DNA methylation profiling to determine the primary sites of metastatic cancers using formalin-fixed paraffin-embedded tissues.

Identifying the primary site of metastatic cancer is critical to guiding the subsequent treatment. Approximately 3-9% of metastatic patients are diagnosed with cancer of unknown primary sites (CUP) even after a comprehensive diagnostic workup. However, a widely accepted molecular test is still not available. Here, we report a method that applies formalin-fixed, paraffin-embedded tissues to construct reduced representation bisulfite sequencing libraries (FFPE-RRBS). We then generate and systematically evaluate 28 molecular classifiers, built on four DNA methylation scoring methods and seven machine learning approaches, using the RRBS library dataset of 498 fresh-frozen tumor tissues from primary cancer patients. Among these classifiers, the beta value-based linear support vector (BELIVE) performs the best, achieving overall accuracies of 81-93% for identifying the primary sites in 215 metastatic patients using top-k predictions (k = 1, 2, 3). Coincidentally, BELIVE also successfully predicts the tissue of origin in 81-93% of CUP patients (n = 68).

[Application of Real-time Quantitative PCR in Detecting Atypical BCR/ABL mRNA Transcripts in Chronic Myelocytic Leukemia].

OBJECTIVE: To detect atypical BCR/ABL mRNA transcript by real-time quantitative PCR in CML patients without e13a2/e14a2,e19a2 or e1a2 transcripts, and investigate its value of clinical application. METHODS: Twelve cases of CML with positive for t(9;22) translocation, but negative for common major and minor breakpoint cluster regions comfirmed by chromosome karyotyping or FISH analysis, were collected from July 2012 to December 2015. These 12 cases were then detected for b2a3(e13a3), b3a3(e14a3), e6a2, e8a2 and e1a3 fusion variants by real-time quantitative PCR. RESULTS: Among 12 cases 4 variant transcripts were detected, including e1a3 in 1 case (8.33%), e8a2 in 2 cases (16.67%), b2a3 in 5 cases (41.67%) and b3a3 in 4 cases (33.33%), with total positivity of 100%, moreover b2a3 and b3a3 were predominant. CONCLUSION: The detecting atypical BCR/ABL mRNA transcripts by real-time quantitative PCR is suitable for the diagnosis of CML negative for P210, P190 and P230 by standard real-time PCR test, and this detection is still the standard and economic method for monitoring minimal residual disease in CML patients with variants of BCR/ABL fusion gene.

[Application of flow cytometry in diagnosis of T-cell rich diffuse large B-cell lymphoma].

OBJECTIVE: To investigate the application of flow cytometry in diagnosis of T-cell rich diffuse large B-cell lymphoma. METHODS: Histopathologic features, immunohistochemical findings and flow cytometry results of three cases of T-cell rich diffuse large B-cell lymphoma were reviewed retrospectively. RESULTS: In CD45-side scatter (SSC) dot plot of the first patient, two different CD45-positive lymphoid cell populations were identified. The bright population consisted of both T and B cells, with a T-cell predominance. The dim population consisted mainly of B cells which showed lambda light chain restriction. In the second patient, CD45-positive cells were subdivided into two groups according to CD45-SSC dot plot. The small population consisted of both T and B cells, with a T-cell predominance. The large population consisted mainly of B cells which showed kappa light chain restriction. In the third patient, CD19-positive cells were subdivided into two groups according to the expression of CD20 in CD19-CD20 dot plot. The CD20-positive population expressed both kappa and lambda light chains, while the CD20-negative population demonstrated kappa light chain restriction. CONCLUSIONS: Neoplastic B cells can be distinguished from reactive lymphoid cells in T-cell rich diffuse large B-cell lymphoma by flow cytometry, according to a number of parameters which include intensity of antigen expression, loss of antigens, expression of non-B-cell lineage antigens, patterns of forward scatter (FSC) and/or SSC, and expression of immature B-cell antigens.

Clinical study on the related markers of blood coagulation in the patients with ANFH after SARS.

The aim of this research was to investigate the blood coagulation function in the patients with avascular necrosis of the femoral head (ANFH) after severe acute respiratory syndrome (SARS). The expression of CD31, CD61, CD62p, CD63 and PAC-1 on platelet membrane was measured respectively by flowcytometry, and the plasma prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (Fbg) were measured by blood clotting instrument in 26 patients with ANFH after SARS and in 17 healthy adults. The expression of CD31, CD61, CD 62p, CD63 and PAC-1 on platelet membrane in 26 patients was all lower than that in 17 healthy subjects (P < 0.01). The levels of PT, APTT, TT and Fbg in 26 patients were all normal. There is no significant difference (P > 0.05) in those markers between patients and 17 healthy adults. The blood may not be in hypercoagulable state in patients with ANFH after SARS.

