Evaluation of miR-15a, miR-16-1, ZAP-70, Ang-2, and Bcl-2 as potential prognostic biomarkers in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a blood cancer characterized by the accumulation of clonal B-lymphocytes. This study evaluated the mRNA gene expression of miR-15a, miR-16- 1, ZAP-70, and Ang-2 by qPCR, as well as the plasma levels of Bcl-2 by Elisa immunoassay, in CLL patients and healthy controls. Significant differences were observed when comparing patients and controls regarding miR-15a (p < 0.001), miR-16-1 (p < 0.001) mRNA, Ang-2 gene expression, and Bcl-2 plasma levels (p < 0.001). When stratified by risk, differences were maintained with a significantly reduced expression in high-risk patients. A positive correlation was observed between miR-15a and platelets (R2 = 0.340; p = 0.009) as well as between Bcl-2 and leukocytes (R2 = 0.310; p = 0.019). Conversely, negative correlations were observed between ZAP-70 and platelets (R2 = - 0.334; p = 0.011), between miR-15a and lymphocytes (R2 = - 0.376; p = 0.004), as well as between miR-16-and lymphocytes (R2 = - 0.515; p = 0.00004). The data suggest that a reduction in miR-15a and miR-16-1 expressions, in addition to an overexpression of Bcl-2, are associated with the reduction in apoptosis and, consequently, to a longer survival of lymphocytes, thus contributing to lymphocyte accumulation and aggravation of the disease. By contrast, Ang-2 expression was significantly higher in A than in B + C Binet groups. This context leads to the speculation that this biomarker should be investigated in more robust studies within populations with a still relevantly indolent form of the disease in an attempt to identify those patients with a greater potential for an aggravation of the disease

Presence of serum antinuclear antibodies correlating unfavorable overall survival in patients with chronic lymphocytic leukemia / 中华医学杂志(英文版)

BACKGROUND@#Serum antinuclear antibodies (ANAs) are positive in some patients with chronic lymphocytic leukemia (CLL), but the prognostic value of ANAs remains unknown. The aim of this study was to evaluate the role of ANAs as a prognostic factor in CLL.@*METHODS@#This study retrospectively analyzed clinical data from 216 newly diagnosed CLL subjects with ANAs test from 2007 to 2017. Multivariate Cox regression analyses were used to screen the independent prognostic factors related to time to first treatment (TTFT), progression free survival (PFS) and overall survival (OS). Receiver operator characteristic curves and area under the curve (AUC) were utilized to assess the predictive accuracy of ANAs together with other independent factors for OS.@*RESULTS@#The incidence of ANAs abnormality at diagnosis was 13.9%. ANAs positivity and TP53 disruption were independent prognostic indicators for OS. The AUC of positive ANAs together with TP53 disruption was 0.766 (95% confidence interval [CI]: 0.697-0.826), which was significantly larger than that of either TP53 disruption (AUC: 0.706, 95% CI: 0.634-0.772, P = 0.034) or positive ANAs (AUC: 0.595, 95% CI: 0.520-0.668, P < 0.001) in OS prediction. Besides, serum positive ANAs as one additional parameter to CLL-international prognostic index (IPI) obtained superior AUCs in predicting CLL OS than CLL-IPI alone.@*CONCLUSION@#This study identified ANAs as an independent prognostic factor for CLL, and further investigations are needed to validate this finding.

ZAP-70 expression is associated with increased CD4 central memory T cells in chronic lymphocytic leukemia: cross-sectional study

