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

PD-1 and PD-L1 gene expressions and their association with Epstein-Barr virus infection in chronic lymphocytic leukemia.

PURPOSE: The  PD-1 (programmed cell death-1) receptor is expressed on the surface of activated T cells. Its ligand, programmed cell death ligand-1 (PD-L1), is expressed on the surface of dendritic cells or macrophages. The PD-1/PD-L1 interaction ensures prevention of autoimmunity by activating the immune system only when needed. In cancers, PD-L1 expressed on the tumour cells binds to PD-1 receptors on the activated T cells, leading to inhibition of the cytotoxic T cells and immunosuppression. PD-1/PD-L1 pathway is upregulated in EBV infection that is known to worsen the CLL prognosis. Therefore, we aimed to study the association between PD-1 and PD-L1 expressions, EBV status and the CLL prognosis. METHODS AND PATIENTS: The study was conducted on 80 newly diagnosed CLL patients and 80 controls. We analyzed PD-1 and PD-L1 expressions and EBV-DNA load by real-time PCR. The cytogenetic abnormalities and expression of ZAP70 and CD38 were detected by FISH and Flow cytometry, respectively. RESULTS: PD-1/PD-L1 expressions were significantly upregulated in CLL patients compared to controls. In addition, their mRNA levels were significantly higher in EBV( +) versus EBV( -) patients. High expression of PD-1/PD-L1 was associated with poor prognostic markers (RAI stages of CLL, del 17p13, ZAP70, and CD38 expression), failure of complete remission, shorter progression-free survival, and overall survival. CONCLUSION: High expression of PD-1 and PD-L1, together with high EBD-DNA load were linked to worse prognosis in CLL. In addition, PD-1 and PD-L1 might represent suitable therapeutic targets for patients suffering from aggressive CLL.

IFI16 reduced expression is correlated with unfavorable outcome in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Its clinical course is typically indolent; however, based on a series of pathobiological, clinical, genetic, and phenotypic parameters, patient survival varies from less than 5 to more than 20 years. In this paper, we show for the first time that the expression of the interferon-inducible DNA sensor IFI16, a member of the PYHIN protein family involved in proliferation inhibition and apoptosis regulation, is associated with the clinical outcome in CLL. We studied 99 CLLs cases by immunohistochemistry and 10 CLLs cases by gene expression profiling. We found quite variable degrees of IFI16 expression among CLLs cases. Noteworthy, we observed that a reduced IFI16 expression was associated with a very poor survival, but only in cases with ZAP70/CD38 expression. Furthermore, we found that IFI16 expression was associated with a specific gene expression signature. As IFI16 can be easily detected by immunohistochemistry or flow cytometry, it may become a part of phenotypic screening in CLL patients if its prognostic role is confirmed in independent series.

A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination.

A T cell mounts an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling.

Mapping cell populations in flow cytometry data for cross-sample comparison using the Friedman-Rafsky test statistic as a distance measure.

Flow cytometry (FCM) is a fluorescence-based single-cell experimental technology that is routinely applied in biomedical research for identifying cellular biomarkers of normal physiological responses and abnormal disease states. While many computational methods have been developed that focus on identifying cell populations in individual FCM samples, very few have addressed how the identified cell populations can be matched across samples for comparative analysis. This article presents FlowMap-FR, a novel method for cell population mapping across FCM samples. FlowMap-FR is based on the Friedman-Rafsky nonparametric test statistic (FR statistic), which quantifies the equivalence of multivariate distributions. As applied to FCM data by FlowMap-FR, the FR statistic objectively quantifies the similarity between cell populations based on the shapes, sizes, and positions of fluorescence data distributions in the multidimensional feature space. To test and evaluate the performance of FlowMap-FR, we simulated the kinds of biological and technical sample variations that are commonly observed in FCM data. The results show that FlowMap-FR is able to effectively identify equivalent cell populations between samples under scenarios of proportion differences and modest position shifts. As a statistical test, FlowMap-FR can be used to determine whether the expression of a cellular marker is statistically different between two cell populations, suggesting candidates for new cellular phenotypes by providing an objective statistical measure. In addition, FlowMap-FR can indicate situations in which inappropriate splitting or merging of cell populations has occurred during gating procedures. We compared the FR statistic with the symmetric version of Kullback-Leibler divergence measure used in a previous population matching method with both simulated and real data. The FR statistic outperforms the symmetric version of KL-distance in distinguishing equivalent from nonequivalent cell populations. FlowMap-FR was also employed as a distance metric to match cell populations delineated by manual gating across 30 FCM samples from a benchmark FlowCAP data set. An F-measure of 0.88 was obtained, indicating high precision and recall of the FR-based population matching results. FlowMap-FR has been implemented as a standalone R/Bioconductor package so that it can be easily incorporated into current FCM data analytical workflows.

