The value of ultrasound-guided fine needle aspiration in the diagnosis of diffuse large B-cell lymphoma: A single center experiment.

OBJECTIVES: Flow cytometry (FC) is a critical diagnostic approach for guiding targeted chemotherapy and cellular immunotherapy for relapsed and refractory lymphoma patients. The aim of the study was to investigate the value of ultrasound-guided fine needle aspiration (FNA) to improve the quality of FC specimens in relapsed and refractory diffuse large B-cell lymphoma (R/R DLBCL). METHODS: Twenty patients with R/R DLBCL after standard treatment were included. The primary lesions of all cases were confirmed by pathology. FNA and core needle biopsy (CNB) were both used for ultrasound-guided puncture, the specimens obtained by FNA are directly examined by FC, and the specimens by CNB were subjected to FC after grinding. The accuracy of FC with the two methods were evaluated using histopathology as the gold standard. RESULTS: Of the 20 R/R DLBCL cases, 19 were diagnosed as DLBCL pathologically and one was diagnosed as inflammatory granuloma. Among the specimens obtained by CNB, 14 cases examined by FC after grinding showed abnormal mature B cells, five cases were missed, all cases are not misdiagnosed. Among the specimens obtained by FNA, 18 cases showed FC-confirmed abnormal mature B cells, one case was missed, all cases are not misdiagnosed. The sensitivity, specificity, and accuracy of FC with CNB and FNA were 73.68 % (14/19) vs 94.73 % (18/19), 100 % (1/1) vs 100 % (1/1), and 75 % (15/20) vs 97.14 % (19/20), respectively. The sensitivity of the two puncture methods of FC of DLBCL was statistically different (p < 0.001). CONCLUSION: Sampling with ultrasound-guided FNA is of great value to improve the quality of FC specimens. FNA can significantly improve the sensitivity and accuracy of FC diagnosis in R/R DLBCL.

[Misdiagnosis of 3 cases lymphoma due to misjudgement of immunohistochemistry].

OBJECTIVE: To recognize the importance of analyzing the result of immunohistochemical staining correctly. METHOD: Review of the three misdiagnosed cases lymphoma and exploring the causes of misdiagnosis through reviewing their clinics, histopathology and immunohistochemistry. RESULTS: Case 1 of lymphocyte rich classical Hodgkin's lymphoma (LRCHL) was misdiagnosed as follicular lymphoma (FL) initially, the RS cells were overlooked morphologically and wrongly determined BCL-2 and CD20-positive cells as tumor cells immunohistochemically; also once misdiagnosed as nodular lymphocyte predominant Hodgkin's lymphoma (NLPHL) because the CD20-negative RS misjudged cells as the positives. Case 2 of AML tumor cells expressed TdT, CD7 and CD43 unspecifically, which misdiagnosed as T-cell lymphoblastic lymphoma (T-LBL). Case 3 of type B1 thymoma was misdiagnosed as T-LBL, because CK wasn't expressed satisfactorily resulting in neglecting neoplastic epithelial cells, and lymphocytes in the background were TdT and CD99-positive. CONCLUSION: The diagnosis of lymphoma should be based on morphology, immunohistochemistry, clinics, and genetics. Moreover, the correct judgment of immunohistochemical staining is essential to make right diagnosis.

[Predictable recurrence by regular monitoring minimal residual disease with flow cytometry in the patients with both AML and ALL: a single-center study of 163 cases].

OBJECTIVE: To study the predictable value of monitoring minimal residual disease (MRD) regularly by flow cytometry (FCM) in patients with acute leukemia (AL) in the first complete remission (CR(1)). METHODS: From April 2005 to July 2009, AL patients who had got CR(1) after chemotherapy were regularly monitored for MRD in bone marrow by FCM to relapse or to July 2010 in Beijing Daopei Hospital (not including those received stem cell transplantation). The special antibody combinations were employed for each patient according to aberrant expression of leukemia cells. MRD(+) was defined as the aberrant cells more than 0.01%. The probability of continuous CR (CCR) was calculated by Kaplan-Meier formula, and the statistical difference between two CCR probabilities was evaluated by log-rank test. RESULTS: A total of 163 AL patients in CR(1) were monitored to relapse or to July 2010. Among 89 AML patients referred to our hospital within 1 year after diagnosis, 30 cases were in MRD(+) and 59 cases MRD(-) till 12 months following chemotherapy, 3/30 patients in MRD(+) and 47/59 remained in CCR to July 2010. The probability of CCR at 24, 36 months was 13%, 13%in MRD(+) group, 94%, 78% in MRD(-) group respectively, the difference between them was statistically significant (P < 0.01). Among 35 ALL referred to our hospital within 5 months after diagnosis, 13 cases were MRD(+) and 22 cases MRD(-) till 5 months following chemotherapy, 0/13 patients in MRD(+) and 20/22 patients in MRD(-) remained in CCR to July 2010. The probability of CCR at 24, 36 months was 0% in MRD(+) group, 96%, 96% in MRD(-) group respectively, the difference between them was statistically significant (P < 0.01). Over the time point above, all patients with MRD(+) or their MRD from negative to positive relapsed finally, and most patients with MRD(-) remained CCR to July 2010. CONCLUSION: It had a clinical prognostic value to monitor MRD regularly by FCM in the patients with AL after CR(1).