ABSTRACT Background: Although chronic lymphocytic leukemia is basically a B cell disease, its pathophysiology and evolution are thought to be significantly influenced by T cells, as these are probably the most important interaction partner of neoplastic B cells, participating in their expansion, differentiation and survival. Chronic lymphocytic leukemia B cells may also drive functional and phenotypic changes of non-malignant T cells. There are few data about the association between memory T cells and prognosis, especially related to ZAP-70, a common reliable surrogate of the gold standard chronic lymphocytic leukemia prognostic markers. Objective: The aim of this study was to investigate whether the expression of ZAP-70 in chronic lymphocytic leukemia patients is associated with abnormal patterns of the distribution of naïve and memory T cells related to crosstalk between these cells. Methods: In this cross-sectional, controlled study, patients with chronic lymphocytic leukemia were compared with healthy blood donors regarding the expression of ZAP-70 and the distribution of naïve and memory T cell subsets in peripheral blood as measured by flow cytometry. Results: ZAP-70 positive patients presented an increased frequency and absolute number of central memory CD4+ T cells, but not CD8+ T cells, compared to ZAP-70 negative patients and age-matched apparently healthy donors. Conclusions: Because central memory CD4+ T cells are located in lymph nodes and express CD40L, we consider that malignant ZAP-70-positive B cells may receive beneficial signals from central memory CD4+ T cells as they accumulate, which could contribute to more aggressive disease.

Expression of Ki-67 in chronic lymphocytic leukemia and its clinical significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of nuclear antigen Ki-67 in CLL patients and realationship of Ki-67 expression with other clinical parameters.</p><p><b>METHODS</b>Twenty-Six confirmed cases of CLL were analysised retrospectively. The immhnohistochemical method was carried out to examine the expression of Ki-67 in bone marrow cells, the flow cytometer was used to detect ZAP70 (Zeta chain-associated protein), CD38 and other markers, additionally, a panel of probes RB1 (13q14), ATM (11q22.3), P53 (17p13.1) and CSP12 (+12) FISH were perfomed to detect the cytogenetic abnormalities.</p><p><b>RESULTS</b>Out of 26 patients, 15 cases (57.7%) showed positive expression of Ki-67, 11 cases (42.3%) showed negative expression of Ki-67, the average rate of Ki-67 positive expression was (10.86±7.36)%. The level of Ki-67 did not relate with sex, age, Hb, platelet, ZAP70, ATM. β2-MG, IgHV and P53, but related to the Rai staging (P=0.01, r=0.517), CD38 (P=0.02, r=0.469), 13q14 (P=0.021, r=-0.48), and there was statistically significant difference (P<0.05).</p><p><b>CONCLUSION</b>The Ki-67 level is higher in progressive stage of CLL and the Ki-67 expression is related with Rai staging, CD38, 13q14. The expression level of Ki-67 may be used as indicator for evaluation of CLL prognosis and guiding treatment for this disease.</p>

Detection of p53 gene deletion in Xinjiang patients with chronic lymphocytic leukemia by fluorescence in situ hybridization and its clinical significance / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the presence of p53 gene deletion in Xinjiang patients with chronic lymphocytic leukemia and its clinical significance.</p><p><b>METHODS</b>Interphase fluorescence in situ hybridization (FISH) was used to detect the p53 gene deletion in 77 patients with CLL. Presence of the deletion and its association with clinical and laboratory features as well as prognostic factors were analyzed. Kaplan-Meier method was used to calculate survivals, and the results were compared using a Log-rank test.</p><p><b>RESULTS</b>p53 gene deletion was found in 10 (12.9%) of the patients but none from the control group (P<0.05). The deletion was found in 12.5% (4/32) of ethnic Hans and 13.3% (6/45) of ethnic Uyghurs (P>0.05). No significant different distribution of p53 gene deletion was found in regard to sex, age, ethnicity, peripheral blood cell count (except for Hb) or the levels of lactate dehydrogenase, β2-micro globulin and CD38 (P>0.05). The deletion rate was higher in the group with high expression of ZAP-70 and patients with advanced stage disease than that in the group of low expression and early-stage CLL (P<0.05). Among 20 patients who received fludarabine therapy, the overall remission rate for those with p53 gene deletion (20%) was lower than those without (75%) (P<0.05). With a median follow-up time of 39.0 (8.0-136.0) months, 11 cases had died (14.3%), among them, 7 cases died from CLL and related complications, and all of them were founded p53 gene deletion. In patients with p53 gene deletion, the progression-free survival (18 months) was shorter than those without the deletion (55 months) (P<0.05).</p><p><b>CONCLUSION</b>The p53 gene deletion has been found in more than 10% of patients with CLL, and the deletion rate did not significantly differ between ethnic Han and Uyghur patients. The deletion is associated with advanced stage of the disease. High-level ZAP-70 expression and the presence of p53 deletion are associated with shorter survival and poor response to fludarabine containing therapy. Therefore, drugs affecting the p53 signaling pathway should be avoided.</p>

Prevalence of ZAP-70 and CD 38 in Indian chronic lymphocytic leukemia patients.