Peripheral blood lymphocyte phenotype of ZAP-70⁺ and ZAP-70⁻ patients with B-cell chronic lymphocytic leukaemia.

BACKGROUND: Up to now, the immune status of chronic lymphocytic leukemia (CLL) patients in association with the expression of zeta-chain-associated protein kinase 70 (ZAP-70) in leukemic cells has not been evaluated. AIM: The aim of this work was the study of the peripheral blood (PB) T-lymphocyte phenotypes in ZAP-70-positive (ZAP-70(+)) and ZAP-70-negative (ZAP-70(-)) untreated patients with CLL. MATERIALS AND METHODS: ZAP-70-, CD25-, CD3-, CD4-, and CD8-positive lymphocytes were enumerated by flow cytometry in PB of 120 untreated CLL patients. CD8+, CD3+CD4+ and CD3+CD25+ cells were counted for the non-leukemic lymphocytes. RESULTS: The patients were distributed into two groups: the ZAP-70(+) group of high CLL progression (n = 61), and the ZAP-70(-) group of low CLL progression (n = 59). In the ZAP-70(+) group, the ratio CD4/CD8 (0.33 ± 0.62; p = 0.001) and the numbers of the CD3+ (34.8 ± 8.1%; p = 0.01), CD3+CD4+ (24.4% ± 4.8; p = 0.001), and CD3+CD25+ (6.2 ± 0.91%; p = 0.001) lymphocytes were reduced and the percentage of the CD8+ cells (73.1 ± 4.6%; p = 0.0001) was above the norm. In the ZAP-70(-) group, the number of the CD3+CD4+ cells (36.9 ± 6.1%; p = 0.001) was within the norm, but the numbers of the CD8+ (11.3 ± 1.1%; p = 0.0001) and CD3+ (41.2 ± 5.3%; p = 0.05) lymphocytes were reduced; the ratio CD4/CD8 (3.26 ± 0.88; p = 0.001) and the percentage of the CD3+CD25+ cells (27.1 ± 3.4%; p = 0.0001) were above the norm. CONCLUSIONS: Our data show that the increased CD4/CD8 ratio, caused by the reduced number of the CD8+ lymphocytes, and the increased number of CD3+CD25+ cells are characteristic for the ZAP-70(-) group (slow progressing) of untreated CLL patients. In ZAP-70(+) patients, the CD4/CD8 ratio was significantly below the norm indicating an active disease process. Results of our study contribute to identification of CLL patients with different prognosis in routine diagnostic/prognostic procedures.

Thalidomide regulation of NF-κB proteins limits Tregs activity in chronic lymphocytic leukemia.

BACKGROUND: Thalidomide may represent a novel therapeutic strategy in the treatment of chronic lymphocytic leukemia (CLL). Since the activation of nuclear factor kappa B (NF-κB) causes not only malignant transformation and tumor progression, but also allows tumor cells to evade immune surveillance, NF-κB signaling components might constitute a potential target for future therapy in CLL. OBJECTIVES: The current study is an attempt to characterize proteins regulated by thalidomide. Thalidomide's influence on NF-κB proteins and on regulatory T cells (Treg) in CLL was investigated. MATERIAL AND METHODS: A total of 15 patients with CLL were treated with a combined thalidomide/fludarabine regimen. Peripheral blood mononuclear cells were separated by Ficoll density gradient centrifugation. To evaluate glucocorticoid induced tumour-necrosis-factor-receptor-related protein (GITR) expression in regulatory T cells, cells incubated with anti-CD3, ani-CD4 and anti-CD25 were permeabilized and then stained with anti-FOXP3 and analyzed using flow cytometry. Human TNF enzyme-linked immunosorbent assay (ELISA) was used to determine the tumor necrosis factor (TNF) levels in the serum. To evaluate NF-κB activity, chemiluminescent oligonucleotide-based ELISA was performed. RESULTS: It was found that thalidomide regulates NF-κB activity differentially, and the activity of certain NF-κB components correlated with TNF levels and T regulatory cell (CD4 + CD25 high GITR + ). CONCLUSIONS: These results might indicate that thalidomide not only regulates TNF but also directly interferes with NF-κB components.

Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP-70 protein expression in neoplastic B-cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting. METHODS: In this study, we characterized the IgVH status and ZAP-70 expression by molecular techniques in a cohort of 108 patients with CLL, and correlated these results with three different methods of ZAP-70 expression by flow cytometry. We then assessed the results of these methods in terms of prognostic power as characterized by time to first treatment (TTFT). RESULTS: By comparing three different flow cytometry methods using receiver­operator curve (ROC) analysis, we identified that by utilizing a corrected mean fluorescence intensity (CorrMFI) algorithm for assessing ZAP-70 expression, there was good correlation with both IgVH mutational status, and ZAP-70 expression as assessed by qPCR. We were also able to show that ZAP-70 expression, as assessed by both qPCR and the CorrMFI method, was prognostic of TTFT. CONCLUSIONS: While confirmation in a larger patient cohort, with longer follow-up is required, we believe that the CorrMFI represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients.

Molecular biomarkers in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World and remains incurable with conventional chemotherapy treatment approaches. CLL has a highly varied clinical course. The substantial clinical variability in the clinical course of CLL has motivated intense efforts at identifying molecular markers that can be used for CLL prognostication. While many such markers have been proposed, few have stood the test of time; this is due to various reasons outlined in detail in this chapter.Of the reasons that have affected the usefulness and broad applicability of CLL biomarkers a few stand out as recurrent: lack of independent effects of individual markers on prognosis; the use of arbitrary cutoffs when using continuous variables; technical challenges in validity, reproducibility, and reliability (classical test characteristics); and lack of marker validation in prospectively identified CLL patient cohorts.Nonetheless, a few useful prognostic markers (CLL interphase FISH, immunoglobulin heavy chain variable region mutation status) have been identified, and others are still in transition to widespread clinical applications (TP53 mutations, SNP array-based elevated genomic complexity).As CLL therapy transitions from genotoxic combination therapies to targeted therapies, it will be of importance to reestablish the usefulness of our current understanding of individual CLL traits in CLL prognosis. Finally, the identification of predictive markers remains important given the established associations of poor response rates with shortened survival and the ongoing need for more personalized approaches in CLL management.

Pathology consultation on evaluating prognosis in incidental monoclonal lymphocytosis and chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a monoclonal B-cell lymphoproliferative disorder generally characterized by an indolent clinical course. However, some patients with CLL will have more aggressive disease progression, and identifying that subgroup may be important for early, or perhaps more aggressive, intervention. In addition, monoclonal B-cell lymphocytosis is often found on routine laboratory evaluation, and it is important to distinguish this entity from overt CLL. Moreover, since many patients with CLL are discovered incidentally and before significant disease progression, prognostic laboratory evaluation may become increasingly efficacious as therapeutic options replace the older strategy of expectant observation. Prognostication may be especially critical if it correctly identifies patients with early stage CLL who are at high risk of clonal evolution and/ or resistance to chemoimmunotherapy. Laboratory studies include surface CD38 and intracellular ZAP-70 expression by flow cytometry, serum ß2-microglobulin, and immunoglobulin heavy-chain variable gene mutational status. Cytogenetics for targeted chromosome alterations may similarly aid in predicting outcome and guiding early intervention. This article concisely reviews the utility of commonly performed prognostic markers and addresses the laboratory evaluation in patients with incidentally discovered early stage CLL.

Quantitative detection of zeta-chain-associated protein 70 expression in chronic lymphocytic leukemia.

Overexpression of zeta-chain-associated protein 70 (ZAP-70) was recently recognized as an independent prognostic marker for the aggressive form of chronic lymphocytic leukemia (CLL). The objective of this study was to demonstrate the feasibility and implementation of quantitative detection of ZAP-70 protein in B cells to clearly distinguish patients with CLL with the aggressive form of the disease. B cells were isolated from patient blood and lysed. Released ZAP-70 protein was detected using an immunomagnetic fluorescence assay. The assay protocol was developed using Jurkat cells and recombinant ZAP-70 (rZAP-70). The limit of detection was determined to be lower than 125 Jurkat cells and 39 pg of rZAP-70 protein. The signal response was linear over a wide dynamic range, from 125 to 40 000 Jurkat cells per test (R(2) = 0.9987) and from 0 to 40 000 pg rZAP-70 protein per test (R(2) = 0.9928). The results from 20 patients with CLL correlated strongly with flow cytometry analysis. Concordance between the two methods for positive and negative results was 100% (7/7) and 92% (12/13), respectively, while the overall concordance between the two methods was 95%. The assay reported here is a simple, reliable and reproducible method for quantitative detection of ZAP-70 in patient leukemic cells, without the need for cell fixation or permeabilization. The ZAP-70 signal was linear over a wide dynamic range, which we believe enables quantitative assessment of small changes in ZAP-70 expression over the course of the disease and in response to therapeutic intervention.

