Clarithromycin Synergistically Enhances Thalidomide Cytotoxicity in Myeloma Cells.

Clarithromycin (CAM) is a macrolide antibiotic that is widely used in the treatment of respiratory tract infections, sexually transmitted diseases and infections caused by the Helicobacter pylori and Mycobacterium avium complex. Recent studies showed that CAM was highly effective against multiple myeloma (MM) when used in combination with immunomodulatory drugs and dexamethasone. However, the related mechanism is still unknown. As 3 immunomodulatory agents are all effective in the respective regimen, we postulated that CAM might enhance the effect of immunomodulatory drugs. We evaluated the interaction effects of CAM and thalidomide on myeloma cells. Taking into consideration that thalidomide did not affect the proliferation of myeloma cells in vitro, we cocultured myeloma cells with peripheral blood monocytes and evaluated the effects of CAM and thalidomide on the cocultured cell model. Data showed that thalidomide and CAM synergistically inhibited the proliferation of the cells. On this same model, we also found that thalidomide and CAM synergistically decreased the secretion of tumor necrosis factor-α and interleukin-6. This might be caused by the effect of the 2 drugs on inhibiting the activation of ERK1/2 and AKT. These data suggest that the efficacy of CAM against MM was partly due to its synergistic action with the immunomodulatory agents.

[Clinical and Pathological Characteristics of Aggressive Natural Killer Cell Leukemia Patients].

OBJECTIVE: To investigate the clinicopathologic characteristics, diagnosis and therapy of aggressive natural killer cell leukemia (ANKL) patients. METHODS: Clinical manifestations, cellular morphology, immunophenotypic analysis by flow cytometry (FCM), TCR gene rearrangement, pathology and Immunohistochemical analysis of bone marrow (BM) were combined to diagnose the six patients with ANKL. RESULTS: The median age of the patients were 35.5 years old. All the patients with fever, cytopenia and liver dysfunction. Imageological examination presented hepatosplenomegaly (6/6), and PET/CT presented diffusely increased metabolism in liver, spleen and BM (3/3). BM cytologic examination presented increased hematophagocyte at the early stage and 1%-42% leukemic cell were detected in BM with the progression of diseases. FCM showed the leukemic cells were positive for CD2(6/6), CD56(5/6), CD16(2/6), CD94(3/6), CD38(3/6), cCD3(1/5), CD8(1/6), CD7(2/6), CD57(1/6) and negative for CD3, CD4, TdT, cMPO, TCR α/ß, TCR γ/δ. The neoplastic cells were negative for TCR gene rearrangement. Five cases showed increased quantitation of whole blood Epstein-Barr virus (EBV) DNA. CONCLUSION: ANKL is a highly aggressive disease. Prompt and repeating BM examination is important to patient with fever, cytopenia and liver dysfunction. The diagnosis of ANKL relies mainly on the integration of clinical, morphologic, immunophenotypic finding and EBV-DNA increasement.

[Effect and Clinical Significance of Bortezomib and Thalidomide on the Memory T Cells Subsets and Regulatory T Cells in Peripheral Blood of Patients with Multiple Myeloma].