Aim of Study: Chronic lymphocytic leukemia (CLL) is the most common chronic lympho-proliferative disorder. This study was undertaken to know the prevalence of ZAP-70 and CD 38 in the treatment naive patients of CLL seen at a tertiary care centre of north India. Materials and Methods: ZAP-70 and CD 38 were tested by flow cytometry on peripheral blood samples. ZAP-70 positive and CD 38 positivity was defined as positive expression on 20% and 30% of CLL cells, respectively. Clinico-hematological profile and its correlation with ZAP-70 and CD 38 were assessed in consecutive 80 CLL patients. Results: There were 64 males and median age of the group was 58 years. Sixteen patients (20%) were asymptomatic and diagnosed incidentally. Median total lymphocyte count (TLC) at presentation was 62 × 10 9 /L. Rai stage distribution was: Stage 0-6, stage I-20, stage II-36, stage III-5, and stage IV-13. ZAP-70 and CD 38 positivity were detected in 20 patients (25%) and 29 patients (36%), respectively. Eleven patients were positive and 34 were negative for both ZAP-70 and CD 38 yielding a concordance rate of 56%. There was no statistically significant difference between ZAP-70 and CD 38 positivity and negativity with regard to age, sex, Lymphocyte count, lymphadenopathy, organomegaly, and Rai staging. Conclusion: ZAP-70 and CD 38 positivity were detected 25% and 36%, respectively, with concordance rate of 56%, which is higher than Western literature. There was no correlation of ZAP-70 and CD 38 positivity with age, sex, lymphadenopathy, organomegaly, and Rai staging.

Construção e expressão de anticorpo humanizado a partirdo anticorpo monoclonal contra proteína de 70 kDa de Sporotrix schenckii (P6E7) / Construction and expression of humanized antibody from the monoclonal antibody against the protein of 70 kDa Sporotrix schenckii (P6E7)

Sporothrix shenckii é o agente etiológico da esporotricose, micose de carater crônico e de ampla distribuição mundial. No Brasil, vem crescendo o número de casos da doença, bem como a incidência de formas clínicas graves ou atípicas. Nosso grupo de pesquisa desenvolveu um anticorpo monoclonal contra o componente antigênico proteíco de 70 kDa, secretado pelas células leveduriformes de S. schenckii, denominado anticorpo monoclonal (AcMo) P6E7. Este AcMo apresentou atividade profilática e terapêutica na esporotricose experimental, no entanto, este anticorpo possui origem murina, o que pode gerar uma resposta imunogênica quando administrado em humanos, impossibilitando sua utilização por tempo prolongado. Visando sua possível utilização no tratamento da esporotricose humana, a nossa proposta foi a humanização do AcMo P6E7 na forma de FvFc (Fv-linker- Fc) através de engenharia genética. Inicialmente construimos duas versões uma humanizada e outra quimérica do AcMo contra a fração de 70 kDa do antigeno de S. schenckii. Os anticorpos foram expressos em vetores de expressão dicistrônicos e produzidos com eficiência e estabilidade estrutural em células de mamíferos da linhagem CHO. Posteriormente, esses anticorpos foram purificados por cromatografia de afinidade e analisados quanto a sua capacidade de ligação ao fungo e função efetora. Verificamos que os FvFcs construídos foram capazes de se ligar a porção de 70 kDa do antígeno de S. schenckii. Através de ensaios de fagocitose, constatamos que os fragmentos FvFc do P6E7 humanizado e quimérico foram capazes de opsonizar as leveduras de S. schenckii aumentando, assim, o índice fagocítico em macrófagos humanos. Esses dados em conjunto, sugerem a possível utilização do anticorpo construído no tratamento da esporotricose humana