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B-cells due to ZAP70 overexpression in patient T-cells.

Flow cytometry is the reference technique for assessing ZAP70 expression, a marker of poor prognosis in CLL. One of the most common methods is to assess ZAP70 levels in CLL cells by calculating the ratio between ZAP70 mean fluorescence intensities (MFIs) in residual T-cells and CLL B-cells (ZAP70 T/B ratio). In this study, we developed a new method for ZAP70 labeling. Cells were labeled with a combination of anti ZAP70 phycoerythrin-conjugated SBZAP monoclonal antibody (mAb) and mAbs against CD45, CD19, and CD5. The latter three were used to specifically gate on different lymphocyte subsets. Staining was performed in absence (test) or in presence of excess unconjugated SBZAP mAb (isoclonic control). A so-called ZAP70 isoclonic ratio between SBZAP MFIs in the test and isoclonic control was calculated. A series of 32 patients with CLL and 10 normal controls were studied. Prediction of IGHV mutation status by ZAP70 isoclonic and T/B ratios was similar. By using the ZAP70 isoclonic ratio, we showed that ZAP70 expression was increased in T-cells from CLL patients. Nearly all cases with increased ZAP70 expression in CLL cells were associated with high ZAP70 expression in cognate T-cells. Therefore, the ZAP70 isoclonic ratio was more likely to closely reflect the biology of ZAP70 dysregulation rather than the T/B ratio. These results also explained why ZAP70 T/B ratios were artefactually close to normal in cells from CLL patients with high levels of ZAP70.

Expression level of DEK in chronic lymphocytic leukemia is regulated by fludarabine and Nutlin-3 depending on p53 status.

Human oncogene DEK has been shown to be upregulated in a number of neoplasms. The purpose of this study was to investigate DEK expression level in chronic lymphocytic leukemia (CLL), analyze the correlation between DEK expression and CLL prognostic markers, and characterize the role of DEK in the response to either chemotherapeutic drugs or nongenotoxic activators of the p53 pathway. DEK mRNA was evaluated by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR), and primary CLL samples were treated in vitro with either fludarabine or Nutlin-3 to explore the interaction of p53 status and DEK mRNA expression. The median expression levels of DEK mRNA were 6.792 × 10 (-2) (1.438 × 10 (-2) -3.201 × 10 (-1) ) in 65 patients with CLL. A marked increase of DEK mRNA expression was observed in the CLL patients with unmutated immunoglobulin heavy chain variable (IGHV) gene (p = 0.025), CD38-positive (p = 0.047), del(17p13) (p = 0.006). Both fludarabine and Nutlin-3 significantly downregulated DEK in the primary CLL cells which were with normal function of p53, or without deletion or mutation of p53 (p = 0.042, p = 0.038; p = 0.021, p = 0.017; p = 0.037, p = 0.017). However, the downregulation of DEK was not observed in the primary CLL cells which were with dysfunction of p53, or with deletion or mutation of p53 (p = 0.834, p = 0.477; p = 0.111, p = 0.378; p = 0.263, p = 0.378). These data show that DEK might be applied for the assessment of prognosis in patients with CLL, and fludarabine and Nutlin-3 regulate DEK expression depended on p53 status.

[The prognostic value of smudge cells (Gumprecht shadows) in chronic lymphocytic leukaemia]. / A Gumprecht-rögök arányának prognosztikus értéke krónikus lymphoid leukaemiában.

