and 0% vs 57.1%, P = 0.002 respectively). It is concluded that NOTCH1 mutation in adult T-ALL has different characteristics and clinical significance from pediatric patients, and the difference between Chinese patients and patients in Western countries is also indicated.

[Genetic characteristics and its prognostic significance for 217 adult patients with acute lymphoblastic leukemia].

OBJECTIVE: To evaluate cyto- and molecular genetic characteristics of adult patients with acute lymphoblastic leukemia (ALL) and its prognostic significance. METHODS: Two hundred and seventeen adult patients with ALL were analyzed for cyto- and molecular genetic characteristics with combined conventional cytogenetics, fluorescence in situ hybridization (FISH), real-time quantitative PCR (qPCR) and nested PCR. Significance of genetic findings for prognosis was evaluated. RESULTS: t(9;22)(q34;q11)/BCR-ABL has been the most frequent abnormality found in the cohort (56.3%). And 22.4% of cases with BCR-ABL detected by FISH was negative by cytogenetic analysis. Ratio of patients in high-risk group increased with age; Patients with B-ALL had a higher risk group than the average-risk group (98.40% vs. 65.70%, P=0.000). The overall survival (OS) rates at 3-month (67.30% vs. 85.10%, P=0.042), 6-month (55.1% vs. 80.4%, P=0.008), 12-month (34.0% vs. 59.1%, P=0.017) and 24-month (13.0% vs. 36.6%, P=0.010) were lower in high-risk group than in average-risk group, with medium OS time (11 months, 95% CI 8.0-13.9) being significantly shorter compared with the average-risk group (19 months, 95%CI 10.8-27.1). CONCLUSION: Adult patients with ALL have unique cyto- and molecular genetic characteristics, which has important value for prognosis and guiding treatment. Moreover, combined cytogenetic and molecular genetic techniques can precisely define sub-groups of ALL patients.

The effect of phloretin on human γδ T cells killing colon cancer SW-1116 cells.

OBJECTIVE: To explore the effect and mechanism of Phloretin on human γδ T cells killing colon cancer SW-1116 cells. METHODS: γδ T cells were amplified in vitro from human peripheral blood mononuclear cells through isopentenyl pyrophosphate method (IPP). After cocultured different concentrations of Phloretin with γδ T cells or SW-1116 cells for 48h respectively, MTT assay was used to test the growth curve of these two cells; Flow cytometry to test the expression of Granzyme B (GraB), perforin (PFP), CD107a and IFN-γ of γδ T cells; Lactate dehydrogenase (LDH) release assay to test the cytotoxic activity of the γδ T cells on SW-1116 cells; and Western blot to test the Wnt3a expression of the γδ T cells. RESULTS: After cultured with IPP for ten days, the percentage of γδ T cells increased from 3.31±3.00% to 78.40±10.30%. Compared to the control group, when the concentration of Phloretin increased from 2.35µg/ml to 18.75µg/ml, it could significantly proliferate the γδ T cell growth (P<0.05) and inhibit the growth of SW-1116 cells in dose-response, and the expression of GraB, PFP, CD107a and Wnt3a significantly increased (P<0.05). Significant positive relationships were observed among CD107a and PFP, GraB, cytotoxicity (P<0.05). The percentage of IFN-γ producing γδ T cells treated with Phloretin was significantly higher than control group. CONCLUSION: Phloretin can enhance the killing effect of γδ T cells on SW-1116 cells; the mechanism may be that Phloretin could proliferate the γδ T cell growth, increase the expression of PFP and GraB, activate the Wnt signaling pathway, and produce higher level of IFN-γ. Indeed CD107a expression probably correlates quite well with antitumor activity.

[Detection and clinical features of MLL gene rearrangement in adult patients with acute leukemia].