[The clinical and laboratory features of 9 cases with gammadeltaT cell lymphoma or leukemia].

OBJECTIVE: To analyze the clinical and laboratory features of 9 cases of gammadeltaT cell lymphoma or leukemia. METHODS: From 2007 to 2011, 9 patients with gammadeltaT-cell lymphoma/leukemia were diagnosed in our hospital. The immunophenotype of the abnormal cells were detected by flow cytometry, clonal gene rearrangement of IgH, TCRgamma, TCRdelta by PCR, chromosome karyotype analysis by G banding, acute leukemia gene and the DNA of type 1 - 8 human herpes virus by multiple nested PCR, The gammadeltaT cells were determined by T cell with TCR gammadelta chain, the malignant gammadelta T cells by the abnormal expression of T cell antigens and the precursor malignant gammadelta T cells by the expression of CD34, TDT, CD99, CD1 a or acute leukemia genes. RESULTS: In the 9 patients with gammadeltaT cell lymphoma leukemia, significant malignant gammadeltaT cells infiltration of bone marrow were found in 8 with blast morphology. 5 were diagnosed as T-ALL/LBL (gammadeltaT type) and 4 HSgammadelta TCL. The clonal gene rearrangement of TCRgamma and/or TCRB were detected in 6/6 patients. Patients either did not achieve complete remission(CR) after induction therapy or relapsed quickly after CR. Only 4/5 patients remained continuous CR(CCR) at 2, 2, 3,12 months respectively, after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the fifth T-ALL (gammadeltaT) relapsed 1 month after allo-HSCT. CONCLUSIONS: The incidence of gammadelta T cell lymphoma or leukemia may be higher than reported, part of them were T-ALL/LBL with poor prognoses. FCM and clonal gene rearrangement of TCRgamma and/or TCRdelta are helpful to diagnosis. Allo-HSCT may be the only curative approach.

[Evaluation of polymorphic post-allotransplant lymphoproliferative disorder by flow cytometry.].

OBJECTIVE: To study the role of flow cytometry (FCM) in detection of polymorphic post-transplant lymphoproliferative disorders (PTLD). METHODS AND RESULTS: Two patients presented with fever and multiple lymphadenopathy on day 46 and day 50 respectively after successful allogeneic hematopoietic stem cell transplantation (allo-HSCT). The symptoms couldn't be controlled by antibiotics. The polymorphic PTLD was diagnosed based on the elevation of bone marrow EB virus DNA and detection of subsets of light chain restricted B cells and/or plasma cells in peripheral blood (PB) samples. The lymphocyte immunophenotypes from PB and/or bone marrow (BM) samples were serially tested by FCM after lowering the dose of immunosupressive agents and treating with antivirus drugs, anti-CD20 antibodies, and cytotoxic T cell infusion. B cells were undetable in two patient, but monoclonal plasma cells appeared or maintained. One patient died after two weeks. Another patient was still on treatment. B cells and plasms cells couldn't be detected in her PB, but there were monoclonal plasma cells in her BM. FCM have a prominent advantage in detect polymorphic PTLD, since it can effectively recognize different cell groups in blood and identify monoclonal subsets. Besides, the immunophenotype of plasma cells in polymorphic PTLD might be different from that in typical plasma cell myeloma. CONCLUSION: Polymorphic PTLD can be detected and followed up by FCM. BM is more suitable than PB for monitoing the disease. Besides lymph node biopsy, B cell abnormaliity could be detected in PB in allo-HSCT patients.