INTRODUCTION: Smudge cells (Gumprecht shadows) are chronic lymphocytic leukaemic cells ruptured during peripheral blood smear preparation. It has been demonstrated to be linked to reduced expression of the cytoskeletal protein vimentin and its inverse correlation with the clinical outcome of the disease. AIMS: Investigation of the percentage of smudge cells, CD38-, ZAP-70-positive cells and the time to treatment in patients with chronic lymphocytic leukaemia. METHODS: Authors investigated the percentage of smudge cells, CD38- and ZAP-70-positive cells in the peripheral blood of 50 patients with chronic lymphocytic leukaemia and their correlation with the time to treatment. RESULTS: 21 patients required treatment in the follow-up period. Their median smudge cell percentage was 9.9%, while it was 26.8% in the non-treated group. The cut-off value of smudge cell positivity was set to 20%. 59.3% of the patients with less than cut-off had to be treated in the follow-up time compared to 21.7% of patients with more smudge cells. These findings were similar to the prognostic value of CD38 and ZAP-70. The necessity of treatment increased to 75-77.8% with the combination of investigated markers. The time to treatment was 19 months when smudge cells were less than 20%, but above 20% it was 36.15 months. In case of low smudge cell percentage and CD38 positivity the time to treatment was 14.14 months and in case of high smudge cell percentage and CD38 negativity it was 32.92 months. In discordant cases the time to treatment was 18.43 months. The authors also present a case report that demonstrates the relationship between the percentage of smudge cells and apoptotic cells with annexin V and 7-AAD staining. CONCLUSIONS: Estimation of smudge cells on a blood smear could be a simple and cheap prognostic test in chronic lymphocytic leukaemia with sensitivity similar to CD38 and ZAP-70 estimation. Combination of these tests raised the sensitivity of their prognostic value.

Diagnosis and classification of B-cell non-Hodgkin lymphomas. The role of multiparameter flow cytometry.

Immunophenotyping is a routine method to evaluate B-cell non-Hodgkin lymphomas. Flow cytometry plays a complementary role in diagnosis and classification of these types of lymphomas, since combination of morphologic, immunophenotypic and genotypic features is needed to correctly classifying each disease entity. Multiparameter flow cytometry, which is now carried out with routine combinations of six to eight monoclonal antibodies, allows identifying even small lymphomatous cell populations on the basis of aberrant B-cell marker expression and clonality. The immunophenotypic patterns obtained by multiparameter flow cytometry are useful to correctly diagnose most of cases of specific subtypes of B-cell non-Hodgkin lymphomas and to discover peculiar clinical presentations, such as discordant and composite lymphomas. Immunophenotypic variability, however, characterizes B-cell lymphomas. Therefore, flow cytometry should always be used in combination with other techniques to correctly classify each disease entity. Finally, multiparameter flow cytometry is characterized by high sensitivity in detecting residual disease.

Comprehensive assessment of prognostic factors predicting outcome in Chinese patients with chronic lymphocytic leukemia treated with fludarabine and cyclophosphamide.

To determine whether prognostic factors remain relevant to chronic lymphocytic leukemia (CLL) patients treated with fludarabine and cyclophosphamide (FC), we prospectively evaluated 86 Chinese CLL patients who received FC in first-line therapy. Twenty-four patients (27.9%) achieved complete remission (CR), and overall response rate was 75.6%. With a median follow-up of 41 months, the median progression-free survival (PFS) was 36.0 months and median overall survival (OS) has not been reached. The strong correlations of lower CR rate with advanced Binet stage, unmutated IGHV, cytogenetic abnormalities of del(17p13) or del(11q23), and p53 mutations were observed by univariable analyses. Stepwise logistic regression identified that unmutated IGHV and p53 abnormality (p53 deletion or mutation) were associated with a decreased odds of achieving CR. The less cycles of treatment, not achieving CR, advanced Binet stage, and p53 abnormality significantly correlated with a shortened PFS. Furthermore, in a multivariate analysis, p53 abnormality and advanced Binet stage were identified as being significant risk factors for early relapse. Not achieving CR, advanced Binet stage, ZAP-70-positive, and p53 abnormality were the adverse factors in determining OS. Only p53 aberration was independently associated with significantly shorter OS by a multivariate analysis. These results suggest that patients with p53 abnormality should be considered for alternative therapies.

Detection methods of ZAP-70 in chronic lymphocytic leukemia.