This study was purposed to investigate the incidence of mixed lineage leukemia (MLL) gene rearrangement and partner gene types as well as the clinical features and prognosis of acute leukemia (AL) with this rearrangement through detection in adult AL using combination of 3 techniques, and to evaluate the clinical value of this combination detection. The MLL gene rearrangement in 183 cases of adult AL was detected by combination of conventional cytogenetics, split signal FISH and multiplex nested PCR. The results showed that the incidence of MLL rearrangements in adult patients with AL was low (8.2%), and MLL-AF4 fusion gene was most common and predominant in acute lymphoblastic leukemia (ALL), while the MLL-AF6 and MLL-AF9 were most frequent in acute myeloid leukemia (AML). Extramedullary involvements were found in 40% of MLL-rearranged AL patients, and 33.3% of patients with MLL-rearranged AL reached to complete remission within 30 days during induction chemotherapy. In addition, in this cohort of MLL-rearranged adult AL patients, the 3-month relapse rate and 6-month overall survival rate were 50.0% and 50.0% respectively. It is concluded that the rate of missed diagnosis of CC technique for patients with MLL-rearranged AL reached to 60% in this study, while the combination of CC, FISH and multiplex nested PCR has been confirmed to have important significance for evaluating prognosis and conducting clinical therapy of patients with MLL-rearranged AL.

Protection of ghrelin postconditioning on hypoxia/reoxygenation in gastric epithelial cells.

AIM: To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells. METHODS: The model of H/R injury was established in gastric epithelial cell line (GES-1) human gastric epithelial cells. Cells were divided into seven groups: normal control group (N); H/R postconditioning group; DMSO postconditioning group (DM); ghrelin postconditioning group (GH); D-Lys3-GHRP-6 + ghrelin postconditioning group (D + GH); capsazepine + ghrelin postconditioning group (C + GH); and LY294002 + ghrelin postconditioning group (L + GH). 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect GES-1 cell viability. Hoechst 33258 fluorochrome staining and flow cytometry were conducted to determine apoptosis of GES-1 cells. Spectrophotometry was performed to determine release of lactate dehydrogenate (LDH). Protein expression of Bcl-2, Bax, Akt, and glycogen synthase kinase (GSK)-3ß was determined by western blotting. Expression of vanilloid receptor subtype 1 (VR1), Akt and GSK-3ß was observed by immunocytochemistry. RESULTS: Compared with the H/R group, cell viability of the GH group was significantly increased in a dose-dependent manner (55.9% ± 10.0% vs 69.6% ± 9.6%, 71.9% ± 17.4%, and 76.3% ± 13.3%). Compared with the H/R group, the percentage of apoptotic cells in the GH group significantly decreased (12.38% ± 1.51% vs 6.88% ± 0.87%). Compared with the GH group, the percentage of apoptotic cells in the D + GH group, C + GH group and L + GH groups significantly increased (11.70% ± 0.88%, 11.93% ± 0.96%, 10.20% ± 1.05% vs 6.88% ± 0.87%). There were no significant differences in the percentage of apoptotic cells between the H/R and DM groups (12.38% ± 1.51% vs13.00% ± 1.13%). There was a significant decrease in LDH release following ghrelin postconditioning compared with the H/R group (561.58 ± 64.01 U/L vs 1062.45 ± 105.29 U/L). There was a significant increase in LDH release in the D + GH, C + GH and L + GH groups compared with the GH group (816.89 ± 94.87 U/L, 870.95 ± 64.06 U/L, 838.62 ± 118.45 U/L vs 561.58 ± 64.01 U/L). There were no significant differences in LDH release between the H/R and DM groups (1062.45 ± 105.29 U/L vs 1017.65 ± 68.90 U/L). Compared with the H/R group, expression of Bcl-2 and Akt increased in the GH group, whereas expression of Bax and GSK-3ß decreased. Compared with the GH group, expression of Bcl-2 decreased and Bax increased in the D + GH, C + GH and L + GH groups, and Akt decreased and GSK-3ß increased in the L + GH group. The H/R group also upregulated expression of VR1 and GSK-3ß and downregulated Akt. The number of VR1-positive and Akt-positive cells in the GH group significantly increased, whereas the number of GSK-3ß-positive cells significantly decreased. These effects of ghrelin were reversed by capsazepine and LY294002. CONCLUSION: Ghrelin postconditioning protected against H/R-induced injury in human gastric epithelial cells, which indicated that this protection might be associated with GHS-R, VR1 and the PI3K/Akt signaling pathway.

[Expression characteristics of isoforms of Ikaros and Helios in patients with leukemia and their mechanism].