[A study of cellular telomerase activity in different stages of multiple myeloma cells].

OBJECTIVE: To evaluate the clinical significance of telomerase in multiple myeloma (MM) and explore its relationship with cell cycle. METHODS: Marrow plasma cells were collected form 21 patients with newly diagnosed or relapsed MM, 2 patients with monoclonal gammopathy of undetermined significance (MGUS), 15 MM patients in complete remission and 10 donors as control. PC isolation from the marrow mononuclear cell fraction was performed using immunomagnetic bead selection with CD(138) antibodies. Telomerase activity was detected with TRAP (Telomerase repeats amplification protocol)-fluorescence. Cell cycle was assayed by flow cytometry. RESULTS: (1) The results showed that the positive rate of telomerase in all the newly diagnosed and relapsed patients was 90.5%, which was higher than that in the control group (10.0%) and the patients in remission (13.3%) (P < 0.01). There was no significant difference between the patients in remission and control group (P > 0.05), as well as between newly diagnosed and relapsed patients (P > 0.05). (2) The average percentage of S period in the telomerase positive patients was (18.78 +/- 8.02)%, while that in the negative patients was (5.64 +/- 4.03)%; there was statistics difference between them (P < 0.05). CONCLUSIONS: (1) Telomerase plays an important role in the development of MM, it may be used to evaluate disease progression, therapeutic curative effect and prognosis; (2) Telomerase activity is expressed in the proliferation period of MM cells. Telomerase activity is correlated with cell cycle.

[Study of anti-myeloma activity of interleukin-2 activated bone marrow in vitro].

OBJECTIVE: To study the anti-myeloma activity of interleukin-2 activated bone marrow (ABM). METHODS: Bone marrow mononuclear cells (BMMNC) from multiple myeloma and iron-deficiency anemia patients were cultured in the presence of rIL-2. The anti-myeloma activity of ABM against U266 cells, cells expressing surface CD45, CD38, CD138, the levels of TNF-alpha and IFN-gamma in ABM culture supernatant were measured with MTT method, flow cytometry and ELISA method respectively after bone marrow was activated with rIL-2 for 24 and 72 hours. RESULTS: The tumor-killing activities against U266 cells of ABM were significantly increased compared with that of non-activated bone marrow (NBM) at 72 hours [(69.70 +/- 26.57)% vs (43.20 +/- 12.39)%, P < 0.05] and 24 hours [(34.25 +/- 11.93)% vs (26.53 +/- 5.48)%]. The CD45(-)CD38(+)CD138(+) cells of ABM from myeloma group at 72 hours were decreased from (8.46 +/- 3.66)% to (4.79 +/- 1.56)% (P < 0.05). TNF-alpha and IFN-gamma were detectable after cultured for 24 hours in both normal control group and myeloma group and went higher at 72 hours. The level of TNF-alpha and IFN-gamma were significantly increased in ABM compared with that in NBM (P < 0.05). Meanwhile, there was a positive relationship between the level of TNF-alpha, IFN-gamma and cytotoxicity of ABM from normal control group at 24 hours and 72 hours (P < 0.05), and was a negative relationship between TNF-alpha and IFN-gamma levels and the CD45(-)CD38(+)CD138(+) cells in myeloma group at 72 hours (P < 0.05). CONCLUSION: Normal BMMNCs activated with rIL-2 have tumor-killing activities against U266 cells. Myeloma cells and tumor burden were decreased in myeloma bone marrow after the marrow was activated with rIL-2. Production of TNF-alpha and IFN-gamma from bone marrow cells including T cells, monocyte-macrophages and NK cells activated with rIL-2 might be involved in anti-myeloma activity of ABM.

Relationship between endothelial dysfunction and serum homocysteine in patients with coronary lesions.

OBJECTIVE: To investigate the relationship between vascular endothelial dysfunction and serum homocysteine (HCY) level in patients with coronary lesions. METHODS: Serum HCY, serum nitric oxide (NO), plasma endothelin-1 (ET-1), and circulation endothelial cell (CEC) were measured in 76 patients who received coronary angiography. Fifty-four patients with a stenosis of 50% or more at least in one coronary atery were as coronary artery disease (CAD) group. Other 22 cases with no recognizable plaque and/or stenosis were as control group. HCY level was detected using an enzyme immunoassay kit. NO concentration was measured using a nitrate reductase kit. Radio-immunoassay was applied to analyse the ET-1 level, and CEC was measured by flow cytometry. RESULTS: The levels of HCY, ET-1, and CEC in patients with coronary lesions were significantly increased in comparison with control group (P < 0.01), while NO level in CAD group was significantly lower compared with that in control (P < 0.01). Using a multivariate stepwise regression analysis, HCY level had a positive correlation with ET-1 level (r = 0.420, P < 0.05) and CECs number (r = 0.423, P < 0.05); and had a negative correlation with NO/ET-1 (r = -0.403, P < 0.05). But there was no significant correlation between HCY and NO levels. CONCLUSIONS: HCY might lead to endothelial cell injury, which would provide a plausible mechanism for the relationship between hyperhomocysteinemia and development of coronary artery disease. HCY can be considered as a predictor for preliminary or active coronary lesion.