Sporothrix shenckii is the etiological agent of sporotrichosis, a chronical fungal infection that shows a worldwide distribution. In Brazil, there is a growing number of cases of sporotrichosis, as well as the incidence of severe or atypical clinical forms. Our research group developed a monoclonal antibody (mAb) against the fungal antigenic component a protein of 70 kDa, secreted by S. schenckii yeasts called P6E7. This mAb showed prophylactic and therapeutic activity in experimental sporotrichosis, however, this antibody has murine origin, which can generate an immune response when administered to humans, precluding their use for prolonged time. For its possible use in the treatment of human sporotrichosis, our proposal is the humanization of mAb P6E7 through genetic engineering. Initially, we built two versions of the original antibody: an humanized version and a chimeric antibody both against the 70 kDa fraction from S. schenckii antigen. The antibodies were expressed in dicistronic expression vectors and were efficiently produced in mammalian cells CHO strain, showing good structural stability. Subsequently, these antibodies were purified by affinity chromatography and assayed for their binding ability to the fungus and effector function. We found that the built os FvFcs (Fv-linker- Fc) fragments were capable of binding to the 70 kDa portion of S.schenckii antigen. Through phagocytosis assays, we found that the FvFc fragments from the humanized and chimeric P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human macrophages. Together, These data suggest the potential use of the antibodies constructed in the treatment of human sporotrichosis

Recent advances in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) was largely considered to be a disease of slow progression, standard treatment with Chlorambucil and having almost similar prognosis. With the introduction of molecular methods for understanding the disease pathophysiology in CLL there has been a remarkable change in the approach towards the disease. The variation in B-cell receptor response and immunoglobulin heavy chain variable region (IGHV) mutation, genetic aberration and defect in apoptosis and proliferation has had an impact on therapy initiation and prognosis. Early diagnosis of molecular variant is therefore necessary in CLL.

Clinical analysis on 40 patients with chronic lymphocytic leukemia / 中国实验血液学杂志

This study was aimed to analyze the clinical and laboratorial characteristics of patients with chronic lymphocytic leukemia (CLL), as well as their relationship with outcomes of patients. The clinical and laboratorial data of 40 CLL patients admitted from 2004 to 2010 in our hospital were analyzed retrospectively. The results indicated that the most of CLL attacked the elderly male patients with median age 66 (from 42 to 80). Flow cytometric analysis showed that 25 cases were positive for typical immunophenotype of CLL. On the other hand, all the patients clearly expressed CD19 and CD5, 7 cases (17.5%) and 14 cases (35%) were positive for the expression of CD38 and Zap70 respectively. 8 cases harbored a mutated immunoglobulin heavy-chain (VH) gene, among them 4 cases belong to VH3 family. Interphase FISH analysis showed that P53 deletion, RB1 deletion, trisomy 12 and normal chromosome were detected in 6, 3, 1, and 5 cases, respectively. The median PFS in 31 patients received treatment of fludarabine based chemotherapy was 48 months (95%CI: 39 - 57 months), among them 27 cases (87.1%) achieved CR + PR. While PFS was 14 months (95%CI: 10 - 18 months, P < 0.001) in 9 patients received other treatment regimen, out of them only 3 cases (33.3%) achieved CR + PR. Patients with normal level of serum β2-microglobulin at diagnosis showed significantly higher overall survival (78%, 95%CI: 69% - 87%) in 36 months than those with abnormal level of serum β2-microglobulin (47%, 95%CI: 35% - 59%, P = 0.004). Significant difference in the rate of CR + PR was noted in the Zap70 positive group (50%) and in negative group (88.5%, P = 0.006). All of 8 patients with IgVH mutation displayed CR after treatment, while 4 cases (66.7%) archived CR among 6 patients without IgVH mutation. It is concluded that CLL is characterized by high heterogeneity in both clinical features and molecular markers, which are associated with prediction of outcomes for patients. The treatment with fludarabine-based chemotherapy results in a major benefit and long survival for patients with CLL.