This study was aimed to investigate the expression characteristics of two transcriptional factors in Ikaros family, Ikaros and Helios isoforms and their mechanism, as well as their correlation with clinical parameters, which play important roles in transcriptional regulation of hematopoiesis. Expression of Ikaros and Helios isoforms in a total of 163 patients with leukemia and correlations between Ikaros and Helios isoforms were analyzed by PCR. The results showed that different expression patters of Ikaros and Helios isoforms existed in leukemia patients, that is, Ikaros isoform (Ik-6) was predominantly expressed in acute lymphoblastic leukemia (ALL) with BCR/ABL fusion gene, while Helios isoform (He-i) was overexpressed in T-cell ALL patients. The results of cloning and sequencing demonstrated that the isoforms of Ikaros and Helios had different genetic alterations. The statistical correlation between these two isoforms not was found in this study, although interaction between Ikaros and Helios has been reported. It is concluded that although Ikaros and Helios belong to the same family with similar structure of zinc fingers, their isoforms have different expression profile, specific genetic alterations, and different clinical relevance in patients with leukemia. The connection and interaction between Ik-6 and He-i needs further research.

[Study of immunoglobulin and T-cell receptor gene rearrangements in patients with non-Hodgkin's lymphoma].

OBJECTIVE: To investigate immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements in bone marrow or peripheral blood of patients with non-Hodgkin's lymphoma (NHL), and to explore the potential clinical significance. METHODS: The Ig/TCR gene rearrangements in bone marrow or peripheral blood of 139 NHL patients were analyzed by using BIOMED-2 multiple primers system and Multiplex PCR assay. RESULTS: Ig clonality was detected in 85.4% (70/82) of chronic lymphocytic leukemia (CLL), including 46.3% (38/82) IgH rearrangement, 62.2% (51/82) IgK rearrangement and 1.2% (1/82) IgL rearrangement, and in 39.4% (13/33) of other categories of B-lineage NHL (B-NHL), including 33.3% (11/33) IgH and 39.4% (13/33) IgK rearrangements. TCR clonality was detected in 50.0% (12/24) of all definite T-lineage NHL (T-NHL), including 8.3% (2/24) TCRB and 45.8% (11/24) TCRG, no TCRD was detected. The detection rate of gene rearrangements of NHL diversed in different clinical stages \[36.4% in early stage (Ann Arbor stage I and II) and 45.6% in late stage (III and IV)\] and in different degrees of malignancy (40.0% indolent NHL and 45.6% aggressive NHL), but no obvious statistical significance was obtained (P > 0.05). The detection rate of bone marrow invasions of NHL (except CLL) with examinations of bone marrow smear under the microscope was 12.3% (7/57), much lower than the clonality testing (43.9%, 25/57) (P < 0.05). Sensitivity test showed that the sensitivity of malignant clonality testing by multiplex PCR was 3.12% - 6.25%. CONCLUSIONS: The detection rate of gene rearrangements diverses in different clinical stages and degrees of malignancy of NHL, but the correlation has not been proved in this research. The sensitivity of multiplex PCR-based clonality testing is enhanced with the combination of BIOMED-2 primers system. It is more sensitive than the morphological examinations of bone marrow smear in detecting bone marrow invasions, and may provide a powerful strategy in the routine diagnosis and assessment after treatment.

Heterogeneous leukemic clones identified by NPM1 mutation analysis in patient with acute monocytic leukemia.

NPM1 mutation is the most common molecular abnormality in patients with acute myeloid leukemia (AML), especially normal karyotype AML (NK-AML), and is associated with a favorable prognosis in the absence of concomitant FLT3-ITD. Like other molecular abnormalities such as FLT3-ITD, C/EBPα and c-Kit mutation, NPM1 mutation normally presents as a recurrent molecular abnormality. The NPM1 mutation is generally used as a molecular marker in the prognosis evaluation of a patient with AML. Here, we report a different case. He was first diagnosed with NPM1 mutation-positive acute monocytic leukemia. However, he achieved no remission, but the NPM1 mutation dramatically became negative after induction chemotherapy. Finally, he achieved complete remission after salvage chemotherapy and the NPM1 mutation was still negative. To our knowledge, this is a rare case according to the worldwide published literature.

[Clinical value of interphase fluorescent in situ hybridization in diagnosis of core-binding factor acute myelocytic leukemia].