OBJECTIVE: To investigate the effects of bortezomib(BTZ) and thalidomide(TM) on peripheral blood memory T-cells (Tm) and regulatory T cells(Tregs) in patients with multiple myeloma(MM). METHODS: Eighty-six MM patients received 2 courses of chemotherapy were divided into effective (partial response at least) group (63 cases) and ineffective (no partial response) group (17 cases) according to therapeutic efficacy; these 80 patients were divided into BTZ group (38 cases) and TM group (42 cases) yet according to therapeutic regimens, 20 newly diagnosed MM patients were used as baseline group, 30 healthy volunteers were used as healthy control group. The Tm subsets and Treg in peripheral blood of each groups were detected by flow cytometry. RESULTS: The CD4+ central memory T cells (CD4+ TCM) percentage of CD4+ Tm, the CD18+ TCM percentage of CD18+Tm and ratio of CD8+ TCM and CD8+ effector memory T cells (TEM) (CD8+ TCM/TEM) in baseline group were all significantly lower than those in healthy control group (P<0.05). After treatment with BTZ regimen or TM regimen, the CD8+TCM percentage of CD8+ Tm in effective group significantly increased to level of healthy control group (P<0.05); the Treg cell level in effective and in effective groups was not significantly different from that in baseline group(P>0.05), but the Treg percentage of CD4+ cells ineffective group was significantly higher than that in baseline group and ineffective group (P<0.05). According to ROC curve, the critical value of CD8+TCM/TEM for predicting chemotherapeutic response was 0.27 with sensitivity of 57.1% and specificity of 94.1%. CONCLUSION: When MM patients are in an immuno-exhanstive status, the treatment with BTZ or TM both can reverse the immuno-inhibitory status of MM patients, moreover, does not affect the Treg cell count; the Treg percentage in BTZ and TM effective groups both are significantly higher than that in baseline group and ineffective group. The ratio of CD8+TCM/TEM contributes to evaluating the chemotherapeutic efficacy.

[Cost-Effectiveness Analysis of Different Chemotherapy Regimens in the Treatment of Patients with Multiple Myeloma].

OBJECTIVE: To compare the pharmaco-economic effect of 3 chemotherapeutic regimens in the treatment of patients with multiple myeloma(MM). METHODS: One hundred and thirty-eight newly diagnosed cases of MM in our hospital were analyzed retrospectively, and then MM patients were divided into group A, B and C group according to therapeutic regimen. Group A was treated with VCD therapeutic regimen (bortezomib + cyclophosphamide + dexamethasone, 63 cases), The patients in group B was treated with BiCTD therapeutic regimen (clarithromycin+cyclophosphamide+thalidomide+dexamethasone, 44 cases), The patients in group C was treated with CTD therapeutic regimen (cyclophosphamide+ thalidomide+dexamethasone, 33 cases). The clinical efficacy, adverse reaction, cost-effectiveness were observed and analysed after 4 courses of treatment among 3 groups. RESULTS: The overall response rates of group A, B and C were 96.83%, 81.82% and 64.52% with statistical significant difference (P<0.01). The high efficiency response rates of 3 groups were 82.5%, 59.09%, 32.26% with very significant statistical difference (P<0.01). The infection rate of group A was statistically and significantly higher than other 2 groups (P=0.048), and the constipation rate in group A was statistically and significantly higer than that in group B and C (P<0.05). The cost-effectiveness ratios of 3 groups were 69567.44, 20765.12 and 21475.48, respectively. The incremental cost-effectiveness ratio of group A and B were 183933.21 and 22259.09, as compared with group C. The result was in accordance with sensitivity test. CONCLUSION: Clinicial efficacy of group A is the best,but group B has advantages on cost-effectiveness ratio as compared with other groups, otherwise, group B has low incidence of adverse reaction. In the view of safety, therapeutic efficacy and pharmacoeconomics for treatment of patients with MM, the BiCTD regimen has been confirmed to be superior to the other 2 groups.

[Establishment and Management of Multiple Myeloma Specimen Bank Applied for Molecular Biological Researches].

OBJECTIVE: To establish a multiple myeloma specimen bank applied for molecular biological researches and to explore the methods of specimen collection, transportation, storage, quality control and the management of specimen bank. METHODS: Bone marrow and blood samples were collected from multiple myeloma patients, plasma cell sorting were operated after the separation of mononuclear cells from bone marrow specimens. The plasma cells were divided into 2 parts, one was added with proper amount of TRIzol and then kept in -80 °C refrigerator for subsequent RNA extraction, the other was added with proper amount of calf serum cell frozen liquid and then kept in -80 °C refrigerator for subsequent cryopreservation of DNA extraction after numbered respectively. Serum and plasma were separated from peripheral blood, specimens of serum and plasma were then stored at -80 °C refrigerator after registration. Meantime, the myeloma specimen information management system was established, managed and maintained by specially-assigned persons and continuous modification and improvement in the process of use as to facilitate the rapid collection, management, query of the effective samples and clinical data. RESULTS: A total of 244 portions plasma cells, 564 portions of serum, and 1005 portions of plasma were collected, clinical characters were documented. CONCLUSION: A multiple myeloma specimen bank have been established initially, which can provide quality samples and related clinical information for molecular biological research on multiple myeloma.