Prognostic significance of telomere length in patients with chronic lymphocytic leukemia / 中国实验血液学杂志

This study was aimed to explore the prognostic significance of telomere length in patients with chronic lymphocytic leukemia (CLL) and to analyze relation of telomere length with Binet stage, IgVH mutation status, CD38, ZAP-70 expression as well as other clinical features. 35 CLL patients who contained 80% or more tumor cells in the peripheral blood or bone marrow samples were selected as objects studied, while 13 healthy donors were served as normal controls. The telomere relative length was detected by using a real-time fluorescent quantitative polymerase chain reaction method (qPCR); the expression of CD38 and ZAP-70 protein were detected by flow cytometry, the IgVH mutation was detected by multiplex PCR. The results showed that the mean telomere relative length in CLL patients and normal controls were 0.384 and 0.443 respectively, but the difference between them was not significant (p > 0.05). The telomere length was significantly correlated with Binet stages and IgVH mutation status. Patients in Binet stage B and C showed significantly shorter telomeres than those in Binet stage A (p = 0.001). Mean telomere relative lengths in patients without IgVH mutation were shorter than those in patients with IgVH mutation (p = 0.015). No relation of telomere length with sex, age, ZAP-70 protein and CD38 were found (p > 0.05). It is concluded that telomere length may have a prognostic significance for CLL patients. Combining telomere length and IgVH mutation status may achieve a better prognostic subclassification for CLL patients.

Bag3 gene expression in chronic lymphocytic leukemia and its association with patients' prognosis / 中国实验血液学杂志

The aim of this study was to investigate bag3 gene expression in chronic lymphocytic leukemia (CLL)patients and its association with clinical prognosis. A total of 46 blood samples from untreated CLL patients were collected, SYBR Green-based real-time PCR was used to detect the bag3 mRNA expression, and its association with prognostic index was analyzed by statistical software. The results showed that the median values of bag3 level detected by real-time PCR in 46 CLL patients and normal controls were 0.021 (0.0007 - 1.124) and 0.0025 (0.0005 - 0.014) respectively, the former was significantly higher than the latter. The bag3 level in drug-resistant group was obviously higher as compared with the drug-responsive group. No association was found between bag3 expression and patient clinical baseline information (gender and age) as well as established prognostic factors (lymphocyte count, disease stage, IgVH mutation status, cytogenetics analysis and CD38, ZAP 70 expression). It is concluded that the bag3 expression in CLL patients is markedly higher than that in normal controls, while the high bag3 level in CML patients is probably related with drug resistance, but is not related with clinically established prognostic factors.

Detection of puma mRNA levels by real-time quantitative RT-PCR in chronic lymphocytic leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the expression level of puma (p53 up-regulated modulator of apoptosis) mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis. The puma mRNA expressions in 100 CLL patients and 11 normal controls were measured by relative quantification RT-PCR with fluorescent dye SYBR Green I, the beta-actin was used as internal reference. The difference of puma expression rate between groups with different prognostic factors was described using the Mann-Whitney U test. The relative quantitative value of puma expression was calculated by means of 2 (-ΔCt). The results indicated that the correlation coefficients of the standard curves in qRT-PCR were ≥ 0.99. The coefficients of variations (CV) within group or between groups were < 5%, and the sensitivity reached 10² copies/microg RNA. The median puma mRNA expression level was 1.038 x 10⁻³ (4.106 x 10⁻⁴ - 2.806 x 10⁻³) in CLL patients, which was 1.220 x 10⁻³ (7.233 x 10⁻⁴ - 1.405 x 10⁻³) in normal controls. There was no difference of puma mRNA expression between CLL patients and normal controls (U = 544.5, p = 0.957). Puma expression was significantly correlated with Binet stages (p < 0.001), expression of CD38 (p = 0.002), ZAP-70 protein (p = 0.012), LDH levels (p = 0.009) and beta₂-MG (p = 0.046). The puma expression level in patients with earlier Binet stage (Binet stage A) was obviously higher than that in patients with later Binet stage (Binet stage B, C). The puma expression levels in patients with positive expression of CD38 and ZAP-70 protein, elevating levels of LDH and beta₂-MG were sharply lower than those in patients without above-mentioned unfavorable factors. The puma expression was also correlated with molecular cytogenetic abnormalities, the puma expression levels in patients with trisomy 12 (p = 0.003) and 14q32 translocation (p = 0.045) detected by FISH were significantly lower than those in patients without above-mentioned molecular cytogenetic abnormalities. It is concluded that the qRT-PCR assay is reliable and sensitive. Puma mRNA expression is significantly correlated with a great deal of prognostic factors, and may be a prognostic marker of CLL.