Natural killer/T cell lymphoma initiating with pleural effusion: the significance of MICM combined techniques for the diagnosis.

Natural killer (NK)/T-cell lymphomas represent a rare type of lymphoma derived from either activated NK cells or cytotoxic T cells. They are most commonly extranodal and tend to present as destructive lesions within the midline facial structures. Other than the nasal cavity and Para nasal sinuses, several other extra nodal sites of involvement have been reported, including the pharynx, gastrointestinal tract, and testis. Occasionally, pleural effusion has also been observed. Here, a case of lymphoma of NK/T-cell type presented as pleural effusion was reported. The patient was previously misdiagnosed as B cell non-Hodgkin's lymphoma by pathological and immunohistochemistry (IH) analysis for pleural membrane biopsy specimen. After the analysis of the pleural fluid cells by a combination of morphologic, immunophenotypic, cytogenetic and molecular (MICM) methods in Beijing Dao-Pei hospital, some lymphoblasts were found morphologically, which expressed cytoplasmic CD3 (cCD3) and CD56 by flow cytometry analysis and had a clonal T-cell receptor gamma (TCR-gamma) gene rearrangement by molecular analysis, so that the diagnosis was finally corrected as NK/T-cell lymphoma and an allogeneic stem cell transplantation was successfully performed. In conclusion, this unusual case highlights the significance of MICM combined techniques for the diagnosis of lymphoma, as well as an unusual presentation of a rare disease and the successful treatment.

[Analysis of variant translocation der ins (17; 15) in patient with APL by G-banding technique and interphase fluorescence in situ hybridization].

To investigate the biological characteristics of the variant translocation der ins (17;15) in a patient with acute promyelocytic leukemia (APL), the conventional G-banding technique, interphase fluorescence in situ hybridization (int-FISH), RT-PCR, gene scanning, gene sequence and flow cytometry were performed. The results indicated that the variant translocation der ins (17, 15) observed by G banding technique was a rare type, the int-FISH assay by using dual-color pml/raralpha fusion probes confirmed the cytogenetic findings. The detection results of other molecular methods demonstrated the existence of the whole pml/raralpha fusion gene, while this case had insertion variant translocation. This patient got complete remission by using combined chemotherapy, and survives with continuous complete remission during following up for 1 year. In conclusion, the variant translocation der ins (17; 15) is rare type in APL, its incidence is lower, several signal types in detection of int-FISH were observed and the combination chemotherapy for this patient showed more obvious efficacy.

[Phenol butyrate inhibits cell cycle of leukemia cell lines through up-regulation of p21WAF1/CIP1 gene].

OBJECTIVE: To elucidate effects of histone deacetylase inhibitors on cell cycle of leukemia cell lines and investigate its molecular mechanisms. METHODS: Kasumi-1, U937 and NB4 cell lines were exposed to a histone deacetylase inhibitor, phenyl butyrate (PB), for 24, 48 and 72 hrs. Cells were harvested for cell cycle analysis by flow cytometry. Gene expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, was determined by semi-quantitative reverse transcriptase polymerase chain reaction (semi-quantitative RT-PCR). Promoter activity of p21WAF1/CIP1 was determined by luciferase-reporter assay in 293T cell line. RESULTS: PB inhibited cell cycle of Kasumi-1, U937 and NB4 cell lines, showing G(0)/G(1) phase arrest and S-phase fraction reduction with a dose and time dependent manner. After Kasumi-1, U937 or NB4 cells exposed to 3 mmol/L PB for 72 hrs, G(0)/G(1)-phase fraction was increased by 42.03%, 44.36% and 26.82%, and S-phase fraction was decreased by 31.86%, 38.9% and 26.77%, respectively. After Kasumi-1, U937 and NB4 cell lines exposed to PB, the expression of p21WAF1/CIP1 gene was increased by (2.06 +/- 0.27), (2.78 +/- 0.40) and (1.78 +/- 0.20) times at its maximum, respectively. PB could stimulate p21WAF1/CIP1 promoter activity (by luciferase-reporter assay) and the effect was dose dependent. The promoter activity was increased by 5.74 times after the cells exposed to 3 mmol/L PB for 48 hrs. PB stimulating p21WAF1/CIP1 promoter activity was mainly mediated by a 101 base pairs fragment upstream of transcription start site. CONCLUSION: PB could inhibit cell cycle of leukemia cell lines. The effects were mainly through up-regulation of p21WAF1/CIP1 expression.