Activity of fibroblast growth factor receptor inhibitors TKI258, ponatinib and AZD4547 against TPR­FGFR1 fusion.

8p11 myeloproliferative syndrome (EMS) is a rare disease characterized by the constitutive activation of fibroblast growth factor receptor 1 (FGFR1). To date, four cases of EMS with the chromosomal translocation, t(1;8)(q25;p11.2), have been reported. In the present study, TPR­FGFR1­expressing Baf3 cells were established and confirmed by polymerase chain reaction. To identify the most promising drug for EMS, the activities and associated mechanism of three tyrosine kinase inhibitors (TKIs), TKI258, ponatinib and AZD4547, against TPR­FGFR1 were tested by MTT assay, flow cytometry and western blot. The data demonstrated that TPR­FGFR1 was localized in the cytoplasm, and was able to transform interleukin-3-dependent hematopoietic Baf3 cells into growth factor­independent cells. All of the three TKIs markedly inhibited the proliferation of TPR­FGFR1­expressing Baf3 cells, and the activation of FGFR1 and the downstream signaling molecules, extracellular signal­regulated kinase 1/2, phospholipiase Cγ and signal transducer and activator of transcription 5. AZD4547 was the most efficient drug, and TKI258 was the least. By contrast, no significant difference was found among the three drugs on their effect on cell apoptosis. Taken together, the data obtained in the present study suggested that AZD4547 had increased potency, compared with TKI258 and ponatinib, for the treatment of EMS.

[Clinical and Immunophenotypic Properties of Small Cell Variant of T-cell Prolymphocytic Leukemia].

OBJECTIVE: To investigate the clinical, morphologic and immunophenotypic properties of the patients with small cell variant of T-cell prolymphocytic leukaemia(T-PLL). METHODS: Peripheral blood and bone marrow cytomorphologic and immunophenotypic examination, and T-cell receptor(TCR) gene rearrangement detection were used to verify the diagnosis for 2 patients with lymphocytosis. Two patients were treated with combined chemotherapeutic protocol based on fludarabine. RESULTS: At diagnosis of case 1, the main lymphocytes of peripheral blood smear were the small mature lymphocytes without nucleoli. The immunophenotype of the cells was CD3+CD5+CD7+CD4+CD8+TCRα/ß+. The patient achieved complete remission after treatment with combined with CTX of fludarabine. The disease relapsed at 32 months after diagnosis. The abnormal lymphocytes were medium-sized ones with a visible nucleolus. Immunophenotyping showed that the leukemic cells were predominantly CD8 positive(CD3+CD5+CD7+CD4-CD8+TCRα/ß+). Both the peripheral blood and bone marrow cells of case 2 were predominanthy the mature lymphocytes, and their immunophenotype was HLA-DR+CD7+CD5+CD4+CD3+CD2+CD56+cCD3+TCRα/ß+. The combined fludarabine therapy was ineffective. CONCLUSION: Immunophenotypical switch from CD4+CD8+ to CD4-CD8+ may be associated with a poor response to chemotherapy. CD56 expression is an independent poor prognostic factor for primary refractory disease in T-PLL and may be considered for implementing risked-adapted therapeutic strategies.

[Acute Promyelocytic Leukemia Developed during Imatinib Therapy for Gastrointestinal Stromal Tumors].