Research progress on prognostic markers of chronic lymphocytic leukemia / 浙江大学学报·医学版

Chronic lymphocytic leukemia (CLL) remains the most common adult leukemia. The recent progress on research of molecular and cellular genetics of CLL promotes the development of the diagnosis, treatment and prognosis for CLL patients. IGVH gene mutation status is the most important prognostic marker for CLL patients. Zeta-chain-associated protein kinase (ZAP-70) can be used as a surrogate marker for IGVH mutation status. CD38 is a type II transmembrane glycoprotein promoting B cell activation and proliferation, which can improve the survival of CLL cells and enhance their proliferation, so it also can be used as an independent prognostic indicator for CLL. Chromosome aberrations are found in more than 80% of CLL cases. The most frequent abnormalities are losses of chromosomal material, with deletions in band 13q14 being the most common. The most common gains of chromosomal material are trisomies 12q. Human leukocyte antigen G (HLA-G) is a non-classical HLA-I gene. Increased expression of HLA-G leads to the malignant progression of CLL, significantly shortens survival, indicating HLA-G might serve as a prognostic marker in CLL. Toll-like receptors (TLA) are important component of natural immunity. The combination of TLR agonists and release chemotherapy, monoclonal antibodies and tumor vaccines would bring a breakthrough for the treatment of CLL.

Evaluation of standardized ZAP-70 expression detection in CLL--review / 中国实验血液学杂志

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical course. ZAP-70 expression shows a high concordance with IgVH gene mutational status and the method of detection for ZAP-70 expression is relatively simple, thus the ZAP-70 is used as a surrogate marker for IgVH gene mutational status. In recent years, ZAP-70 expression evaluation of different methods have shown difference that restrict the clinical application of ZAP-70. Therefore, the standardization of ZAP-70 expression detection has become a focus. The current review summarizes bio-characteristics, clinical application and detection method of ZAP-70. The detection methods of ZAP-70 are divided into analytical methods and experiment techniques. The analytical methods include percentage method, fluorescent quantitation and ratio method, in which the most important procedure is setting control. The experiment techniques include sample storage, time from blood draw to detection, fixation method, the antibody and the selection of fluorescence. Plenty of literatures have compared the variables, but the standardization of ZAP-70 expression detection method has not yet been decided.

Clinical and biological features of 8 patients with Richter's syndrome / 中国实验血液学杂志

In order to evaluate the clinical, biological features and prognostic factors of Richter's syndrome (RS), 8 RS patients were analyzed retrospectively. The serological test, multiplex parameter flow cytometry, conventional cytogenetic analysis, FISH technique and PCR combined with sequence detection were used to detect the LDH, β(2)-MG, TK1, SF, CA125, ZAP-70, chromosome karyotype, ATM and p53 gene deletion, as well as +12 abnormality and IgVH mutation. The results indicated that 7 out of 8 patients transformed to diffuse large B cell lymphoma (DLBCL) and 1 patient transformed to Hodgkin lymphoma (HL). Among 8 patients, LDH level in 7 patients, β(2)-MG level in 4 patients, SF level in 7 patients, CA-125 level in 4 patients and TK1 level in 1 patient exceeded the normal range. Meanwhile, ZAP-70 and CD38 were expressed positively in 4 and 7 out of 8 patients respectively. Unmutated IgVH was found in 5 patients, and 4 patients had the complex chromosome abnormalities. +12 and p53 deletion was found in 1 patient. 8 patients were divided into two groups (Binet A + B and Binet C), the mean time from diagnosis to progression was 98.5 months in Binet A + B group, compared with 38.3 months in Binet C group, there was significant difference between two groups (p = 0.021). Mean overall survival was 123.8 months and 49.8 months in two groups, respectively (p = 0.049). The mean survival after transformation was 34.5 months in Binet A + B group and 10.3 months in Binet C group. In conclusion, the level of LDH, β(2)-MG and SF are higher in RS patients in Binet C group, and so are the incidence of high expressed ZAP-70 and CD 38, unmutated IgVH. The clinical stage may be the risk and prognostic factors for RS transformation.