The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous, with some patients experiencing rapid disease progression and others living for decades without requiring treatment. Immunoglobulin heavy chain variable region (IGHV) mutation status is a powerful prognostic factor in patients with CLL. The presence or absence of IGHV mutation status is currently the gold-standard prognostic factor, but this technique is labor-intensive and costly. The expression of ζ-chain-associated protein kinase 70 kDa (ZAP-70), detected by flow cytometry, has been served as a potential surrogate for the absence of IGHV mutation status since 2003. Given the current problems with the standardization of ZAP-70 assessment by flow cytometry, this chapter reviews the analysis of ZAP-70 expression by flow cytometry and the relative experimental conditions using the associated publications for "ZAP-70" and "detection method" on Medline since 2003. Thus, mean fluorescence intensity (MFI) ratio method yielded a more reproducible and easily adaptable method to routine use in the clinical diagnostic laboratory. In conclusion, though ZAP-70 is a valuable prognostic factor in CLL, detection methods of ZAP-70 remain to be fully standardized.

Combined normal donor and CLL: Single tube ZAP-70 analysis.

INTRODUCTION: Zeta-chain-associated protein kinase 70 (ZAP-70) has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment. METHODS: Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP/PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ND B-cell ratio; and modified Z-index. RESULT: Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and immunoglobulin heavy chain variable (IGHV) mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, and clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (P < 0.0001). CONCLUSION: The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.

Improved flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia using experimentally optimized isotypic control antibodies.

INTRODUCTION: Expression of ZAP-70 by chronic lymphocytic leukemia (CLL) is associated with more aggressive disease and can help differentiate CLL using mutated immunoglobulin heavy chain variable genes (VH) from cases expressing unmutated VH genes. However, flow cytometric detection of ZAP-70 in CLL shows considerable variability and may be of questionable significance because most laboratories cannot correlate their results to clinical outcome or VH mutational data. METHODS: Seventy cases of CLL were evaluated for ZAP-70 using a previously optimized staining procedure and two different methods to eliminate nonspecific background staining. One method, not previously reported, used isotypic control antibodies, where the concentrations were adjusted/optimized so that normal B-cells stained negatively for ZAP-70. The other used ZAP-70 stained peripheral blood B-cells from normal donors. The percentages of ZAP-70 stained CLL cells above the two thresholds were compared. RESULTS: Concentrations of isotypic control antibodies had to be increased from manufacture's recommendations to insure normal B-cells were ZAP-70 negative. ZAP-70 levels among the CLL cases formed a bimodal distribution using the optimized isotypic control threshold, with 30 having low values (0-32% positive) and 40 high values (60-99% positive). In contrast, a continuous distribution was obtained with the ZAP-70 stained B-cell threshold. VH mutational status strongly correlated with the optimized control values as 29/30 low ZAP-70 cases had mutated VH genes and 37/40 high ZAP-70 cases used unmutated VH genes. CONCLUSIONS: Use of an optimized isotypic control threshold could increase the reliability of flow based ZAP-70 detection and correlates well with VH mutational status.

Augmentation of T-cell immune responses and signal transduction proteins in oral cancer patients: potential for IL-2-mediated immunotherapy.

PURPOSE: Immune impairment is hypothesized to be one of the reasons for the dismal treatment response in oral cancers. This study evaluates the immune impairment in patients with primary squamous cell carcinoma of the oral cavity and the effect of IL-2 administration on restoration of the immune responses. METHODS: T-cell populations were enumerated by flow cytometry; T-cell function by MTS proliferation assay to PHA and anti-CD3, expression of T-cell signaling proteins ZAP-70, TCRζ, p(56)lck, PKC and CD-ε in T cells with and without activation by IL-2 using Western blot and statistical analysis using X (2) test and bivariate correlation analysis in 112 patients. RESULTS: Reduction in proportion of CD3(+) and CD4(+) T lymphocytes, decrease in the CD4(+)/CD8(+) T-cell ratios, reduced lymphocyte transformation to PHA and anti-CD3 and reduced production of interleukin-2(IL-2) were observed in the patient group. Lymphocyte proliferation to anti-CD3 could be augmented in 59.5% of non-responders by IL-2 (range 10-90%) along with significant increase in the expression of TCR-ζ and ZAP-70, CD3ε, p(56) LCK and PKC to varying degrees. The expression of ZAP-70 and TCR-ζ was found to be closely related to treatment response and could be augmented by IL-2 in terms of proliferation and IL-2 production. CONCLUSIONS: The results suggest IL-2 to augment T-cell responses in a proportion of oral cancer patients with poor response to conventional therapy. IL-2 immunotherapy can be thought of as a personalized adjuvant therapy for oral cancer following the in vitro identification of IL-2 responders using the expression of TCRζ and ZAP-70 as biomarkers.