[Application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression].

OBJECTIVE: To investigate the application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression. METHODS: The results of flow cytometry of 30 lymphoma/leukemia cases with aberrant antigen expression, of which 3 cases being lymphomas, 8 B-cell leukemia, 1 T-cell leukemia, 17 acute non-lymphoid leukemia and 1 acute non-lymphoid leukemia involving lymph nodes were analyzed. Immunohistochemistry (EnVision) for CD79a, CD3 and MPO was performed on all cases. RESULTS: Eleven cases of B-cell lymphoma/leukemia were cytoplasmic CD79a (cCD79a)-positive, cytoplasmic CD3 (cCD3epsilon) and cytoplasmic MPO (cMPO)-negative. Five of these cases were positive for CD5 and 2 for CD5, 1 or 2 for myeloid marker(s). The T-cell leukemia cases were cCD3epsilon-positive, cCD79a and cMPO-negative, they also co-expressed CD13 and CD33. The mantle cell lymphoma cases were positive for CD3, CD13 and CD33. Of the 8 B-cell leukemia cases, 4 were positive for CD5, 3 for CD13 and 1 for CD13 and CD33. The 18 acute non-lymphoid leukemia cases (including 1 acute non-lymphoid leukemia case involving lymph nodes) were cMPO-positive and cCD79a and cCD3epsilon-negative. Eight of the 18 expressed T-cell markers (including 1 case of acute non-lymphoid leukemia involving lymph nodes), 8 expressed B-cell markers, 2 expressed both T and B-cell markers. CONCLUSIONS: Flow cytometry can demonstrate aberrant antigen expression in lymphoma/leukemia cells and is helpful in delineating their cell origin. The technique is thus useful in the differential diagnosis of lymphoma/leukemia.

[B cell subsets and the expression of CD40 in peripheral blood of patients with severe acute respiratory syndrome].

OBJECTIVE: To explore the changing patterns of B cell subsets and the expression of CD40 on B cells in the course of severe acute respiratory syndrome (SARS) and their relationship to the severity of the disease. METHODS: Peripheral blood from 162 cases of SARS, 30 cases of mycoplasma pneumonia and 30 healthy adult volunteers were measured for the number of total B cells (CD19+ B cell), CD5 positive B cells (B1 cell) and mean fluorescent intensity of CD40 on B cell (CD40MF) using tri-color flow cytometry to analyze the distribution of the above parameters in SARS, mycoplasma pneumonia and healthy adult volunteers and their relation to the severity of SARS. RESULTS: The number of total B cells of clinically diagnosed SARS was (292 +/- 181) x 10(6)/L, significantly greater than that of healthy adults which was (200 +/- 65) x 10(6)/L (F = 6.17, P < 0.05) but smaller than that of patients infected with mycoplasma pneumonia which was (359 +/- 168) x 10(6)/L (F = 6.28, P < 0.05). During day 11 - 20 of the disease, the number of B1 cells of SARS patients was (24 +/- 14) x 10(6)/L smaller than that of healthy adults which was (39 +/- 20) x 10(6)/L (F = 4.23, P < 0.05). In the early stage of SARS (within 10 days), the expression of CD40 decreased significantly as compared with healthy adults (F = 5.13, P < 0.05). The number of B cells in severe SARS and mild cases were (347 +/- 156) x 10(6)/L and (268 +/- 211) x 10(6)/L respectively. The difference was statistically significant (F = 7.11, P < 0.01). CONCLUSIONS: B cells, B1 cells and the expression of CD40 on B cells were involved in the pathogenesis of SARS. The B cell count correlated with the severity of SARS. The monitoring of B cells, B1 cells and the expression of CD40 on B cells is useful in the differential diagnosis of SARS.

[The relationship between the peripheral blood of CD61, CD63, PAC-1 and the transplant kidney function].