clinical utility of LPL and ZAP-70 expression in assessment of prognosis in patients with B-cell chronic lymphocytic leukemia

It was found that B-CLL patients with unmutated immunoglobulin heavy chain variable region [IgVH] have poor prognosis and clinical outcome. The difficulty of performing the mutation status of IgHV in the routine diagnostic workup of B-CLL patients, prompts the search for surrogate markers particularly gene expression profile in B-CLL cells. To investigate the role of LPL and ZAP-70 expression in assessment of prognosis and survival in a group of B-CLL patients and correlate the results with other prognostic variables. The study included 47 B-CLL patients who were subjected to clinical staging which was done by Binet and Rai scoring systems. The expression of LPL and ZAP-70 was measured by real-time quantitative PCR [RQ-PCR] and flow-cytometric analysis respectively. The patients were followed over 24 months for proper estimation of treatment free survival [TFS] and disease free survival [DPS]. The results of our study showed positive LPL and ZAP-70 expression in 46.8% and 48.9% of B-CLL patients respectively. There was significant correlation between LPL and ZAP-70 positive expression in our patients [p<0.0001]. The positive expression of both genes is associated with advance in clinical staging with significant correlation between LPL and ZAP-70 positive expression and shortening of the TFS and DPS and subsequent classification of most of the LPL+ and ZAP-70+ cases as patients with poor prognosis. Our study showed that expression of LPL and ZAP-70 has a significant role in determining the prognosis in B-CLL patients, being positive expression of LPL and ZAP-70 is associated with poor prognosis with shortening of TFS and DPS. Also, both genes expression is of great value in selection of B-CLL patients in early clinical stages that are in need to start chemotherapy to avoid progression to aggressive forms of the disease

Abnormality of serum immunoglobulin in peripheral blood of patients with chronic lymphocytic leukemia / 中国实验血液学杂志

In order to investigate the relation of serum immunoglobulin (Ig) level to age, sex, disease stages and prognosis in chronic lymphocytic leukemia (CLL) patients, the levels of serum IgG, IgA and IgM in 83 CLL patients were detected by immune rate turbidimetry. the expressions of CD38 and ZAP-70 in CLL cells were determined by multi-parameter flow cytometry (FCM). The results showed that among the 83 CLL patients, the IgG, IgA and IgM levels were reduced in 12 (14.5%), 26 (31.3%) and 34 cases (41.0%) respectively. The incidence of Ig reduction was higher in Binet C stage than that in Binet A and B (p=0.011). There was higher incidence of Ig reduction in high-risk group of Rai stage than that in low-risk group (p=0.011). The positive rate of CD38 or ZAP-70 was significantly higher in Ig reduction group than that in normal Ig level group (p=0.033 and p=0.038 respectively). The positive rate of CD38 and ZAP-70 was also higer in advanced disease, and among them the positive rate of ZAP-70 expression in Binet C stage was obviously higer than Binet A and B (p=0.047). Nonetheless, there was no significant relationship of Ig level with gender and age (p>0.05). It is concluded that the function of humoral immunity in CLL patient is closely related to the disease stages. The serum Ig detection seems important for evaluating immunologic state and prognosis of CLL patients.