OBJECTIVE: To investigate the clinicopathologic and molecular characteristics of acute promyelocytic leukemia(APL) developed during imatinib therapy for gastrointestinal stromal tumors(GIST). METHODS: A 49-year-old woman was hospitalized for abdominal pain. The abdominal CT revealed a gastric mass. Laparoscopic resection of the tumor was performed. The histopathologic analysis showed poorly differentiated malignant cell infiltration with epithelioid features. Immunohistochemistry staining of these cells was positive for CD117 and CD34. GIST was confirmed and imatinib treatment was given. RESULTS: After 1 year,the patient developed progressive pancytopenia. Bone marrow aspirate showed marked hyperplasia of bone marrow cells with 92.5% promyelocyte, consistent with APL. Cytogenetic analysis demonstrated t(15;17)(q22;q21) as the sole abnormality. PML/RARα fusion gene was positive and Kit mutation was negative. After combined treatment with ATRA, arsenic trioxide and idarubicin, patient achieved cytogenetic and molecular remission. CONCLUSION: The metachronous coexistence of GIST with APL is uncommon. The potential nonrandom association and causal relationship between these malignancies remained to be investigated. Further studies would be necessary to clarify the relationship between imatinib and secondary malignancies in GIST patients.

[Application Value of CD138 MACS-FISH in the Genetic Diagnosis of Plasma Cell Dyscrasia].

OBJECTIVE: To investigate the cytogenetic characteristics of the various plasma cell dyscrasia using the CD138 MACS-FISH, to elucidate the application value of MACS-FISH in the genetic diagnosis of plasma cell dyscrasia, and to explore the standardization of FISH detection for plasma cell dyscrasia. METHODS: A total of 232 patients with newly diagnosed plasma cell dyscrasia were collected, including 203 cases of MM, 24 cases of AL amyloidosis and 5 cases of MGUS, whose cytogenetic abnormalities were detected by MACS-FISH, and the differences of the positive detection rates of chromosome karyotype analysis, C-FISH and MACS-FISH in MM cytogenetic abnormality were compared. The sensitivity of C-FISH and MACS-FISH were analyzed and compared according to the proportion of bone marrow plasma cells. The correlation between the positive cell rates of C-FISH and MACS-FISH and the proportion of plasma cells were analyzed respectively. The differences in the clone size detected by C-FISH and MACS-FISH were compared. RESULTS: The incidence of cytogenetic abnormality of MM, AL amyloidosis and MGUS detected by MACS-FISH were 85.9%, 62.5%, 60%, respectively. The incidence rate of MM cytogenetic abnormality detected by Chromosome karyotype analysis and C-FISH were 20.0% and 64.7%, respectively, which were significantly lower than that of MACS-FISH(P<0.001). The positive rate of 14q32 translocation, del(14q32), t(11;14), +17p13 and the coexistence of 2 and ≥3 kinds of cytogenetic abnormalities detected by MACS-FISH were significantly higher than that detected by C-FISH(P<0.05). When the plasma cell ratio was less than or equal to 5%, the positive detection rate of MACS-FISH was significantly higher than that of C-FISH (P=0.001), and there was no significant difference in different plasma cell proportion group of MACS-FISH. However, when the plasma cell ratio was less than or equal to 5%, the positive detection rate of C-FISH detection was significantly lower than that of the other 3 groups (P=0.013,P=0.001,P<0.001). The positive cell rates of all cytogenetic abnormalities in C-FISH group and +1q21 and 14q32 translocation in MACS-FISH group were significantly positively correlated with the proportion of plasma cells(P<0.05). The clone size of various cytogenetic abnormalities in MACS-FISH group were significantly higher than that in C-FISH group(P<0.001). CONCLUSION: MACS-FISH may significantly enhance the detection rate of cytogenetic abnormalities in various plasma cell dyscrasia, and it can better reflect the cytogenetic abnormality of plasma cell dyscrasia and its clone size. MACS-FISH may be recommended as a standard method for the genetic diagnosis of plasma cell dyscrasia, the risk stratification of MM and SMM, as well as the genetic diagnosis and research of MGUS and AL amyloidosis.

[Clinicopathologic Characteristics and Outcome of Isolated Ovarian Relapse in Adolescent with Acute Lymphoblastic Leukemia].