The purpose of this study was to evaluate the clinical value of interphase fluorescence in situ hybridization (I-FISH) in diagnosis of core-binding factor acute myelocytic leukemia (CBF AML). The cytogenetic characteristics in leukemia cells from 82 cases of AML-M(2) and 43 cases of AML-M(4)/M(5) were detected by using I-FISH with AML1-ETO double color double fusion probe and double color break point isolated gene probe CBFß-MYH11, and the detected results were compared with results detected by conventional cytogenetic R banding technique (CC). The results indicated that AML1-ETO fusion gene was detected in 30.5% cases (25/82) by FISH, and t(8;21)(q22;q22) karyotypic aberrations was found in 28.0% cases (23/82) by CC method. Among 25 FISH positive cases, typical FISH positive signal pattern (1R1G2F) was displayed in 22 cases and atypical signal pattern (1R2G1F and 2R1G2F) was found in the other 3 cases. Among all 43 AML-M(4)/M(5) cases, the CBFß-MYH11 fusion gene was detected in 23.3% cases (10/43) by FISH, which sensitivity was significant higher than that by CC method (2/43) (p < 0.05). It is concluded that some insufficiency of CC technique can be compensated by FISH, and combination of I-FISH with CC technique play a crucial role in diagnosis of CBF AML and in monitoring of minimal residual disease.

Differential expression of Bcl-2 and Bax during gastric ischemia-reperfusion of rats.

AIM: To investigate expression of Bcl-2 and Bax in gastric ischemia-reperfusion (GI-R) and involvement of extracellular signal-regulated kinase (ERK) 1/2 activation. METHODS: The GI-R model was established by ligature of the celiac artery for 30 min and reperfusion in Sprague-Dawley rats. Rats were assigned to groups in accordance with their evaluation period: control, 0, 0.5, 1, 3, 6, 24, 48, and 72 h. Expression and distribution of Bcl-2 and Bax proteins were analyzed by immunohistochemistry and western blotting in gastric tissue samples after sacrifice. RESULTS: Compared with controls, the percentage of positive cells and protein levels of Bcl-2 decreased in the early phases of reperfusion, reached its minimum at 1 h (P < 0.05); it then increased, reaching its peak at 24 h of reperfusion (P < 0.05). The pattern of Bax expression was opposite to that of Bcl-2. Bax expression increased after reperfusion, with its peak at 1 h of reperfusion (P < 0.05), and then it decreased gradually to a minimum at 24 h after reperfusion (P < 0.05). On the other hand, inhibition of activation of ERK1/2 induced by PD98059, a specific upstream MEK inhibitor, had significant effects on Bcl-2 and Bax in GI-R. Compared with GI-R treatment only at 3 h of reperfusion, PD98059 reduced the number of Bcl-2 positive cells (0.58% of R3h group, P < 0.05) and Bcl-2 protein level (74% of R3h group, P < 0.05) but increased the number of Bax-positive cells (1.33-fold vs R3h group, P < 0.05) and Bax protein level (1.35-fold of R3h group, P < 0.05). CONCLUSION: These results indicated that the Bcl-2 and Bax played a pivotal role in the gastric mucosal I-R injury and repair by activation of ERK1/2.

Identification of the STAT5B-RARα fusion transcript in an acute promyelocytic leukemia patient without FLT3, NPM1, c-Kit and C/EBPα mutation.

T(15;17) is the most common chromosomal aberration in patients with acute promyelocytic leukemia (APL), leading to the formation of PML-RARα fusion gene. In a small subset of patients with APL, the RARα gene is fused with different partners. Here, we report a rare APL case with STAT5B-RARα fusion transcript. Cytomorphologic and immunophenotypic analyses showed typical features of APL. However, cytogenetic analysis showed normal karyotype, and interphase fluorescence in situ hybridization (FISH) showed PML-RARα negative. Quantitative RT-PCR also showed PML-RARα negative but STAT5B-RARα positive and sequencing analysis confirmed the result. Molecular markers including FLT3, NPM1, c-Kit and C/EBPα mutation were all negative. To our knowledge, this is the first APL patient with STAT5B-RARα in Chinese population and the fifth patient around the world according to published paper.