Lipoprotein lipase and serum thymidine kinase level in chronic lymphocytic leukemia and their correlations with other prognostic factors / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate lipoprotein lipase (LPL) and serum thymidine kinase (TK) levels in chronic lymphocytic leukemia (CLL) and their correlations with other prognostic factors.</p><p><b>METHODS</b>LPL expression level in peripheral blood samples of 58 CLL patients was detected by semi-quantitative reverse transcription PCR (RT-PCR). Serum TK1 level in 39 CLL patients was detected by enhanced chemiluminescence (ECL) and TK monoclonal antibody (Anti-TK mAb). IgVH mutation status was detected by multiplex PCR and sequencing of purified PCR products. The expression of ZAP-70 protein and CD38 were determined by flow cytometry . Panel probes and fluorescence in situ hybridization (FISH) were used to detect cytogenetic aberrations.</p><p><b>RESULTS</b>The median LPL expression level in CLL was 0.26 (0 -6.29), while undetectable in normal controls. LPL expression level was significantly correlated with IgVH mutation status, Binet stages, CD38 and cytogenetic aberrations. Patients with unmutated IgVH genes had higher LPL than those with IgVH mutations (P = 0.010). Patients in Binet stage B and C had higher LPL than those in stage A (P = 0.011). LPL level was higher in patients with CD38 > or = 30% (P = 0.001). Higher LPL level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22) than those with favorable cytogenetics (deletion in 13q as the sole abnormality) (P = 0.002). LPL level was not significantly correlated with sex, age, and ZAP-70 protein (P > 0.05). The level of TK1 was higher in CLL patients than that in normal control (P < 0.05). Patients with higher level of absolute lymphocyte count (ALC), lactate dehydrogenase (LDH), unmutated IgVH genes and ZAP-70 had higher levels of TK1 than those with lower level of ALC, LDH, mutated IgVH genes and ZAP-70 (P = 0.018, P = 0.018, P = 0.030 and P = 0.038, respectively). TK1 level had no correlation with sex, age, Binet stages, CD38, and cytogenetic aberrations (P > 0.05).</p><p><b>CONCLUSIONS</b>LPL expression and serum TK1 levels significantly correlate with other CLL prognostic factors and could be predictive markers for IgVH mutation status. LPL and serum TK1 might be applied to the assessment of prognosis in CLL patients.</p>

Prognostic factors for chronic lymphocytic leukemia with typical and atypical immunophenotype / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the prognostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, absolute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations.</p><p><b>METHODS</b>According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophenotype CLL, 16 with score 4 or 3 were atypical immunophenotype CLL. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, sIg, CD20, CD79b expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations.</p><p><b>RESULTS</b>There was no significant difference between the two groups in sex, age, ZAP-70 and IgVH mutation status (P=0.398, P=0.189, P=0.268 and P=0.131, respectively). The incidence of ALC> or =50 x 10(9)/L, Binet B + C, CD38> or =30% in atypical CLL patients (43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P=0.026, P<0.01 and P=0.026, respectively). The proportion of typical patients (26.8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P=0.022).</p><p><b>CONCLUSION</b>There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.</p>

Clinical significance of ZAP-70 protein expression in chronic lymphocytic leukemia/small lymphocytic lymphoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features and prognostic significance of ZAP-70 protein expression in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).</p><p><b>METHODS</b>The histologic features of 52 cases of CLL/SLL with lymph node and/or bone marrow biopsies performed were retrospectively reviewed. Immunohistochemical study using EliVision for ZAP-70 protein was adopted.</p><p><b>RESULTS</b>The lymph nodes of the 12 cases studied showed effacement of the nodal architecture and was replaced by a monotonous infiltration of small lymphoid cells. Among them, proliferation centers were identified in 6 cases. Similar morphologic pattern was seen in the 40 bone marrow biopsy samples, but no proliferation center formation obtained. The infiltration pattern of tumor cells in the bone marrow were further subdivided into nodular (n = 9), interstitial (n = 3), mixed (n = 9) and diffuse types (n = 19). There was no significant difference found on survival rates between the diffuse infiltration and non-diffuse infiltration groups (Fisher's exact test, P = 0.199). ZAP-70 protein was mainly located in the cytoplasm and nuclei of lymphoma cells. There were 21 cases (40.4%) positive for ZAP-70 and among them, 11 died of this disease or the related infections. On the other hand, ZAP-70 was negative in 31 cases (59.6%) and only 4 of them died of this disease or related infections. The overall survival in ZAP-70-negative group was higher than that of the ZAP-70-positive group (59 months versus 39 months, chi(2) = 6.991, P = 0.008). Follow-up information was available in 51 patients. Among the 21 dead cases, 15 died of CLL/SLL or the related infection.</p><p><b>CONCLUSION</b>A positive expression of ZAP-70 protein in CLL/SLL suggests a poor prognosis.</p>