OBJECTIVES: To explore the relationships between the peripheral blood levels of CD61, CD63, PAC-1 and the incidence of acute rejection and tubular necrosis after renal transplantation, and recovery of the graft function. METHODS: The peripheral blood levels of CD61, CD63, and PAC-1 of 86 patients with uremia in different stages before and after transplantations were analyzed by flow cytometry. The patients were divided into three groups: (1) twenty-nine patients with normal grafts function, (2) hirty with acute rejection and (3) twenty-seven with acute tubular necrosis. The patients with acute rejection were randomly divided into treatment group with anticoagulants and cntrol group. RESULTS: The peripheral blood levels of CD61, CD63 and PAC-1 significantly increased (P < 0.05) in the patients with acute rejection, in comparison with those with normal grafts function and those with acute tubular necrosis. The peripheral blood levels of CD61, CD63 and PAC-1 in patients with acute rejection in anticoagulants therapy was lower, recovery time of the grafts function was shorter, one-year survival rates of patients and grafts were higher, as compared with those of controls. CONCLUSIONS: The patients with acute rejection have significantly high peripheral blood levels of CD61, CD63 and PAC-1 before transplantation, however, these values in patients with acute tubular necrosis are not high, this suggesting that acute rejection might relate to platelet activation, while acute tubular necrosis might not relate to it. After anticoagulants therapy in patients with acute rejection, the grafts function might recover faster and their one-year survival rates and grafts might be higher in those with CD61, CD63 and PAC-1 decreasing remarkably.

[Study of platelet membrane glycoprotein expression in mice with decompression sickness].

OBJECTIVE: To investigate the role of expression of platelet membrane glycoprotein CD31, CD61 and CD62p in the pathogenesis of decompression sickness. METHODS: Mice were randomly divided into decompression sickness group and normal control group. The animals in decompression sickness group were exposed to 600 kPa compressed air for 60 minute, then they were rapidly decompressed to normal pressure in one minute. At 60th minute after reducing to normal pressure, the expression of CD31, CD61 and CD62p on platelet membrane in mice was measured by flow cytometry. RESULTS: The mean fluorescence intensity of CD31, CD61 and positive percentage of CD62p on platelet membrane [(18.64 +/- 1.01), (271.06 +/- 24.25), (4.48% +/- 0.43%) respectively] in decompression sickness group were significantly increased compared with normal control group [(16.89 +/- 1.69), (234.09 +/- 15.96), (3.00% +/- 0.66%) respectively] (P < 0.05, P < 0.01). CONCLUSION: Inadequately rapid decompression may induce up regulation of platelet membrane glycoprotein CD31, CD61 and CD62p expression in mice, which may lead to thrombosis.

[Expression of vascular endothelial growth factor in colorectal cancer and its clinical significance].

OBJECTIVE: To evaluate the relationship between vascular endothelial growth factor (VEGF) and colorectal cancer. METHODS: Samples of cancer and adjacent normal mucosa were taken from 34 patients with colorectal cancer. The percentage and average fluorescence intensity pf VEGF positive cells in these samples were examined by using flow cytometry. RESULTS: The percentage of VEGF positive cells was 57% +/- 29% in cancer tissue and 42% +/- 24% in normal tissue (P < 0.05). The average fluorescence intensity of VEGF positive cells was 24% +/- 11% in cancer tissue and 16% +/- 7% in normal tissue (P < 0.01). The percentage of VEGF positive cells was 30% +/- 22% in cancer of Dukes stage B and 72% +/- 18% in cancer of Dukes stage C (P < 0.01). The average fluorescence intensity was 18% +/- 25% in cancer of Dukes stage B and 27% +/- 12% in cancer of Dukes stage C (P < 0.01). CONCLUSION: VEGF is associated with the development and prognosis of colorectal cancer. Its relation with degree of differentiation of colorectal cancer remains to be studied.

Methylation of p16 and p15 genes in multiple myeloma.

OBJECTIVE: To investigate the frequency of p16 and p15 gene methylation in multiple myeloma (MM), and its relationship with bone marrow cell apoptosis and clinical outcome. METHODS: Twenty-two patients with MM were studied to detect p16 and p15 gene methylation. Methylation-specific polymerase chain reaction (MSP) was used to detect gene methylation, and terminal transferase-mediated dUTP nick end-labeling (TUNEL) was used to detect cell apoptosis. RESULTS: p16 and/or p15 gene methylatoin was detected in 10 of 22 patients (45.4%). There were 3 patients with p16 gene methylation, 9 patients with p15 gene methylation, and 2 patients with both genes methylation. The incidence of methylation of p15 gene was higher than that of p16 gene (P < 0.05). The patients with p16 and/or p15 gene methylation had a delayed cell apoptosis, poor response to chemotherapy, and a short over-all survival (OS). CONCLUSION: The methylation of p16 and/or p15 gene plays a key role in MM apoptosis pathogenesis. The patients with both p16 and p15 gene methylation had a poor prognosis.