OBJECTIVE: To investigate the clinicopathologic characteristics,diagnosis and treatment of isolated ovarian relapse of acute lymphoblastic leukemia(ALL). METHODS: A 16-year-old girl presented with complaints of bone and joint pain. The peripheral blood and bone marrow(BM) smears showed 32% and 72% blasts, respectively, which were myeloperoxidase-negative. The blasts were positive for HLA-DR, TdT, CD10, CD19, CD22 and cCD79a and negative for CD34, CD5, CD7, CD13, CD33, CD56 and MPO detected by flow cytometry. BM cytogenetic analysis and fusion gene screening revealed t(1;19)(q23;p13) and E2A/PBX1. She was diagnosed as B-cell acute lymphoblastic leukemia (B-ALL) and was treated with CALGB8811 protocol. She presented lower abdominal pain with intermittent colick at 7 months after complete remission. The pelvic ultrasound showed a lobulated mixed echogenic mass in the right ovary, and an exploratory laparotomy was performed. RESULTS: Pathologic examination and immunohistochemistry of resected ovarian tumor revealed extensive infiltration by lymphoblasts with positive for TdT, CD20, CD43 and CD79a. Further investigations failed to reveal any other extramedullary involvement. Hemogram, peripheral blood and bone marrow smear examination were unremarkable at the same time. The isolated extramedullary ovarian relapse of ALL was confirmed. Simultaneous, the detection of minimal residual disease by multiparametric flow cytometry showed positive with 5.0×10-4. The reinduction chemotherapy including a high-dose methotrexate and cytarabine was given to the patients. She experienced the second ovarian relapse after 1 year and refused further treatment. CONCLUSION: Although uncommon, ovarian recurrence after chemotherapy for ALL should be considered in the patients with suggestive symptoms. Screening by pelvic ultrasonography may be valuble for early detection of pelvic disease in ALL.

[Clinico-pathologic Characteristics of Adult Patients with Atypical Infectious Mononucleosis].

OBJECTIVE: To investigate the clinicopathologic characteristics of adult patients with atypical infectious mononucleosis(IM). METHODS: From January 2003 to December 2013, a total of 5 cases of atypical IM misdiagnosed as lymphoma were selected, and the clinico-pathological characteristics and efficacy of treatment were analyzed. Biopsy of lymph node or tonsil was performed to evaluate the possibility of lymphoma. Peripheral blood EBV antibody and EBV-DNA were examined by ELISA and real-time fluorescence quantitative PCR, respectively. RESULTS: All the cases were considered as lymphoma on the basis of morphological features in initial evaluation before relapse. These features included a florid immunoblastic proliferation, distortion of the underlying nodal or tonsillar architecture and the presence of necrosis. The immunophenotypic features, EBV encoded RNA (EBER) in situ hybridization and the gene rearrangement of immunoglobulin or T cell receptor may be helpful for the distinction of atypical IM from lymphoma. CONCLUSION: IM as EBV-related lymphoproliferative process shows marked clinical and histological diversity. Atypical case of IM may mimic many different type of lymphoma in clinical and pathologic features, and the misdiagnosis should be avoided by using molecular and pathological examination.

[Relationship between the catalysis of Bence Jones protein and renal impairment in patients with multiple myeloma].

This study was purposed to investigate the relationship between the catalysis of Bence Jones protein (BJP) in urine of patients with multiple myeloma(MM) and toxicity on the renal proximal tubular cells in vitro, and to explore the potential mechanism for the toxicity of BJP to renal impairment in patients with MM. The Michaelis-Menten constant (K(m)) and catalytic constant (k(cat)) of the amidase activity of BJP was calculated by Hanes equation. The LLC-PK1 cells were cultured with different concentration of BJP for 24 h, then proliferation of the cells were determined by MTT method and apoptosis were determined by flow cytometry. The results showed that the BJP from the MM patients with renal impairment significantly inhibited cell proliferation, as compared with that from MM patients without renal impairment. The BJP with higher k(cat) had higher toxicity to LLC-PK1 cells. BJP could induce apoptosis and necrosis of LLC-PK1 cells when reached a certain concentration and this effect enhanced with increase of BJP concentration. It is concluded that the catalysis of BJP and its toxicity to renal tubular epithelial cells has a positive correlation, and toxic effect of BJP on renal tubular epithelial cells results from inhibiting proliferation and inducing apoptosis and necrosis of the cells, which may be one of renal impairment mechanisms in MM patients.